Presented at ASMS 2015

IntroductionValidations of LC-MS/MS assays include assessments of matrix effects, and hemolysis is a special category of matrix effect. Hemolysis is caused by the rupture of blood cells, resulting in the release of hemoglobin and other cellular components into plasma or serum samples. These components can sometime impact the performance of optical methods due to visible color and density changes1,2. Additionally, they can also impact the accuracy and precision of LC-MS/MS assays due to matrix effects or/and other interferences. Here we present two case studies to demonstrate how LC coupled with high resolution, accurate mass MS (HRAM) helped identify the source and minimize the impact of matrix effects caused by hemolysis on two separate LC-MS/MS assays for the quantitation of LipoTP and loxapine in human plasma.

Results and DiscussionLipoTP LipoTP is a small amphiphilic peptide consisting of a hydrophilic tripeptide and an aliphatic acyl group. The molecular weight of LipoTP is 2875.6.

A sensitive LC-MS/MS assay was initially developed for quantitative analysis of LipoTP in human plasma (K2EDTA) with a dynamic range of 1.00-1,000 ng/mL on an AB SCIEX™ 6500 LC-MS/MS system. While validating this assay, it met the acceptance criteria for all required tests set forth in FDA guidelines.

However, for the hemolysis evaluation, an interfering peak was observed which eluted just prior tothe analyte peak; the intensity of the interfering peak increased with the % hemolysis (Figure 1). If the fronting peak was included in the peak integration, the % difference is 20.9% higher (0.5% hemolysis) and 86.2% higher (2.0% hemolysis) than in the control sample with no hemolysis (Table 1). If the fronting peak is excluded, the % difference is -14.5% and -10.2%, respectively, compared to the control (Table 2).

To identify the unknown interference, the same sample extracts were analyzed under the same LC conditions on a Thermo Scientific™ Q Exactive™ Plus in targeted SIM and full scan modes at a resolution of both 70K and 140K. Interestingly, the interfering peak was not observed by HRAM analysis and the difference in instrument response from the test group was acceptable (< 15%), indicating that the endogenous compound had no impact on the quantification of LipoTP using LC-HRAM.

Mining of the LC-HRAM (SIM) data indicated that there were trace amounts of endogenous compounds which eluted at retention times near the analyte peak in hemolyzed samples but not in the non-hemolyzed control samples (Figures 2 and 3). Further investigation determined that the interference peak was primarily due to hemoglobin alpha chains. These previously “unknown” interferences were successfully identified by intact mass measurement and confirmed by top down experiments (Figures 4 and 5).

MethodologyLipoTPSample Preparation and Extraction1. Aliquot 25.0 µL plasma sample into the corresponding wells of a

96-well plate2. Add 25.0 µL of internal standard working solution 3. Add 500 µL of hydrolysis solution to all samples4. Incubate and shake the plate at 37 °C for 60 min5. Extract sample by HLB 96-well SPE plate

Chromatographic ConditionsColumn: XSelect CSH C18 (Waters) 3.5 µm, 2.1 x 30 mmMobile Phase: A: 10mM ammonium formate, pH unadjusted B: 1% formic acid in MeCNColumn temperature: 45 °CFlow rate: 0.45 mL/minute

Mass Spectrometer Conditions (MS/MS)Instrument AB SCIEX™ 6500 Ionization mode: Electrospray, positive ion modeSource Temperature: 500 °C SRM transitions: LipoTP m/z 719.7→884.0 LipoTP IS m/z 728.7→893.2

Mass Spectrometer Conditions (HRAM)Instrument: Thermo Scientific™ Q Exactive™ PlusIonization mode: Electrospray, positive ion modeSource Temperature: 500 °C Resolution: 70,000/140,000

Target SIM: LipoTP / Sum of m/z 719.455, 719.705, 719.957, 720.206 LipoTP IS / Sum of m/z 728.2273, 728.4764, 728.7269

Unknown ID: Full scan; Full scan + data dependent Top 10 MS/MS; HCD MS/MS for Top Down analysis

LoxapineSample Preparation and Extraction1. Aliquot 100 µL of plasma sample into a 96-well plate2. Add 50.0 µL of internal standard working solution

[10.0 ng/mL Loxapine-d8]3. Add 50.0 µL of extraction buffer to all samples4. Extract samples by Oasis µHLB, 30 µm plate

