28.46 13.86 Method Plasma1 Plasma2 Plasma3 Plasma4 … · and SPE, for Quantitative LC-MS Analysis...

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Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA Poster Note 64801 ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research. INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst. MATERIALS AND METHODS Calibrators and QC Samples •1.0 mg/mL MMA and 100 μg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1. Table 1. Concentrations of MMA in QC samples Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer Data Analysis Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing. Precipitation-Derivatization Method Sample Preparation • Mix: 100 μL of plasma sample + 200 μL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 μl of supernatant to disposable culture tube containing 50 μl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly recommended for the nitrogen flow. • Add 200 μL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to dryness at room temperature. (No reaction time is needed) • Add 300 μL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into an HPLC vial with limited volume insert if needed. • Inject 20 μL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 μm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583 Table 2. SRM Experimental Parameters Analyte Precursor (m/z) Product Ion (m/z) Collision Energy (V) Qualification / Qualification Polarity MMA 231.2 175.2 6 Qualifier Positive MMA 231.2 119.2 11 Quantifier Positive d3-MMA 234.2 178.2 6 Qualifier Positive d3-MMA 234.2 122.2 11 Quantifier Positive Table 3. LC Gradient Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 μL methanol followed by 200 μL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing • Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 μL for LC/MS analysis Time (min) Flow rate (mL/min) %A %B 0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2 Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4) Table 4. SRM Experimental Parameters (SPE) Analyte Precursor (m/z) Product Ion (m/z) Collision Energy (V) Qualification / Qualification Polarity MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 μm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3) SPE Method

Transcript of 28.46 13.86 Method Plasma1 Plasma2 Plasma3 Plasma4 … · and SPE, for Quantitative LC-MS Analysis...

Page 1: 28.46 13.86 Method Plasma1 Plasma2 Plasma3 Plasma4 … · and SPE, for Quantitative LC-MS Analysis RT: 1.54 of Methylmalonic Acid in Plasma for Clinical Research ... 1.74 0.5 60 40

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta KozakThermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

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NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

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This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

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CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

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NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

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NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

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NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

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NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

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NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

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NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

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NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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RT: 1.89 2.45 2.582.01

1.70

RT: 1.90RT: 2.27

2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1001.31

1.32

1.83

0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.55

1.30

RT: 1.56

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RT: 1.54

RT: 1.54

NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.56

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RT: 1.54

RT: 1.54

NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.58

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.86

RT: 2.22

1.86

RT: 2.21

NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

0

10

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100RT: 2.22

RT: 2.22

1.86

RT: 2.20

NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

0

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40

50

60

70

80

90

100RT: 2.25

RT: 2.24

1.88

RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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RT: 1.89 2.45 2.582.01

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2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

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2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

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NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

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NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

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NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

Page 2: 28.46 13.86 Method Plasma1 Plasma2 Plasma3 Plasma4 … · and SPE, for Quantitative LC-MS Analysis RT: 1.54 of Methylmalonic Acid in Plasma for Clinical Research ... 1.74 0.5 60 40

2 Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research

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NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

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This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

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CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1001.31

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1.83

0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.54

NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.54

RT: 1.54

NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.86

RT: 2.22

1.86

RT: 2.21

NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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10

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RT: 2.22

1.86

RT: 2.20

NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 2.24

1.88

RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

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2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

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NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

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NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

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NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

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NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

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NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

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CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

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MMA Qualifier

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NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

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1.86

RT: 2.22

1.86

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NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.86

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NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.88

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NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

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NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

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0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.86

RT: 2.22

1.86

RT: 2.21

NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 2.22

1.86

RT: 2.20

NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 2.24

1.88

RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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RT: 1.89 2.45 2.582.01

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2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

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1001.31

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0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

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NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

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NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

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RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

Page 3: 28.46 13.86 Method Plasma1 Plasma2 Plasma3 Plasma4 … · and SPE, for Quantitative LC-MS Analysis RT: 1.54 of Methylmalonic Acid in Plasma for Clinical Research ... 1.74 0.5 60 40

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

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2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

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NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

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NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

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NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

