Chapter Clinical, Forensic and Pharmaceutical Applications
Transcript of Chapter Clinical, Forensic and Pharmaceutical Applications
Clinical, Forensic and Pharmaceutical Applications
• Page 4Rapid development of analytical method for anti-epileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
• Page 11Determination of ∆9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
• Page 17Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample prepa-ration
• Page 23Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
• Page 29Simultaneous screening and quantitation of amphetamines in urine by on-line SPE-LC/MS method
• Page 36Single step separation of plasma from whole blood without the need for centrifugation ap-plied to the quantitative analysis of warfarin
• Page 42Development and validation of direct analysis method for screening and quantitation of amphetamines in urine by LC/MS/MS
• Page 48Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
• Page 54Application of a sensitive liquid chromatography-tandem mass spectrometric method to pharma-cokinetic study of telbivudine in humans
• Page 60Accelerated and robust monitoring for immu- nosuppressants using triple quadrupole mass spectrometry
• Page 66Highly sensitive quantitative analysis of felodip-ine and hydrochlorothiazide from plasma using LC/MS/MS
• Page 73Highly sensitive quantitative estimation of geno-toxic impurities from API and drug formulation using LC/MS/MS
• Page 80Development of 2D-LC/MS/MS method for quan-titative analysis of 1a,25-Dihydroxylvitamin D3 in human serum
• Page 86Analysis of polysorbates in biotherapeutic prod-ucts using two-dimensional HPLC coupled with mass spectrometer
• Page 93A rapid and reproducible Immuno-MS platform from sample collection to quantitation of IgG
• Page 99Simultaneous determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
• Page 103Low level quantitation of loratadine from plasma using LC/MS/MS
PO-CON1452E
Rapid development of analyticalmethod for antiepileptic drugs inplasma using UHPLC method scoutingsystem coupled to LC/MS/MS
ASMS 2014 ThP 672
Miho Kawashima1, Satohiro Masuda2, Ikuko Yano2,
Kazuo Matsubara2, Kiyomi Arakawa3, Qiang Li3,
Yoshihiro Hayakawa3
1 Shimadzu Corporation, Tokyo, JAPAN,
2 Kyoto University Hospital, Kyoto, JAPAN,
3 Shimadzu Corporation, Kyoto, JAPAN
2
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
IntroductionMethod development for therapeutic drug monitoring (TDM) is indispensable for managing drug dosage based on the drug concentration in blood in order to conduct a rational and ef�cient drug therapy. Liquid chromatography coupled with tandem quadrupole mass spectrometry is increasingly used in TDM because it can perform selective and sensitive analysis by simple sample pretreatment. The UHPLC method scouting system coupled to tandem
quadrupole mass spectrometer used in this study can dramatically shorten the total time for optimization of analytical conditions because this system can make enormous combinatorial analysis methods and run batch program automatically. In this study, we developed a high-speed and sensitive method for measurement of seventeen antiepileptics in plasma by UHPLC coupled with tandem quadrupole mass spectrometer.
Figure 1 Antiepileptic drugs used in this assay
Experimental
UHPLC based method scouting system (Nexera X2 Method Scouting System, Shimadzu Corporation, Japan) is configured by Nexera X2 UHPLC modules. For the detection, tandem quadrupole mass spectrometer (LCMS-8050, Shimadzu Corporation, Japan) was used. The system can be operated at a maximum pressure of 130 MPa, and it enables to automatically select up to 96 unique combinations of eight different mobile phases and six different columns. A
dedicated software was newly developed to control the system (Method Scouting Solution, Shimadzu Corporation, Japan), which provides a graphical aid to configure the different type of columns and mobile phases. The software is integrated into the LC/MS/MS workstation (LabSolutions, Shimadzu Corporation, Japan) so that selected conditions are seamlessly translated into method files and registered to a batch queue, ready for analysis instantly.
Instruments
N
O NH2
Carbamazepine Carbamazepine- 10,11-epoxide
N
O NH2
O
Diazepam
N
N
O
CH3
Cl
Ethomuximide
NHCH3
CH3
O
O
Felbamate
O ONH2
O
NH2
O
Gabapentin
NH2
OH
O
N
N
NCl
Cl
NH2
NH2
Lamotrigine Levetiracetam
N O
CH3
NH2
O
Phenobarbial
NH
NH
O
O
O
CH3
Primidone
NH
NH
O
CH3 O
Phenytoin
NH NH
O
O
Tiagabine
SCH3
NS OH
O
CH3
Zonisamide
ON
S
O
O
CH3O
O
OO
O
CH3
CH3
CH3
CH3
OS
O
ONH2
Topiramate Vigabatrin
CH2
NH2
OH
O
Clonazepam
NH
N
N+
O
O O
Cl
-
NH
N
N+
O
O O
Nitrazepam
-
3
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Figure 2 Nexera Method Scoutuing System and LCMS-8050 triple quadrupole mass spectrometer
Result
The MS condition optimization was performed by flow injection analysis (FIA) of ESI positive and negative ionization mode, and the compound dependent parameters such as CID and pre-bias voltage were adjusted using automatic
MRM optimization function. The transition that gave highest intensity was used for quantification. The MRM transitions used in this assay are listed in Table 1.
The main standard mixture was prepared in methanol from individual stock solutions. The calibration standards were prepared by diluting the standard mixture with methanol. QC sample was prepared by adding 4 volume of acetonitrile to 1 volume of control plasma, thereby precipitating proteins, and subsequently adding the standard mixture to the supernatant to contain plasma concentration equivalents stated in Table 4. The QC samples were further diluted 100 times (10 μL sample
added to 990μL methanol) before injection. Next step of preparation procedure was divided into three groups by the intensity of each compound. For ethomuximide, phenobarbial and phenytoin, the supernatant was used for the LC/MS/MS analysis without further dilution. For zonisamide, 10 μL supernatant was further diluted with 990 μL methanol. For others, 100 μL supernatant was further diluted with 900 μL methanol. The diluted solutions were used for the LC/MS/MS analysis.
MRM condition optimization
Calibration standards and QC samples
4
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Figure. 3 Schematic representation and features of the Nexera Method Scouting System.
Table 1 Compounds, Ionization polarity and MRM transition
Retaintion (min)Compound Polarity Precursor m/z Product m/z
3.84
3.24
3.93
4.79
2.50
2.86
2.27
2.96
2.32
3.90
3.06
3.64
2.83
4.28
3.14
0.82
2.58
+
+
+
+
+
+
+
+
+
+
+
+
+
-
-
-
-
237.1
253.1
316.1
284.9
239.3
172.2
256.2
171.2
281.9
219.2
376.2
130.2
213.1
140.0
231.0
337.9
143.1
194.2
180.15
269.55
154.15
117.20
154.25
211.05
126.15
236.20
162.15
111.15
71.15
132.10
42.00
42.05
78.00
143.10
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
36 analytical conditions, comprising combinations of 9 mobile phase and 4 columns, were automatically investigated using Method Scouting System. Schematic representation of scouting system was shown in Figure 3. From the result of scouting, the combination of 10 mM
ammonium acetate water and methanol for mobile phase and Inertsil-ODS4 for separation column were selected. Using this combination of mobile phase and column, the gradient condition was further optimized. The final analytical condition was shown in Table 2.