Chromatographic ConditionsColumn: Luna μm PFP(2) 100A, 50x2 mmMobile Phase: A: 0.2% FA in 10 mM ammonium acetate in water, pH unadjusted B: MeCNColumn temperature: 35 °CFlow rate: 0.50 mL/minute

Assessing Hemolysis Failures for LC-MS/MS Assays Using LC-HRAM on a Q-Exactive™ Mass Spectrometer Laixin Wang1, Alicia Pietrasiewicz1, Chad Moore1, Carrie Pederson1, Scott Reuschel1, Min Meng1, Hongxia Wang2, David Horn2 and Jonathan Josephs2 1Covance, Salt Lake City, Utah, 2Thermo Fisher Scientific, San Jose, CA

Covance is the drug development business of Laboratory Corporation of America® Holdings (LabCorp®). Findings in this poster were developed by scientists who at the time were affiliated with the LabCorp Clinical Trials or Tandem Labs brands, now part of Covance.

Conclusions▶ Some protein components from human hemoglobin co-elute

with LipoTP and interfere with its quantitation under selected LC-MS/MS conditions (AB SCIEX™ 6500); however, these components have no impact on the quantitation of LipoTP using LC-HRAM on a Thermo Scientific™ Q Exactive™ Plus.

▶ No hemoglobin components co-eluted near loxapine under the experimental conditions; however, an endogenous small compound (MW ~ 327.25) did co-elute with loxapine (MW = 327.12). This endogenous molecule likely caused the failure of the hemolysis test with the LC-MS/MS assay on an AB SCIEX™ but had no impact on the quantitation of loxapine using LC-HRAM on a Thermo Scientific™ Q Exactive™ Plus.

Mass Spectrometer Conditions (MS/MS)Instrument AB SCIEX™ 5000 Ionization mode: Electrospray, Positive ion modeSource Temperature: 500 °C SRM transitions: Loxapine m/z 328.3 → 271.0 Loxapine-d8 m/z 336.3 → 276.1 Mass Spectrometer Conditions (HRAM)Instrument Thermo Scientific™ Q Exactive™ PlusIonization mode: Electrospray, Positive ion modeSource Temperature: 500 °C Resolution: 70,000Target SIM: Loxapine m/z+ 328.1213 Loxapine-d8 m/z+ 336.1712Full Scan: m/z+ 150-2,000

Figure 1. Typical chromatograms of LipoTP (left) in 0% (top), 0.5% (middle) and 2.0% (bottom) hemolyzed human plasma analyzed on an AB SCIEX™6500.

Figure 2. LC-HRAM results from 0% hemolyzed LipoTP sample on a Q-Exactive. Both analyte and IS peaks have very clean isotopic peak distribution.

Figure 4. HRAM full scan analysis (270.00-4000.000) of a 2.0 % hemolyzed plasma sample showing a major interference peak from 1.9’ to 2.45’. The peak contains primarily human hemoglobin and serum proteins as further identified by protein deconvolution (Figure 6).

Figure 5. Protein deconvolution results (HRAM at 140K resolution) for 2.0% hemolyzed LipoTP sample. Top down experiment was done by fragmenting m/z 890.2355 at charge 17+ with HCD 22.

Statistics Low Level (0.0% Hemolysis)

Low Level (0.5% Hemolysis)

Low Level (2.0% Hemolysis)

0.0079033 0.0093629 0.0136530.0078137 0.0092123 0.0140080.0077435 0.0092382 0.0139760.0075997 0.0093733 0.0146410.0075089 0.0087821 0.0144630.0073071 0.0094824 0.014701

Mean 0.007646 0.009242 0.01424S.D. 0.000219 0.000246 0.000422

%CV 2.9Precision Passes

2.7Precision fails

3.0Precision fails

%Difference N/A 20.9 86.2

n 6 6 6

% Difference = ([Average Low Hemolysis - Average Low Control] / Average Low Control) * 100

Statistics Low Level (0.0% Hemolysis)

Low Level (0.5% Hemolysis)

Low Level (2.0% Hemolysis)

0.0062899 0.0051529 0.00543520.0060518 0.0049557 0.00561470.0051779 0.00445 0.00506020.0059897 0.0054412 0.00481770.0059093 0.0047888 0.00556040.0055905 0.005133 0.0049744