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RT: 1.90RT: 2.27

2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1001.31

1.32

1.83

0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.55

1.30

RT: 1.56

1.29

RT: 1.54

RT: 1.54

NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.56

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RT: 1.54

RT: 1.54

NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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RT: 1.58

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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50

60

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80

90

100RT: 2.22

1.86

RT: 2.22

1.86

RT: 2.21

NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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10

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0

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40

50

60

70

80

90

100RT: 2.22

RT: 2.22

1.86

RT: 2.20

NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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40

50

60

70

80

90

100RT: 2.25

RT: 2.24

1.88

RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

a RT: 1.58 - 2.71 SM: 5B

1.6 1.8 2.0 2.2 2.4 2.6Time (min)

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RT: 1.89 2.45 2.582.01

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2.472.04 2.642.151.74

RT: 2.26

NL: 1.04E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082615_cal1_25

NL: 6.07E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082615_cal1_25

NL: 9.89E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082615_cal1_25

MMA Quantifier

MMA qualifier

D3-MMA

This is the butylated succinic acid peak. It is an endogenous compound always present in plasma samples. Succinic acid is isobaric with MMA and has to be chromatographically separated.

25 nM

a

25 nM

b

CONCLUSIONS •Both sample preparation methods allow for LC-MS analytical performance meeting clinical lab requirements. •Both sample preparation methods can be automated. •The use of the newly discovered additive-catalyst is critical in the MMA derivatization method. It is proven to be highly effective in approving derivatization reaction completion, and consequently, the reproducibility of the derivatization reaction itself (without internal standard correction) is achievable (it is not achievable for most published MMA derivatization methods). •Selection of specific sample preparation method method by the lab will depend on other then analytical performance factors e.g. consumables cost or sample preparation workflows already used in the lab.

TRADEMARKS/LICENSING © 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others. For research use only. Not for use in diagnostic procedures.

Mindy Gao, Joe DiBussolo, Xiaolei Xie, Kristine Van Natta, Marta Kozak, Thermo Fisher Scientific, 355 River Oaks Pkwy, San Jose, CA 95134, USA

METHOD PERFORMANCE EVALUATION

Figure 1. Calibration curves (25 nM – 100,000 nM) and lowest calibration standard peaks for quantifying and qualifying product ions (a: Deriv. method, b: SPE method)

ABSTRACT To evaluate two sample preparation methods, precipitation-derivatization method (APCI in positive mode), and SPE method (HESI in negative mode), for the quantitative LC/MS analysis of methylmalonic acid (MMA) in human plasma samples using a triple quadrupole mass spectrometer for use in clinical research.

INTRODUCTION Liquid chromatography-mass spectrometry (LC-MS) analytical methods are widely used for analysis of methylmalonic acid (MMA) in clinical research laboratories. Simple sample preparation, low limits of quantitation and separation from the naturally occurring structural isomer succinic acid (SA) are among the analytical method requirements. Here we evaluated the performance of two sample preparation methods for LC-MS analysis that fulfilled all of these requirements for clinical research needs. The SPE method (SPE) utilized new Thermo Scientific™ SOLAµ™ extraction plates which use small volumes of eluting solvent resulting in elimination of the evaporation and reconstitution steps needed in conventional SPE methods. The protein precipitation-derivatization method (Deriv) was optimized to improve efficacy and reproducibility of derivatization reaction using a new additive-catalyst.

MATERIALS AND METHODS

Calibrators and QC Samples •1.0 mg/mL MMA and 100 µg/mL internal standards (d3-MMA) were purchased form Cerilliant (Round Rock, TX) •Since analyte free matrix was not available calibration standards were prepared in aqueous solution with 2% acetonitrile. •QC samples Level I and Level II were purchased from RECIPE Chemicals (Munich, Germany). •Level 0 QC was prepared from pooled donor plasma samples. •Concentrations of QC samples and calibrators are specified in Table 1.