UHPLC condition optimization
Auto SamplerLPGE Unit
Column Oven
LCMS-8050
Pump A
Pump B
1 2 3 4
1 2 3 4
(A) 1 – 10mM Ammonium Acetate 2 – 10mM Ammonium Formate 3 – 0.1%FA - 10mM Ammonium Acetate(B) 1 – Methanol 2 – Acetonitrile 3 – Methanol/Acetonitrile=1/1
Kinetex XB-C18 (Phenomenex)
Kinetex PFP (Phenomenex)
InertsilODS-4 (GL Science)
Discovery HS F5-5 (SPELCO)
2.1 x 50 mm
2.1 x 50 mm
2.1 x 50 mm
2.1 x 50 mm
5
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Table.2 UHPLC analytical conditions
Figure. 4 Chromatogram of 17 AEDs calibration standards
Column : Inertsil ODS-4 (50 mmL. x 2.1mmi.d., 2um)
Mobile phase : A) 10mM Ammonium Acetate
B) Methanol
Binary gradient : B conc. 3% (0.65 min) → 40% (1.00 min) → 85% (5.00 min)
→ 100% (5.01-8.00 min) → 3% (8.01-10.00 min)
Flow Rate : 0.4 mL/min
Injection vol. : 1 μL
Column Temp. : 40 deg. C
Figure 4 shows MRM chromatograms of the 17 AEDs. It took only 10 minutes per one UHPLC/MS/MS analysis, including column rinsing.
Precision, accuracy and linearity of AEDs
0.0 1.0 2.0 3.0 4.0 5.0 min
Vigabatrin130.20>71.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Gabapentin172.20>154.25(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Levetiracetam171.20>126.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Ethomuximide140.00>42.00(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Zonisamide213.10>132.10(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Primidone 219.20>162.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Felbamate239.30>117.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Lamotrigine256.20>211.05(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Phenobarbial231.00>42.05(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Topiramate337.85>78.00(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Carbamazepine-10,11-epoxide253.10>180.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Phenytoin251.00>208.20(-)
0.0 1.0 2.0 3.0 4.0 5.0 min
Carbamazepine237.10>194.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Nitrazepam 281.90>236.20(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Clonazepam 316.10>269.55(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Tiagabine376.20>111.15(+)
0.0 1.0 2.0 3.0 4.0 5.0 min
Diazepam284.90>154.15
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
6
Table.3 Linearity of 17 AEDs QC sample
Compound Linarity (ng/mL) r2
0.25
0.25
0.005
0.01
25
0.5
2
0.25
0.5
0.005
5
5
0.25
0.25
0.5
0.5
0.5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
50
50
2.5
5
2500
100
50
50
100
1
500
500
10
50
100
50
20
0.999
0.998
0.998
0.999
0.998
0.998
0.999
0.999
0.999
0.999
0.996
0.998
0.996
0.998
0.998
0.998
0.996
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
Table 3 illustrates linearity of 17 AEDs and Table 4 illustrates accuracy and precision of the QC samples at three concentration levels. Determination coefficient (r2) of all calibration curves was larger than 0.995, and the precision
and accuracy were within +/- 15%. Excellent linearity, accuracy and precision for all 17 AEDs were obtained at only 1 μL injection volume.
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Rapid development of analytical method for antiepileptic drugs in plasma using UHPLC method scouting system coupled to LC/MS/MS
Table.4 Accuracy and precision of 17 AEDs QC sample
Compound
Plasma concentrationequivalents (µg/mL)
Precision (%) Accuracy (%)
HighMiddleLow
1.8
1.8
0.04
0.1
18
3.6
18
1.8
3.6
0.04
3.6
3.6
1.8
1.8
3.6
8.9
36
71
71
1.8
2.9
714
179
143
71
179
1.4
143
143
45
71
143
89
179
2.2
2.4
3.3
3.2
7.8
1.7
1.3
10.5
2.1
3.3
3.5
7.8
3.2
1.8
12.5
1.4
3.3
0.9
1.9
0.7
1.7
1.5
0.4
0.7
1.2
0.5
1.4
6.2
1.9
0.7
1.8
1.5
1.1
1.3
18
18
0.9
0.7
446
89
36
45
89
0.4
71
89
18
18
36
18
89
0.9
1.3
0.5
1.4
1.4
0.8
0.7
1.7
1.1
1.5
1.6
1.2
0.7
1.0
1.2
2.1
1.6
106.1
104.2
106.7
105.8
104.3
97.1
85.8
107.7
99.5
105.0
100.9
103.2
99.5
107.6
105.4
105.9
111.7
103.9
105.0
102.1
106.6
99.9
106.3
98.8
98.4
104.9
105.2
108.4
100.1
112.6
105.7
101.6
101.6
100.4
95.8
98.2
90.1
100.6
97.0
91.7
89.5
99.2
90.4
97.9
95.8
96.2
97.1
97.5
96.1
88.8
95.2
Carbamazepine
Carbamazepine-10,11-epoxide
Clonazepam
Diazepam
Ethomuximide
Felbamate
Gabapentin
Lamotrigine
Levetiracetam
Nitrazepam
Phenobarbial
Phenytoin
Primidone
Tiagabine
Topiramate
Vigabatrin
Zonisamide
HighMiddleLowHighMiddleLow
Conclusions• We could select the most suitable combination of mobile phase and column from 36 analytical condition without
time-consuming investigation.• We have measured plasma sample as it is after 100-10,000 times dilution by methanol without making tedious sample
pretreatment. Excellent linearity, precision and accuracy for all 17 AEDs were obtained at only 1 uL injection volume.
PO-CON1446E
Determination of Δ9-tetrahydrocannabinoland two of its metabolites in whole blood,plasma and urine by UHPLC-MS/MS usingQuEChERS sample preparation
ASMS 2014 ThP600
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1 CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France
2
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
IntroductionIn France, as in other countries, cannabis is the most widely used illicit drug. In forensic as well as in clinical contexts, ∆9-tetrahydrocannabinol (THC), the main active compound of cannabis, and two of its metabolites [11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC) and 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)] are regularly investigated in biological �uids for example in Driving Under the In�uence of Drug context (DUID) (�gure 1). Historically, the concentrations of these compounds were determined using a time-consuming extraction procedure
and GC-MS. The use of LC-MS/MS for this application is relatively recent, due to the low response of these compounds in LC-MS/MS while low limits of quanti�cation need to be reached. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here a highly sensitive UHPLC-MS/MS method with straightforward QuEChERS sample preparation (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe).
Methods and MaterialsIsotopically labeled internal standards (one for each target compound in order to improve method precision and accuracy) at 10 ng/mL in acetonitrile, were added to 100 µL of sample (urine, whole blood or plasma) together with 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium
citrate dehydrate/Sodium citrate sesquihydrate) and 200 µL of acetonitrile. Then the mixture was shaken and centrifuged for 10 min at 12,300 g. Finally, 15 µL of the upper layer were injected in the UHPLC-MS-MS system. The whole acquisition method lasted 3.4 min.