Mean 0.005835 0.004987 0.005244S.D. 0.000394 0.000342 0.000335

%CV 6.7Precision Passes

6.9Precision Passes

6.4Precision Passes

%Difference N/A -14.5 -10.1

n 6 6 6

% Difference = ([Average Low Hemolysis - Average Low Control] / Average Low Control) * 100

Table 1. Evaluation of Hemolysis for LipoTP (Including Interfering Peak)All results are expressed as instrument response (i.e., analyte area/internal standard area)

Table 2. Evaluation of Hemolysis for LipoTP (Excluding Interfering Peak)All results are expressed as instrument response (i.e., analyte area/internal standard area)

Figure 3. LC-HRAM results from 2.0% hemolyzed plasma sample on a Q-Exactive. In Targeted SIM mode, impurities were observed in the isotopic distributions for the LipoTP peak, but not for IS peak. The impurities were primarily from human hemoglobin alpha chains as identified below.

Pooled_Extract_2%Hemolysis_SIM 12/18/14 16:43:33

RT: 0.00 - 4.51

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)

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RT: 2.02AA: 2551399SN: 246

RT: 0.46AA: 150527SN: 41

RT: 2.01AA: 518829961SN: 52365

RT: 2.36AA: 957026SN: 519

RT: 0.34AA: 69436SN: 50

RT: 1.30AA: 71172SN: 29

RT: 2.76AA: 121219SN: 19

RT: 3.02AA: 20794SN: 16

RT: 0.81AA: 44826SN: 15

RT: 3.62AA: 34387SN: 13

NL: 2.25E5m/z= 719.4530-719.4588+719.7038-719.7096+719.9544-719.9602+720.2051-720.2109 F: FTMS + p ESI SIM ms [717.71-721.71] MS ICIS Pooled_Extract_2%Hemolysis_SIM

NL: 4.33E7m/z= 722.8784-722.8842+728.2245-728.2303+728.4752-728.4810+728.7259-728.7317 F: FTMS + p ESI SIM ms [726.48-730.48] MS ICIS Pooled_Extract_2%Hemolysis_SIM

718 719 720 721 722 723 724 725 726 727 728 729 730m/z

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719.7056z=4

718.1688z=? 719.4546

z=4 720.2067z=4719.1714

z=?720.7092

z=4721.3349

z=21718.0127

z=?728.4767

z=4

728.7272z=4

728.9778z=4727.9762

z=4 729.7278z=4

726.9066z=?

NL: 4.40E4Pooled_Extract_2%Hemolysis_SIM#330-393 RT: 1.82-2.14 AV: 32 T: FTMS + p ESI SIM ms [717.71-721.71]

NL: 9.50E6Pooled_Extract_2%Hemolysis_SIM#329-396 RT: 1.82-2.16 AV: 34 T: FTMS + p ESI SIM ms [726.48-730.48]

718.8 719.0 719.2 719.4 719.6 719.8 720.0 720.2 720.4 720.6m/z

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719.7056z=4

719.9560z=4

719.4545z=4

720.2066z=4718.7811

z=4719.1714

z=?

720.4563z=4

719.2821z=4

719.0133z=?

719.6212z=21

719.5276z=4

720.2851z=21

720.7091z=4

720.1745z=?

719.8122z=21

720.5259z=21

719.3819z=1

720.0131z=1

718.8366z=?

718.7171z=1

LipoTP

IS

Hemoglobin alpha chain

Expanded

Figure 3. LC-HRAM results from 2.0% hemolyzed LipoTP sample on a Q-Exactive. In Targeted SIM mode, impurities were observed in the isotopic distributions for the LipoTP peak, but not for IS peak. The impurities were primarily from human hemoglobin alpha chains as identified below.

Figure 6. HRAM Targeted SIM results (top, m/z+ 328.1210 for loxapine) and full scan (middle, m/z+ 150-2000) for 2.0% hemolyzed plasma sample. No human hemoglobin or serum protein components were observed at the retention times near the analyte/IS peaks under the experimental conditions.