Table 1. Concentrations of MMA in QC samples

Instrument •Thermo Scientific TM Dionex TM UltiMate TM 3000 HPLC system •Thermo Scientific TM OAS-3X00TXRS autosampler •Thermo Scientific TM TSQ Endura™ triple quadrupole mass spectrometer

Data Analysis

Thermo Scientific™ TraceFinder™ software version 3.2 was used for data acquisition and data processing.

Precipitation-Derivatization Method Sample Preparation • Mix: 100 µL of plasma sample + 200 µL of mixture of acetonitrile:methanol (9:1, v:v) containing 250 nM

internal standard (d3-MMA). Vortex, centrifuge • Transfer 100 µl of supernatant to disposable culture tube containing 50 µl of 1M zinc sulfate • Evaporate* to dryness at room temperature under stream of nitrogen. A gas drying unit is highly

recommended for the nitrogen flow. • Add 200 µL of 3N HCl in n-butanol to each sample tube, then again evaporate* under nitrogen to

dryness at room temperature. (No reaction time is needed) • Add 300 µL of 50% methanol to each sample, vortex thoroughly, centrifuge. Transfer supernatant into

an HPLC vial with limited volume insert if needed. • Inject 20 µL onto LC-MS system. • *: Evaporation under elevated temperature will shorten the drying time LC method • Column: Thermo Scientific TM Accucore TM C8, 2.6 µm, 50 x 2.1 mm at ambient • Mobile Phase A: 0.1% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Isocratic separation at 63.5% Mobile Phase B • Flow rate: 0.5 mL/min

Mass Spectrometry Method • APCI in positive ionization mode • SRM method with ion ratio confirmation (Table 2) Table 2. SRM Experimental Parameters

Evaluation of Two Sample Preparation Methods, Precipitation-Derivatization and SPE, for Quantitative LC-MS Analysis of Methylmalonic Acid in Plasma for Clinical Research.

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

Plasma Level 0 (Deriv/SPE) RECIPE Level I RECIPE Level II 81.7 / 110 260 583

Table 3. LC Gradient

Sample Preparation • Conditioning the Thermo Scientific™ SOLAµ WAX 2mg/1mL/96 well plate (P/N: 60209-005) with 100 µL

methanol followed by 200 µL 5 mM ammonium acetate at pH 4 (adjusted with formic acid) • Centrifuge the plasma samples to ensure smooth SPE flow without clogging • Deliver 100uL of calibrator, QC, or plasma sample onto the well which containing 300 uL of 5 mM

ammonium acetate at pH 4 (adjusted with formic acid) with 5 uM d3-MMA internal standard, aspirate and dispense one time using the same pipet tip for better mixing

• Wash well with 100 uL of water (HPLC grade) • Elute 2x 35 uL of 1% NH3 into the collecting plate which containing 30 uL of 10% formic acid • Shake the plate for 30 sec, or transfer eluent into an HPLC vial with limited volume insert if needed. • Inject 15 µL for LC/MS analysis

Time (min) Flow rate (mL/min) %A %B

0 0.3 98 2 1.19 0.3 98 2 1.2 0.5 98 2 1.49 0.5 98 2 1.5 0.5 60 40 1.74 0.5 60 40 1.75 0.5 98 2 1.85 0.7 98 2 2.5 0.7 98 2

Mass Spectrometry Method • HESI in negative ionization mode • SRM method with ion ratio confirmation (Table 4)

Table 4. SRM Experimental Parameters (SPE)

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 117.1 55.3 25 Qualifier Negative MMA 117.1 73.3 10 Quantifier Negative

d3-MMA 120.1 58.3 25 Qualifier Negative d3-MMA 120.1 76.3 10 Quantifier Negative

LC Method • Column: Thermo Scientific TM Accucore TM RP-MS, 2.6 µm, 100 x 2.1 mm at ambient • Mobile Phase A: 0.4% formic acid in water • Mobile Phase B: 0.1% formic acid in methanol • Flow rate: 300 – 700 uL/min (LC gradient: Table 3)

SPE Method

RESULTS

Limit of quantitation (LOQ) were defined as the lowest concentrations that had back-calculated values within 20% of nominal and RSD for 5 replicates within 20%. Figure 1 shows chromatograms of the lowest calibration standards.