Figure 1: Structures of THC and two of its metabolites
OH
O
H
HCH3
CH3
OHO
THC-COOH
OH
O
H
H
CH2
CH3CH3
OH
11-OH-THC
OH
O
H
H
CH3
CH3CH3
THC
3
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
UHPLC conditions (Nexera MP system)
Column : Kinetex C18 50x2.1 mm 2.6 µm (Phenomenex)
Mobile phase A : 5mM ammonium acetate in water
B : CH3CN
Flow rate : 0.6 mL/min
Time program : B conc. 20% (0-0.25 min) - 90% (1.75-2.40 min) - 20% (2.40-3.40 min)
Column temperature : 50 °C
MS conditions (LCMS-8040)
Ionization : ESI, negative MRM mode
Ion source temperatures : Desolvation line: 300°C
Heater Block: 500°C
Gases : Nebulization: 2.5 L/min
Drying: 10 L/min
MRM Transitions:
Compound MRM Dwell time (msec)
THC 313.10>245.25 (Quan) 60
313.10>191.20 (Qual) 60
313.10>203.20 (Qual) 60
THC-D3 316.10>248.30 (Quan) 5
316.10>194.20 (Qual) 5
11-OH-THC 329.20>311.30 (Quan) 45
329.20>268.25 (Qual) 45
329.20>173.20 (Qual) 45
11-OH-THC-D3 332.30>314.40 (Quan) 5
332.30>271.25 (Qual) 5
THC-COOH 343.20>245.30 (Quan) 50
343.20>325.15 (Qual) 50
343.20>191.15 (Qual) 50
343.20>299.20 (Qual) 50
THC-COOH-D3 346.20>302.25 (Quan) 5
346.20>248.30 (Qual) 5
Pause time : 3 msec
Loop time : 0.4 sec (minimum 20 points per peak for each MRM transition)
4
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Figure 1: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 50 µg/L
Results
A typical chromatogram of the 6 compounds is presented in figure 1.
Chromatographic conditions
Figure 2: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only
obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 2.
Extraction conditions
A B
5
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Figure 3: Chromatogram obtained after an injection of a 15 µL whole blood extract spiked at 0.5 µg/L (lower limit of quanti�cation).
One challenge for the determination of cannabinoids in blood using LC-MS/MS is the low quantification limits that need to be reached. The French Society of Analytical Toxicology proposed 0.5 µg/L for THC et 11-OH-THC and 2.0 µg/L for THC-COOH. With the current application, the
lower limit of quantification was fixed at 0.5 µg/L for the three compounds (3.75 pg on column). The corresponding extract ion chromatograms at this concentration are presented in figure 3.
Validation data
The upper limit of quantification was set at 100 µg/L. Calibration graphs of the cannabinoids-to-internal standard peak-area ratios of the quantification transition versus
expected cannabinoids concentration were constructed using a quadratic with 1/x weighting regression analysis (figure 4).
Contrary to what was already observed with on-line Solid-Phase-Extraction no carry-over effect was noted using the present method, even when blank samples were
injected after patient urine samples with concentrations exceeding 2000 µg/L for THC-COOH.
THC11-OH-THCTHC-COOH
Figure 4: Calibration curves of the three cannabinoids
THC11-OH-THCTHC-COOH
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Determination of Δ9-tetrahydrocannabinol and two of its metabolites in whole blood, plasma and urine by UHPLC-MS/MS using QuEChERS sample preparation
Conclusions• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.• Low limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.
PO-CON1445E
Determination of opiates, amphetaminesand cocaine in whole blood, plasmaand urine by UHPLC-MS/MS usinga QuEChERS sample preparation
ASMS 2014 ThP599
Sylvain DULAURENT1, Mikaël LEVI2, Jean-michel GAULIER1,
Pierre MARQUET1,3 and Stéphane MOREAU2
1 CHU Limoges, Department of Pharmacology and Toxicology,
Unit of clinical and forensic toxicology, Limoges, France ; 2 Shimadzu France SAS, Le Luzard 2, Boulevard Salvador
Allende, 77448 Marne la Vallée Cedex 23 Univ Limoges, Limoges, France
2
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
IntroductionThe determination of drugs of abuse (opiates, amphetamines, cocaine) in biological �uids is still an important issue in toxicology, in cases of driving under the in�uence of drugs (DUID) as well as in forensic toxicology. At the end of the 20th century, the analytical methods able to determine these three groups of narcotics were mainly based on a liquid-liquid-extraction with derivatization followed by GC-MS. Then LC-MS/MS was proposed,
coupled with off-line sample preparation. Recently, on-line Solid-Phase-Extraction coupled with UHPLC-MS/MS was described, but in our hands it gave rise to signi�cant carry-over after highly concentrated samples. We propose here another approach based on the QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) sample preparation principle, followed by UHPLC-MS/MS.
Methods and MaterialsThis method involves 40 compounds of interest (13 opiates, 22 amphetamines, as well as cocaine and 4 of its
metabolites) and 18 isotopically labeled internal standards (designed with *) (Table1).
Table 1: list of analyzed compounds with their associate internal standard (*)
Cocaine and metabolitesAmphetamines or related
compounds Opiates
• Anhydroecgonine methylester• Benzoylecgonine*• Cocaethylene*• Cocaine*• Ecgonine methylester*
• 2-CB• 2-CI• 4-MTA• Ritalinic acid• Amphetamine*• BDB• Ephedrine*• MBDB• m-CPP• MDA*• MDEA*• MDMA*• MDPV• Mephedrone• Metamphetamine*• Methcathinone• Methiopropamine• Methylphenidate• Norephedrine• Norfen�uramine• Norpseudoephedrine• Pseudoephedrine
• 6-monoacetylmorphine*• Dextromethorphan• Dihydrocodeine*• Ethylmorphine• Hydrocodone• Hydromorphone• Methylmorphine*• Morphine*• Naloxone*• Naltrexone*• Noroxycodone*• Oxycodone*• Pholcodine
3
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
UHPLC conditions (Nexera MP system, �gure 1)
Column : Restek Pinnacle DB PFPP 50x2.1 mm 1.9 µm
Mobile phase A : 5mM Formate ammonium with 0.1% formic acid in water
B : 90% CH3OH/ 10% CH3CN (v/v) with 0.1 % formic acid
Flow rate : 0.474 mL/min
Time program : B conc. 15% (0-0.16 min) - 20% (1.77 min) - 90% (2.20 min) –
100% (4.00 min) – 15% (4.10-5.30 min)
Column temperature : 50 °C
MS conditions (LCMS-8040, �gure 1)
Ionization : ESI, Positive MRM mode
Ion source temperatures : Desolvation line: 300°C
Heater Block: 500°C
Gases : Nebulization: 2.5 L/min
Drying: 10 L/min
MRM Transitions : 2 Transitions per compounds were dynamically scanned for 1 min except
pholcodine (2 min)
Pause time : 3 msec
Loop time : 0.694 sec (minimum 17 points per peak for each MRM transition)
To 100 µL of sample (urine, whole blood or plasma) were added isotopically labeled internal standards (in order to improve method precision and accuracy) at 20 µg/L in acetonitrile (20 µL), and 200 µL of acetonitrile. After a 15 s shaking, the mixture was placed at -20°C for 10 min. Then approximately 50 mg of QuEChERS salts (MgSO4/NaCl/Sodium citrate dehydrate/Sodium citrate
sesquihydrate) were added and the mixture was shaken again for 15 s and centrifuged for 10 min at 12300 g. The upper layer was diluted (1/3; v/v) with a 5 mM ammonium formate buffer (pH 3). Finally, 5 µL were injected in the UHPLC-MS/MS system. The whole acquisition method lasted 5.5 min.