Figure 7. HRAM Target SIM (top, m/z+ 328.1210 for loxapine) and HRAM selected m/z+ 328.2481 from full scan (middle, m/z+ 150-2000) from 2.0% hemolyzed plasma sample. The unknown interference compound is much more abundant than the analyte loxapine in the hemolyzed plasma sample.

LoxapineLoxapine is a member of the dibenzoxazepine class of compounds. The molecular weight of loxapine is 327.8.

A sensitive LC-MS/MS assay was initially developed for quantitative analysis of loxapine and its four metholites (See ASMS 2015 poster #MP 270 - Simultaneous Quantification of Loxapine and its Four Metabolites in Human Plasma using LC-MS/MS) with a dynamic range of 0.050-50.0 ng/mL on an AB SCIEX™ 6500 LC-MS/MS system. When validating this assay, it met the acceptance criteria for all required tests set forth in FDA guidelines with the exception of the hemolysis test for loxapine (Table 3).

Because only loxapine failed the test, the possibility of a sample preparation error was ruled out. Unlike the LipoTP hemolysis failure which was caused by interfering peak, the hemolysis failure for loxapine was due to a decrease in the analyte/IS peak area, indicating that there was ion suppression in the 2% hemolysis test group (Table 4). To investigate, the impact of hemolysis was also evaluated by targeted SIM and full scan LC-HRAM on a Thermo Scientific™ Q Exactive™ Plus.

Once again, the difference in instrument response between the hemolyzed plasma samples and the non-hemolyzed controls was acceptable (< 15%) under the same extraction and LC conditions using HRAM. No known human hemoglobin or serum protein components were observed to elute at retention times near the loxapine/loxapine-d8 peaks under the experimental conditions (Figure 6); however, an additional intense peak with a m/z of 328.2481 did co-elute with loxapine/loxapine-d8. Loxapine has a m/z of 328.1210 by HRAM (Figure 7). It is likely that this unknown endogenous compound may be contributing to the failure of hemolysis test in the loxapine LC-MS/MS assay due to the similar masses that cannot be effectively distinguished by the mass resolution of the AB SCIEX™ 6500 instrument.

Table 3. Hemolysis Evaluation of Loxapine Assay (all analytes, instrument response)

Analytes Statistics Low Level Low Level Low Level (0.0% Hemolysis) (0.5% Hemolysis) (2.0% Hemolysis) Loxapine Mean 0.057786 0.056829 0.070825 %CV 1.9 1.2 3.7 %Difference N/A -1.7 22.6

Loxapine-N-Oxide Mean 0.106142 0.103407 0.100900 %CV 5.8 6.2 10.0 %Difference N/A -2.6 -4.9 Amoxapine Mean 0.078472 0.078985 0.078790 %CV 6.6 6.9 3.4 %Difference N/A 0.7 0.4 8-Hydroxy Mean 0.097783 0.097742 0.096694 Loxapine %CV 2.9 5.0 5.8 %Difference N/A 0.0 -1.1

7-Hydroxy Mean 0.051374 0.051755 0.053043 Loxapine %CV 2.0 3.4 4.2 %Difference N/A 0.7 3.2

Table 4. Hemolysis Evaluation for Loxapine (peak area and instrument response)

Sample Name Area IS Area Instrument Response

% Hemolysis 37987 673067 0.056439 0% Hemolysis 38396 647135 0.059332 0% Hemolysis 34371 592338 0.058026 0% Hemolysis 37773 657711 0.057431 0% Hemolysis 39353 692523 0.056826 0% Hemolysis 38331 653433 0.058661

0.5% Hemolysis 33695 585387 0.05756 0.5% Hemolysis 33132 575238 0.057597 0.5% Hemolysis 33818 604409 0.055952 0.5% Hemolysis 31791 564669 0.0563 0.5% Hemolysis 32429 574073 0.056489 0.5% Hemolysis 28910 506522 0.057076 2% Hemolysis 31225 439690 0.071016 2% Hemolysis 29191 402880 0.072456 2% Hemolysis 28893 396343 0.072899 2% Hemolysis 28146 384693 0.073165 2% Hemolysis 27810 404286 0.068788 2% Hemolysis 26793 402142 0.066626

Reference ▶ Hugges NC, Bajaj N, Fan J and Wong EY. “Assessing the

matrix effects of hemolyzed samples in bioanalysis”. Bioanalysis 2009, 1(6):1057-1066.

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