Method precision and accuracy was evaluated by analyzing replicates of each QC sample (n=4, 5) in three different days (Table 5 and Table 6) and by analyzing replicate injections of each calibration standard (Table 7).

Matrix effects and procedure recoveries • Absolute % recoveries: Internal standard spiked into 10 (Deriv) or 8 (SPE) processed donor plasma samples. Absolute

% recovery was calculated against the same concentration of internal standard spiked into solvent (n=3) (Table 8).

• Spiked recovery: 100 nM of MMA were spiked into 10 donor plasma samples. Un-spiked and spiked donor plasma samples were analyzed in triplicates. Relative and absolute % recoveries were calculated against data collected for spiked solvent (Table 9).

Reproducibility of derivatization reaction: %RSD of internal standard peak area in processed blank plasma samples from 10 donors (n=5) were calculated (Table 10). Reproducibility may be underestimated due to any possible matrix effects.

Table 5. Intra-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 2.14 - 5.42 1.36 - 2.63 1.81 - 2.36 %RSD SPE (n=4) 2.45 - 5.10 0.81 - 1.99 0.35 - 0.93

% Recovery Deriv. (n=5) 92.5 - 97.6 86.8 - 89.9 94.1 - 94.6 % Recovery SPE (n=4) 89.7 - 101 91.8 - 95.3 96.9 - 104

Table 6. Inter-assay precision and accuracy. Replicates of QC samples analyzed in 3 batches

Analyte Method Level 0 Level I Level II Norminal Conc. (nM) 81.7 / 110 260 583

%RSD Deriv. (n=5) 4.6 2.4 2.0

%RSD SPE (n=4) 6.4 2.2 3.5

% Recovery Deriv. (n=5) 95.7 88.5 94.4

% Recovery SPE (n=4) 96.7 93.1 99.7

Calculated Amt %Diff 28.46 13.86 45.83 -8.34 97.42 -2.58 476.0 -4.8 957.8 -4.22 10338 3.38 25474 1.89 51541 3.08 97716 -2.28

Figure 2. Chromatogram of quantifying ion and confirming ion at LOQ concentration (a: deriv. method, b: SPE method)

b

MMA Quantifier

Blank - SPE

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.50 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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0.98 1.581.11 1.68 2.001.42 2.45

1.03 1.06 1.730.98

2.352.041.46

NL: 8.32E3m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 2.53E1m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_bl

NL: 1.49E2m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

NL: 6.95m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS 110715_MMA_Neg_Endura_bl

RT: 0.00 - 2.51 SM: 7B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 1.10E4m/z= 72.8000-73.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 6.28E1m/z= 54.8000-55.8000 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_std25

NL: 9.04E5m/z= 75.8000-76.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

NL: 3.70E4m/z= 57.8000-58.8000 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_std25

LOQ: 25nM

Succinic acid

Figure 3. Chromatogram of quality control samples (a: deriv. method, b: SPE method)

b Level 0 – 110 nM Level I – 260 nM Level II – 583 nM

MMA Quantifier

d3-MMA Quantifier

MMA Qualifier

d3-MMA Qualifier

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 2.41E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 1.41E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_Level0_2

NL: 6.39E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

NL: 2.60E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_Level0_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.86E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 5.13E2m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 8.80E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

NL: 3.62E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelI_2

RT: 0.00 - 2.51 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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NL: 6.55E4m/z= 73.2996-73.3004 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 1.21E3m/z= 55.2997-55.3003 F: - c ESI SRM ms2 117.100 [55.299-55.301, 73.299-73.301] MS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 9.14E5m/z= 76.2996-76.3004 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

NL: 3.75E4m/z= 58.2997-58.3003 F: - c ESI SRM ms2 120.100 [58.299-58.301, 76.299-76.301] MS ICIS 110715_MMA_Neg_Endura_RECIPE_LevelII_2

Level 0 – 82 nM Level I – 260 nM Level II – 583 nM a

MMA Quantifier

MMA qualifier

D3-MMA

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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1.86