Figure 1: Shimadzu UHPLC-MS/MS Nexera-8040 system
4
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Figure 2: Chromatograms obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L. Order of retention - A: norephedrine and norpseudoephedrine / B: ephedrine and pseudoephedrine
Figure 3: Chromatogram obtained after an injection of a 5 µL whole blood extract spiked at 200 µg/L
Results
The analytical conditions allowed the chromatographic separation of two couples of isomers: norephedrine and norpseudoephedrine; ephedrine and pseudoephedrine
(figure 2). A typical chromatogram of the 58 compounds is presented in figure 3.
Chromatographic conditions
A B
5
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Figure 4: in�uence of QuEChERS salts on urine extraction/partitioning: A: acetonitrile with urine sample lead to one phase / B: acetonitrile, QuEChERS salts and urine lead to 2 phases.
As described by Anastassiades et al. J. AOAC Int 86 (2003) 412-31, the combination of acetonitrile and QuEChERS salts allowed the extraction/partitioning of compounds of interest from matrix. This extraction/partitioning process is not only
obtained with whole blood and plasma-serum where deproteinization occurred and allowed phase separation, but also with urine as presented in figure 4.
Extraction conditions
Among the 40 analyzed compounds, 38 filled the validation conditions in term of intra- and inter-assay precision and accuracy were less than 20% at the lower limit of quantification and less than 15% at the other concentrations.Despite the quick and simple sample preparation, no significant matrix effect was observed and the lower limit of quantification was 5 µg/L for all compounds, while the upper limit of quantification was set at 500 µg/L. The
concentrations obtained with a reference (GC-MS) method in positive patient samples were compared with those obtained with this new UHPLC-MS/MS method and showed satisfactory results.Contrary to what was already observed with on-line Solid-Phase-Extraction, no carry-over effect was noted using the present method, even when blank samples were injected after patient urine samples with analytes concentrations over 2000 µg/L.
Validation data
A B
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Determination of opiates, amphetamines and cocaine in whole blood, plasma and urine by UHPLC-MS/MS using a QuEChERS sample preparation
Conclusions• Separation of two couples of isomers with a run duration less than 6 minutes and using a 5 cm column.• Quick sample preparation based on QuEChERS salts extraction/partitioning, almost as short as on-line Solid Phase
Extraction.• Lower limit of quanti�cation compatible with determination of DUID.• No carry over effect noticed.
PO-CON1442E
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
ASMS 2014 ThP-592
Toshikazu Minohata1, Keiko Kudo2, Kiyotaka Usui3, Noriaki Shima4, Munehiro Katagi4, Hitoshi Tsuchihashi5, Koichi Suzuki5, Noriaki Ikeda2
1Shimadzu Corporation, Kyoto, Japan 2Kyushu University, Fukuoka, Japan 3Tohoku University Graduate School of Medicine, Sendai, Japan 4Osaka Prefectural Police, Osaka, Japan 5Osaka Medical Collage, Takatsuki, Japan
2
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
IntroductionIn Forensic Toxicology, LC/MS/MS has become a preferred method for the routine quantitative and qualitative analysis of drugs of abuse. LC/MS/MS allows for the simultaneous analysis of multiple compounds in a single run, thus enabling a fast and high throughput analysis. In this study, we report a developed analytical system using ultra-high
speed triple quadrupole mass spectrometry with a new extraction method for pretreatment in forensic analysis. The system has a sample preparation utilizing modi�ed QuEChERS extraction combined with a short chromatography column that results in a rapid run time making it suitable for routine use.
Figure 1 Scheme of the modi�ed QuEChERS procedure
[ ref.] (1) Usui K et al, Legal Medicine 14 (2012), 286-296
Methods and Materials
Whole blood sample preparation was carried out by the modified QuEChERS extraction method (1) using Q-sep™ QuEChERS Sample Prep Packets purchased from RESTEK (Bellefonte, PA).
1) Add 0.5 mL of blood and 1 mL of distilled water into the 15 mL centrifugal tube and agitate the mixture using a vortex mixer.
2) Add two 4 mm stainless steel beads, 1.5 mL of acetonitrile and 100 µL of acetonitrile solution containing 1 ng/µL of Diazepam-d5. Then agitate using the vortex mixer.
3) Add 0.5 g of the filler of the Q-sep™ QuEChERS Extraction Salts Packet.
4) Vigorously shake the tube by hand several times, agitate well using the vortex mixer for approximately 20 seconds. Then centrifuge the tube for 10 minutes at 3000 rpm.
5) Move the supernatant to a different 15 mL centrifugal tube and add 100 µL of 0.1 % TFA acetonitrile solution. Then, dry using a nitrogen-gas-spray concentration and drying unit or a similar unit.
6) Reconstitute with 200 µL of methanol using the vortex mixer. Then move it to a microtube, and centrifuge for 5 minutes at 10,000 rpm.
7) Transfer 150 µL of the supernatant to a 1.5 mL vial for HPLC provided with a small-volume insert.
Sample Preparation
Sample0.5 mL
Water 1 mL ACN 1.5 mL Diazepam-d5 (IS) 100ng Stainless-Steel Beads (4mm x 2)
[Shake] [Centrifuge]
Transfer supernatant Add 100uL of 0.1% TFA
Dry
Reconstitution with 200 uL MeOH
LC/MS/MS analysis
Q-sep QuEChERSExtraction Salts(MgSO4,NaOAc)
3
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Analytical Conditions
LC-MS/MS Analysis
HPLC (Nexera UHPLC system)
Column : YMC Triart C18 (100x2mm, 1.9μm)
Mobile Phase A : 10 mM Ammonium formate - water
Mobile Phase B : Methanol
Gradient Program : 5%B (0 min) - 95%B (10 min - 13min) - 5%B (13.1 min - 20 min)
Flow Rate : 0.3 mL / min
Column Temperature : 40 ºC
Injection Volume : 5 uL
Mass (LCMS-8050 triple quadrupole mass spectrometry)
Ionization : heated ESI
Polarity : Positive & Negative
Probe Voltage : +4.5 kV (ESI-Positive mode); -3.5 kV (ESI-Negative mode)
Nebulizing Gas Flow : 3 L / min
Drying Gas Pressure : 10 L / min
Heating gas �ow : 10 L / min
DL Temperature : 250 ºC
BH Temperature : 400 ºC
MRM parameter :
Treated samples were analyzed using a Nexera UHPLC system coupled to a LCMS-8050 triple quadrupole mass spectrometer (Shimadzu Corporation, Japan) with LC/MS/MS Rapid Tox. Screening Database. The Database contains product ion scan spectra for 106 forensic and toxicology-related compounds of Abused drugs, Psychotropic drugs and Hypnotic drugs etc (Table 1) and
provides Synchronized Survey Scan® parameters (product ion spectral data acquisition parameters based on the MRM intensity as threshold) optimized for screening analysis.Samples were separated on a YMC Triart C18 column. A �ow rate of 0.3 mL/min was used together with a gradient elution.
Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy
-27
-34
-24
-41
-23
-30
-24
-37
-30
-19
-24
-36
-24
-39
9.338
8.646
5.378
8.408
9.350
8.786
8.253
Diazepam-d5
Alprazolam
Atropine
Estazolam
Ethyl lo�azepate
Etizolam
Haloperidol
154.05
198.20
281.10
205.10
124.15
93.20
267.15
205.25
259.10
287.15
314.10
138.15
165.15
123.10
290.15
290.15
309.10
309.10
290.15
290.15
295.05
295.05
361.15
361.15
343.05
343.05
376.15
376.15
Analytes Ret. Time Q1 m/z Q3 m/zCollisionEnergy
-28
-55
-27
-25
25
14
21
15
19
14
23
16
7.993
8.573
8.093
5.243
6.762
8.883
Risperidone
Triazolam
Amobarbital(neg)
Barbital(neg)
Phenobarbital(neg)
Thiamylal(neg)
191.05
69.05
315.00
308.20
42.00
182.00
42.10
140.10
42.20
85.10
58.10
101.00
411.20
411.20
343.05
343.05
225.15
225.15
183.10
183.10
231.10
231.10
253.00
253.00
4
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Results and DiscussionEtizolam Risperidone TriazolamAlprazolam
0.1ng/mL
Conc. Area Accuracy %RSD9,0048,2889,51975,23675,98374,023829,519831,098849,597
112.1105.1119.389.689.680.699.999.6
104.2
0.01
0.1
1
6.57
6.04
2.53
Conc. Area Accuracy %RSD4,8655,1094,321
48,03849,15254,497
604,640581,207579,390
114.4119.9105.784.085.187.0103.799.2101.2
0.01
0.1
1
8.71
1.82
2.22
Conc. Area Accuracy %RSD29,83232,43630,461335,202309,273343,172
3,826,3733,718,8543,705,165
108.4116.7110.891.383.785.6102.899.4101.4
0.01
0.1
1
5.14
4.74
1.66
Conc. Area Accuracy %RSD3,0473,0643,35627,99125,54226,317288,776297,332294,788
107.0109.2118.594.885.781.599.0101.5102.9
0.01
0.1
1
5.63
7.83
1.96
negative
positive
Figure 2 LCMS-8050 triple quadrupole mass spectrometer
0.01ng/mL
S/N 39.5
309.10>281.10(+)
309.10>281.10(+)
(x103)
(x104)
2.0
1.0
0.5
0.0
1.0
0.5
0.0
8.0
0.00 0.25 0.75 Conc. Ratio0.50
8.5 9.0 9.5
1.0
0.0
Area Ratio
r2=0.998
0.00 0.25 0.75 Conc. Ratio0.50
7.5
5.0
2.5
0.0
Area Ratio (x0.1)
r2=0.998
0.00 0.25 0.75 Conc. Ratio0.50
Area Ratio
r2=0.9985.0
2.5
0.0
4.0
2.0
3.0
1.0
0.00.00 0.25 0.75 Conc. Ratio0.50
Area Ratio (x0.1)
r2=0.998
8.0 8.5 9.0 9.5 8.0 8.5 9.0 9.57.0 7.5 8.0 8.5
0.0
(x104)0.0
0.5
(x103)
1.0
343.05>314.10(+)
343.05>314.10(+)
S/N 145.5
0.0
(x104)0.0
(x103)
2.5
2.5
411.20>191.05(+)
411.20>191.05(+)
S/N 107.6
0.0
(x103)0.0
(x102)
2.5
2.5
S/N 18.8
343.05>315.00(+)
343.05>315.00(+)
5
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
In this experiment, two different matrices consisting of human whole blood and urine were prepared and 18 drugs were spiked into extract solution. Calibration curves constructed in the range from 0.01 to 1 ng/mL for 12 drugs (Alprazolam, Aripiprazole, Atropine, Brotizolam, Estazolam, Ethyl lo�azepate, Etizolam, Flunitrazepam,
Haloperidol, Nimetazepam, Risperidone and Triazolam) and from 1 to 100 ng/mL for 6 drugs (Bromovalerylurea, Amobarbital, Barbital, Loxoprofen, Phenobarbital and Thiamylal). All calibration curves displayed linearity with an R2 > 0.997 and excellent reproducibility was observed for all compounds (CV < 12%) at low concentration level.
Conc. Area Accuracy %RSD1,8371,8622,04121,68522,16920,654227,698223,480225,079
100.299.1
105.899.6
102.492.5
101.398.3
100.9
1
10
100
4.53
5.30
1.62
Conc. Area Accuracy %RSD521464509
5,0785,0335,424
55,42055,65853,484
108.796.6103.495.695.499.4101.4100.898.7
1
10
100
7.10
2.38
1.42
Conc. Area Accuracy %RSD725693617
7,9098,5647,93981,98783,27482,656
106100.2
9198.8107.596.799.299.7100.8
1
10
100
9.82
5.82
0.85
Conc. Area Accuracy %RSD2,5202,1922,28830,80829,62331,379318,233317,214313,399
10795.397.5101.498.3100.6100.799.3100
1
10
100
8.99
1.68
0.71
Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)
Figure 3 Results of 8 drugs spiked in human whole blood using LCMS-8050
7.5 8.0 8.5 9.0
10ng/mL
1ng/mL
2.5
(x102)
0.0(x103)
2.5
0.0
225.15>42.00(-)
225.15>42.00(-)
Area Ratio (x0.1)
r2=0.999
0.0 25.0 Conc. Ratio50.0
2.0
1.0
0.0
Area Ratio (x0.01)
0.0 25.0 Conc. Ratio50.0
5.0
2.5
0.0
r2=0.999Area Ratio (x0.1)
r2=0.999
0.0 25.0 Conc. Ratio50.00.00
0.25
0.50
0.75
1.00
0.0 25.0 Conc. Ratio50.00.0
1.0
2.0
3.0
4.0Area Ratio (x0.1)
r2=0.999
S/N 40.2 S/N 38.2 S/N 167.95.0
(x10)
0.0(x102)
5.0
2.5
0.0
183.10>42.10(-)
183.10>42.10(-)
S/N 15.3
231.10>42.20(-)
231.10>42.20(-)
1.0
(x102)
0.0
0.5
(x103)
1.0
0.5
0.0
5.0
(x102)
0.0
2.5
(x103)
5.0
2.5
0.0
253.00>58.10(-)
253.00>58.10(-)
4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Simultaneous analysis for forensic drugs in human blood and urine using ultra-high speed LC-MS/MS
Conc. Area Accuracy %RSD1,4681,2331,24517,24120,54618,689211,917251,963234,789
102.286.687.6104.4114.7106.996.810397.9
1
10
100
12.73
5.10
3.34
Conc. Area Accuracy %RSD651695654
4,9895,6135,443
55,39269,48166,327
93.696.189
105.2109.6108.692.6104
101.3
1
10
100
2.77
2.07
5.98
Conc. Area Accuracy %RSD612545609
5,6566,6326,38471,96588,68582,091
103.689.499.397.9106.1104.495.210599.1
1
10
100
8.16
4.24
4.95
Conc. Area Accuracy %RSD3,1423,4703,15327,25734,37732,933365,563431,826390,719
95.1100.591.494.9110.8108.598.5104.196.1
1
10
100
4.54
8.15
4.15
Figure 4 Results of 4 drugs spiked in human urine using LCMS-8050
Conclusions• The validated sample preparation protocol can get adequate recoveries in quantitative works for all compounds ranging
from acidic to basic. • The combination of the modi�ed QuEChERS extraction method and high-speed triple quadrupole LC/MS/MS with a
simple quantitative method enable to acquire reliable data easily.