RT: 2.22

1.86

RT: 2.21

NL: 1.36E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_Level0_4

NL: 8.13E2m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_Level0_4

NL: 7.76E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_Level0_4

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

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60

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RT: 2.22

1.86

RT: 2.20

NL: 3.46E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelI_2

NL: 1.83E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelI_2

NL: 7.24E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelI_2

RT: 0.00 - 2.71 SM: 9B

0.0 0.5 1.0 1.5 2.0 2.5Time (min)

0

10

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50

60

70

80

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100RT: 2.25

RT: 2.24

1.88

RT: 2.23

NL: 8.45E3m/z= 119.1494-119.1506 F: + c APCI SRM ms2 231.150 [119.149-119.151] MS 082815_QC_RECIPE_LevelII_2

NL: 4.25E3m/z= 175.1991-175.2009 F: + c APCI SRM ms2 231.150 [175.199-175.201] MS 082815_QC_RECIPE_LevelII_2

NL: 7.19E3m/z= 122.1994-122.2006 F: + c APCI SRM ms2 234.200 [122.199-122.201] MS ICIS 082815_QC_RECIPE_LevelII_2

Table 7. Replicate injections of each calibration standard-precision and accuracy

Table 8. Matrix effect: Internal standard absolute % recovery compared to samples prepared in solvent (10 donors for deriv. method and 8 donors for SPE method)

Method Cal1 Cal2 Cal3 Cal4 Cal5 Cal6 Cal7 Cal8 Cal9 Cal10

Conc. (nM) 25 50 100 500 1000 2000 10000 25000 50000 100000

%RSD Deriv. (n=5) 2.82 4.14 2.06 2.59 3.9 ---* 2.24 2.04 2.77 3.1

%RSD SPE (n=4) 3.57 2.08 0.89 0.76 0.9 0.46 0.08 0.32 0.2 0.5

Avg. % Rec Deriv. (n=5) 113 101 95.2 93.9 96.5 ---* 94.8 105 102 98.2

Avg. % Rec SPE (n=4) 109 102 105 94.5 94.1 95.1 99.5 101 99.9 100

Table 9. Matrix effect/Spike recovery: Previously analyzed plasma from 10 donors were spiked with 100 nM of MMA

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 2.11 12.6 10.7 1.97 5.07 12.2 1.46 5.50 0.69 0.49 %RSD SPE (n=4) 2.80 0.85 2.82 2.01 4.71 2.39 4.39 2.53 --- --- %Rec Deriv. (n=3) 98.3 85.4 90.8 103 100 101 83.9 103 101 98.0 %Rec SPE (n=4) 93.8 104 107 104 102 98.5 101 89.7 --- ---

Method Plasma1 Plasma2 Plasma3 Plasma4 Plasma5 Plasma6 Plasma7 Plasma8 Plasma9 Plasma10

%RSD Deriv. (n=3) 1.83 3.08 2.82 0.69 2.55 1.17 3.38 1.36 1.35 2.49 %RSD SPE (n=4) 1.66 1.30 1.53 2.35 2.01 1.14 0.55 2.31 0.61 1.60 %Rec Deriv. (n=3) 97.2 94.7 95.9 95.1 94.3 89.9 93.9 92.5 92.0 96.8 %Rec SPE (n=4) 104 107 99.7 106 96.8 104 100 105 100 99.7

Table 10. MMA derivatization reaction process reproducibility

Method Analyte # of Donor Samples # of Replicates %RSD

Deriv. method d3-MMA 10 n = 5 12.3

*: This concentration was not evaluated in derivatization method.

Calculated Amt %Diff 26.81 7.26 48.01 -3.99 104.9 4.95 472.4 -5.52 970.1 -2.99 1952 -2.42

10169 1.69 25309 1.24 50170 0.34 99454 -0.55

Analyte Precursor (m/z)

Product Ion (m/z)

Collision Energy (V)

Qualification / Qualification Polarity

MMA 231.2 175.2 6 Qualifier Positive

MMA 231.2 119.2 11 Quantifier Positive

d3-MMA 234.2 178.2 6 Qualifier Positive

d3-MMA 234.2 122.2 11 Quantifier Positive

PN64801-EN 0616S

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