7.5 8.0 8.5 9.0
Phenobarbital (neg) Thiamylal (neg)Amobarbital (neg) Barbital (neg)
Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999Area Ratio (x0.1)
r2=0.999
2.0
3.0
1.0
0.00.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0 0.0 25.0 Conc. Ratio50.0
0.50
0.75
0.25
0.00
1.0
0.5
0.0
5.0
2.5
0.0
10ng/mL
1ng/mL
2.5
(x102)
0.0(x103)
2.5
0.0
225.15>42.00(-)
225.15>42.00(-)
S/N 14.7 S/N 9.4 S/N 18.3 S/N 97.41.0
(x102)
(x102)
5.0
2.5
0.0
183.10>42.10(-)
183.10>42.10(-)
231.10>42.20(-)
231.10>42.20(-)
253.00>58.10(-)
253.00>58.10(-)
1.0
0.0
(x102)
(x103)
1.0
0.5
0.0
2.5
5.0
0.0
(x102)
(x103)
5.0
2.5
0.0
4.5 5.0 5.5 6.0 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
PO-CON1460E
Simultaneous Screening and Quantitationof Amphetamines in Urine by On-line SPE-LC/MS Method
ASMS 2014 ThP587
Helmy Rabaha1, Lim Swee Chin1, Sun Zhe2,
Jie Xing2 & Zhaoqi Zhan2
1Department of Scienti�c Services, Ministry of Health,
Brunei Darussalam;2Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE
2
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
IntroductionAmphetamines belong to stimulant drugs and are also controlled as illicit drugs worldwide. The conventional analytical procedure of amphetamines in human urine includes initial immunological screening followed by GCMS con�rmation and quantitation [1]. With new SAMHSA guidelines effective in Oct 2010 [2], screening, con�rmation and quantitation of illicit drugs including amphetamines were allowed to employ LC/MS and LC/MS/MS, which usually does not require a derivatization step as used in the GCMS method [1]. The objective of this study was to develop an on-line SPE-LC/MS method for
analysis of �ve amphetamines in urine without sample pre-treatment except dilution with water. The compounds studied include amphetamine (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guideline (group A in Table 1). Four potential interferences (group B in) and PMPA (R) as a control reference were also included to enhance the method reliability in identi�cation of the �ve targeted amphetamines from those structurally similar analogues which potentially present in forensic samples.
ExperimentalThe test stock solutions of the ten compounds (Table 1) were prepared in the toxicology laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and matrix to prepare spiked amphetamine samples were not pre-treated off-line by any means except dilution of 10 times with pure water. An on-line SPE-LC/MS was set up on the LCMS-2020, a single quadrupole system, with a switching valve and a trapping column kit (Shimadzu Co-Sense con�guration) installed in the column oven and controlled by the LabSolutions workstation. The analytical column used was Shim-pack VP-ODS 150 x 2mm (5um) and the trapping column was Synergi Polar-RP 50 x 2mm (2.5um), instead of
a normal SPE cartridge. The injected sample �rst passed through the trapping column where the amphetamines were trapped, concentrated and washed by pure water for 3 minutes followed by switching to the analytical �ow line. The trapped compounds were then eluted out with a gradient program: 0.01min, valve at position 0 & B=5%; 3 min, valve at position 1; 3.01-10 min, B=5% → 15%; 10.5-12 min, B=65%; 12.1 min, B=5%; 14 min stop, valve to position 0. The mobile phases A and B were water and MeOH both with 0.1% formic acid and mobile C was pure water. The total �ow rates of the trapping line and analytical line are 0.6 and 0.3 mL/min, respectively. The injection volume was 20uL in all experiments.
3
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
Figure 1: Schematic diagram of on-line SPE-LC/MS system
Table 1: Amphetamines & relevant compounds
Name Abbr. Name Formula Structure
Amphetamine
Methampheta-mine
3,4-methylene-dioxyamphetamine
3,4-methylene-dioxymetham phetamine
3,4-methylene dioxy-N-ethyl amphetamine
Nor pseudo-ephedrine
Ephedrine
Pseudo-Ephedrine
Phentermine
Propyl-amphetamine
AMPH
MAMP
MDA
MDMA
MDEA
Nor pseudo-E
Ephe
Pseudo-E
Phent
PAMP
No
A1
A2
A3
A4
A5
B1
B2
B3
B4
R
C9H13N
C10H15N
C10H13NO2
C11H15NO2
C12H17NO2
C9H13NO
C10H15NO
C10H15NO
C10H15N
C12H19N
Manual injectorPump A
SPE Trapping Column
5
13
Mixer
Switching Valve
LCMS-2020
Waste
Pump B Auto sampler
Analytical column
Pump C
4
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
Results and Discussion
With ESI positive SIM and scan mode, all of the 10 compounds formed protonated ions [M+H]+ which were used as quantifier ions. The scan spectra were used for confirmation to reduce false positive results. Mixed standards of the ten compounds in Table 1 spiked in urine was used for method development. An initial difficulty encountered was that the normal reusable SPE cartridges
(10-30 mmL) for on-line SPE could not trap all of the ten compounds. With using a 50mmL C18-column to replace the SPE cartridge, the ten compounds studied were trapped efficiently. Furthermore, the trapped compounds were well-separated and eluted out in 8~13 minutes as sharp peaks (Figure 2) by the fully automated on-line SPE-LC/MS method established.
Calibration curves of the on-line SPE-LC/MS method were established using mixed standard samples with concentrations from 2.5 ppb to 500 ppb. Linear calibration
curves with R2> 0.999 were obtained for every compound (Figure 3 & Table 2).
Development of on-line SPE-LC/MS method
Figure 2: SIM chromatograms of urine blank (a) and �ve amphetamines and related compounds (125 ppb each) spiked in urine (b) by on-line SPE-LC/MS.
0.0 2.5 5.0 7.5 10.0 12.5 min0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
(a) Urine blank (b) spiked samples
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
doEp
hedr
ine
Pseu
do
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
Figure 3: Calibration curves of �ve amphetamines and �ve related compounds with concentrations from 2.5 ppb to 500 ppb by on-line SPE-LC/MS method
0 250 Conc.0.0
2.5
5.0
7.5
Area (x1,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
Area (x10,000,000)
0 250 Conc.0.0
1.0
2.0
3.0Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x10,000,000)
0 250 Conc.0.0
2.5
5.0
Area (x1,000,000)
AMPH MAMP
Phent PAMP
MDA MDMA MDEA
Ephedrine Pseudo-ENor pseudo-E
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
5
Table 2: Peak detection, retention, calibration curves and method performance evaluation
NameRec. %
(62.5ppb)RSD%(n=6)(62.5ppb)
LOD/LOQ(ppb)
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP (Ref)
97.3
84.4
78.9
85.6
76.5
71.8
72.2
74.8
74.5
69.5
M.E %(62.5ppb)
69.3
111.0
109.2
71.1
96.8
70.3
116.3
107.1
69.9
96.8
Linearity(r2)
0.9982
0.9960
0.9976
0.9983
0.9968
0.9989
0.9973
0.9908
0.9960
0.9912
1.67
0.54
0.41
0.98
0.94
1.94
1.08
2.18
1.82
5.30
S/N(2.5ppb)
11.3
33.7
28.5
17.5
30.3
18.2
36.6
41.9
12.7
37.7
0.71/2.17
0.25/0.76
0.29/0.88
0.48/1.46
0.26/0.80
0.45/1.36
0.23/0.70
0.19/0.57
0.66/2.01
0.22/0.66
SIM ion(+)
152.1
166.1
166.1
136.1
150.1
180.1
194.1
208.1
150.1
178.1
RT(min)
8.0
8.4
9.0
9.6
10.2
10.4
10.8
12.2
12.4
12.7
Conc. range(ppb)
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
2.5 - 500
The trapping efficiency of the on-line SPE is critical and must be evaluated first, because it determines the recovery of the method. In this study, the recovery of the on-line SPE was determined by injecting a same mixed standard sample from a manual injector installed before the analytical column (by-pass on-line SPE) and also from the Autosampler (See Figure 1). The peaks areas obtained by the two injections were used to calculate recovery value of the on-line SPE method. As shown in Table 2, the recovery obtained with 62.5 ppb mixed standards are at 69.5% ~ 97.3%. The recovery with 250 ppb and 500 ppb mixed samples were also determined and similar results were obtained. Matrix effect was determined with 62.5 ppb and 250 ppb levels of mixed samples in clear solution and in urine. The results (Table 2) show a variation between 69.3% and 116% with compounds. The matrix effect with different
urine specimens did not show significant differences. Repeatability was evaluated with spiked mixed samples of 62.5 ppb and 250 ppb. The results of 62.5 ppb is shown in Table 2, RSD between 0.41% and 5.3%. The sensitivity of the on-line SPE-LC/MS method was evaluated with spiked sample of 2.5 ppb level. The SIM chromatograms are shown in Figure 4. The S/N ratios obtained ranged 11.3~42, which were suitable to determine LOQ (S/N = 10) and LOD (S/N = 3). Since the urine samples were diluted for 10 times with water before injection, the LOD and LOQ of the method for source urine samples were at 1.9~7.1 and 5.7~21.7 ng/mL, respectively. The confirmation cutoff values of the five targeted amphetamines (Group A) in urine enforced by the new SMAHSA guidelines are 250 ng/mL [2]. The on-line SPE-LC/MS method established has sufficient allowance in terms of sensitivity and confirmation reliability for analysis of actual urine samples.
Performance evaluation of on-line SPE-LCMS method
Figure 4: SIM chromatograms of 10 compounds with 2.5 ppb each by on-line SPE-LC/MS method.
7.5 10.0 12.5 min
1.0
2.0
3.0
4.0
5.0
6.0(x10,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do
Ephe
drin
e
Pseu
do
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
6
Figure 5: Durability test of on-line SPE-LC/MS method, comparison of 1st and 200th injections.
The durability of the trapping column was tested purposely by continuous injections of spiked urine samples (125 ppb) for 200 times in a few days. Figure 5 shows the chromatograms of the first and 200th injections of a same
spiked sample. The results show that the variations of peak area and retention time of the 200th injection compared to the 1st injection were at 89.5%~117.8% and 89.5%~99.8% respectively.
Durability of on-line SPE trapping column
Confirmation reliability of LC/MS and LC/MS/MS methods must be proven to be equivalent to the GCMS method according to the SMAHSA guidelines [2]. Validation of confirmation reliability of the on-line SPE-LC/MS method has not be carried out systematically. The high sensitivity of MS detection in SIM mode is a key factor to ensure no false-negative and the scan spectra acquired
simultaneously is used for excluding false-positive. In this work, the confirmation reliability was evaluated using five different urine specimens as matrix to prepare spiked samples of 2.5 ppb (correspond 25 ng/mL in source urine) and above. The results show that false-positive and false negative results were not found.
Con�rmation Reliability
ConclusionsA novel high sensitivity on-line SPE-LC/MS method was developed for screening, conformation and quanti�cation of �ve amphetamines: AMPH, MAMP, MDMA, MDA and MDEA in urines. The recovery of the on-line SPE by employing a 50mmL Synergi Polar-RP column was at 72%~86% for the �ve amphetamines, which are considerably high if comparing with conventional on-line
SPE cartridges. The method performance was evaluated thoroughly with urine spiked samples. The results demonstrate that the on-line SPE-LC/MS method is suitable for direct analysis of the amphetamines and relevant compounds in urine samples without off-line sample pre-treatment.
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0
(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do Ephe
drin
ePs
eudo
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
0.0 2.5 5.0 7.5 10.0 12.5 min
0.0
0.5
1.0
1.5
2.0(x1,000,000)
2:152.10(+)2:166.10(+)2:208.20(+)2:194.10(+)2:180.10(+)2:178.10(+)2:150.10(+)2:136.10(+)
Nor
pseu
do Ephe
drin
ePs
eudo
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
Phen
t
AM
PH
1st injection spiked mixed std 125ppb in urineinj vol: 20 µL
200th injection spiked mixed std 125ppb in urineinj vol: 20 µL
Simultaneous Screening and Quantitation of Amphetamines in Urine by On-line SPE-LC/MS Method
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. SAMHSA “Manual for urine laboratories, National laboratory certi�cation program”, Oct 2010, US Department of
Health and Human Services.
PO-CON1481E
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
ASMS 2014 MP762
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil J. Loftus1
1Shimadzu, Manchester, UK 2WMIC, University of Manchester, UK
2
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
IntroductionDried plasma sample collection and storage from whole blood without the need for centrifugation separation and refrigeration opens new opportunities in blood sampling strategies for quantitative LC/MS/MS bioanalysis. Plasma samples were generated by gravity �ltration of a whole blood sample through a laminated membrane stack allowing plasma to be collected, dried, transported and analysed by LC/MS/MS. This novel plasma separation card (PSC) technology was applied to the quantitative LC/MS/MS analysis of warfarin, in blood samples. Warfarin is a coumarin anticoagulant vitamin-K antagonist used for the treatment of thrombosis and thromboembolism. As a
result of vitamin-K recycling being inhibited, hepatic synthesis is in-turn inhibited for blood clotting factors as well as anticoagulant proteins. Whilst the measurement of warfarin activity in patients is normally measured by prothrombin time by international normalized ratio (INR) in some cases the quantitation of plasma warfarin concentration is needed to con�rm patient compliance, resistance to the anticoagulant drug, or diet related issues. In this preliminary evaluation, warfarin concentration was measured by LC/MS/MS to evaluate if PSC technology could complement INR when sampling patient blood.
Materials and Methods
Warfarin standard was dissolved in water containing 50% ethanol + 0.1% formic acid, spiked (60uL) to whole human blood (1mL) and mixed gently. 50uL of spiked blood was deposited onto the PSC. After 3 minutes, the primary filtration overlay was removed followed by 15 minutes air drying at room temperature. The plasma sample disc was prepared directly for analysis after drying. LC/MS/MS sample preparation involved vortexing the sample disk in
40uL methanol, followed by centrifugation 16,000g 5 min. 20uL supernatant was added directly to the LCMS/MS sample vial already containing 80uL water (2uL analysed). Control plasma comparison was prepared by centrifuging remaining blood at 1000g for 10min. 2.5uL supernatant plasma was taken, 40uL methanol added, and prepared as PSC samples. LCMS/MS sample injection volume, 2uL.
Sample preparation
Warfarin was measured by MRM, positive negative switching mode (15msec).
LC-MS/MS analysis
LC/MS/MS System : Nexera UHPLC system + LCMS-8040 Shimadzu Corporation
Flow rate : 0.4mL/min (0-7.75min), 0.5mL/min (7.5-14min), 0.4mL/min (15min)
Mobile phase : A= Water + 0.1% formic acid
B= Methanol + 0.1% formic acid
Gradient : 20% B (0-0.5 min), 100% B (8-12 min), 20% B (12.01-15 min)
Analytical column : Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
Column temperature : 50ºC
Ionisation : Electrospray, positive, negative switching mode
Desolvation line : 250ºC
Drying/Nebulising gas : 10L/min, 2L/min
Heating block : 400ºC
3
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
Design of plasma separator technology
Plasma separation work�ow
Control Spot:[Determines whether enough blood was placed on the card].
Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].
Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.
Isolation Screen[Precludes lateral wicking along the card surface].
Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].
1 3 42
A NoviPlex card is removed from foil packaging.
Approximately 50uL of whole blood is added to the test area.
After 3 minutes, the top layer is completely removed (peeled back).
The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.
The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.
Figure 1. Noviplex work�ow.
4
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
Figure 3. Comparison between the warfarin response in both positive and negative ion modes for warfarin calibration standards at 2.5ug/mL and 0.4ug/mL extracted from the plasma separation cards and a conventional plasma sample. There is a broad agreement in ion signal intensity between
the 2 sample preparation techniques.
ResultsComparison between plasma separation cards (PSC) and plasma
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
(x100,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.000.250.500.751.001.251.501.752.002.252.502.753.00
(x100,000)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00.10.20.30.40.50.60.70.80.91.01.11.2(x100,000)
1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x100,000)
Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05
Q1 (V) -22Collision energy -15Q3 (V) -15
Plasma separation cardNegative ionWarfarin m/z 307.20 > 161.25
Q1 (V) 14Collision energy 19 Q3 (V) 30
PlasmaNegative ionWarfarin m/z 307.20 > 161.05
Q1 (V) 14Collision energy 19 Q3 (V) 30
Plasma Positive ionWarfarin m/z 309.20 > 163.05
Q1 (V) -22Collision energy -15Q3 (V) -15
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
2.5ug/mL Calibration standard
0.4ug/mLCalibration standard
5
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
The drive to work with smaller sample volumes offers significant ethical and economical advantages in pharmaceutical and clinical workflows and dried blood spot sampling techniques have enabled a step change approach for many toxicokinetic and pharmacokinetic studies. However, the impressive growth of this technique in the quantitative analysis of small molecules has also discovered several limitations in the case of sample
instability (some enzyme labile compounds, particularly prodrugs, analyte stability can be problematic), hematocrit effect and background interferences of DBS. DBS also shows noticeable effects on many lipids dependent on the sample collection process. To compare PSC to plasma lipid profiles the same blood sample extraction procedure applied for warfarin analysis was measured by a high mass accuracy system optimized for lipid profiling.
Plasma separation card comparison
Figure 4. In both ion modes, the calibration curve was linear over the therapeutic range studied for warfarin extracted from PSC’s (calibration range 0-3ug/mL, single point calibration standards at each level with the exception of replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3); r2>0.99 for
PSC analysis [r2>0.99 for a conventional plasma extraction]).
Figure 5. Matrix blank comparison. In both ion modes, the MRM chromatograms for PSC and plasma are comparable. Warfarin ion signals were not detected in the any PSC or plasma matrix blank.
Plasma separation cardNegative ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)
Plasma separation cardPositive ionWarfarin m/z 309.20 > 163.05Replicate calibration points at 2.5ug/mL and 0.4ug/mL (n=3)
Linear regresson analysisy = 246527x + 14796
R² = 0.9986
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
0 0.5 1 1.5 2 2.5 3 3.5
Linear regression analysisy = 133197x + 15795
R² = 0.9954
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
0 0.5 1 1.5 2 2.5 3 3.5
Blood concentration ( ug/mL) Blood concentration ( ug/mL)
0.0 2.5 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75(x10,000)
2.5 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75(x10,000)
Matrix blank comparisonPositive ionPlasma separation card matrix blankPlasma matrix blank
Matrix blank comparisonNegative ionPlasma separation card matrix blankPlasma matrix blank
Single step separation of plasma from whole blood without the need for centrifugation applied to the quantitative analysis of warfarin
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Conclusions• In this limited study, plasma separation card (PSC) sampling delivered a quantitative analysis of warfarin spiked into
human blood.• PSC generated a linear calibration curve in both positive and negative ion modes (r2>0.99; n=5); • The warfarin plasma results achieved by using the PSC technique were in broad agreement with conventional plasma
sampling data.• The plasma generated by the �ltration process appears broadly similar to plasma derived from conventional
centrifugation.• Further work is required to consider the robustness and validation in a routine analysis.
References• Jensen, B.P., Chin, P.K.L., Begg, E.J. (2011) Quanti�cation of total and free concentrations of R- and S-warfarin in
human plasma by ultra�ltration and LC-MS/MS. Anal Bioanal Chem., 401, 2187-2193• Radwan, M.A., Bawazeer, G.A., Aloudah, N.M., Aluadeib, B.T., Aboul-Enein, H.Y. (2012) Determination of free and total
warfarin concentrations in plasma using UPLC MS/MS and its application to patient samples. Biochemical Chromatography, 26, 6-11
Figure 6. Lipid pro�les from the same human blood sample extracted using a plasma separation card (left hand pro�le) compared to a conventional plasma samples (centrifugation). Both lipid pro�les are comparable in terms of distribution and the number of lipids detected (the scaling has been
normalized to the most intense lipid signal).
Conventional plasma samplePositive ionLCMS-IT-TOFLipid pro�ling
Diacylglycero-phosphocholines
Ceramidephosphocholines
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
MonoacylglycerophosphoethanolaminesMonoacylglycerophosphocholines
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
Plasma separation card samplePositive ionLCMS-IT-TOFLipid pro�ling
PO-CON1462E
Development and Validation of Direct Analysis Method for Screeningand Quantitation of Amphetamines in Urine by LC/MS/MS
ASMS 2014 MP535
Zhaoqi Zhan1, Zhe Sun1, Jie Xing1, Helmy Rabaha2
and Lim Swee Chin2 1Shimadzu (Asia Paci�c) Pte Ltd, Singapore, SINGAPORE;2Department of Scienti�c Services, Ministry of Health,
Brunei Darussalam
2
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
IntroductionAmphetamines are among the most commonly abused drugs type worldwide. The conventional analytical procedure of amphetamines in human urine in forensic laboratory involves initial immunological screening followed by GCMS con�rmation and quantitation [1]. The new guidelines of SAMHSA under U.S. Department of Health and Human Services effective in Oct 2010 [2] allowed use of LC/MS/MS for screening, con�rmation and quantitation of illicit drugs including amphetamines. One of the advantages by using LC/MS/MS is that derivatization of amphetamines before analysis is not needed, which was a standard procedure of GCMS method. Since analysis speed and throughput could be enhanced signi�cantly, development and use of LC/MS/MS methods are in
demand and many such efforts have been reported recently [3]. The objective of this study is to develop a fast LC/MS/MS method for direct analysis of amphetamines in urine without sample pre-treatment (except dilution with water) on LCMS-8040, a triple quadrupole system featured as ultra fast mass spectrometry (UFMS). The compounds studied include amphetamines (AMPH), methamphetamine (MAMP) and three newly added MDMA, MDA and MDEA by the new SAMHSA guidelines, four potential interferences as well as PMPA as a control reference (Table 1). Very small injection volumes of 0.1uL to 1uL was adopted in this study, which enabled the method suitable for direct injection of untreated urine samples without causing signi�cant contamination to the ESI interface.
ExperimentalThe stock standard solutions of amphetamines and related compounds as listed in Table 1 were prepared in the Toxicology Laboratory in the Department of Scienti�c Services (MOH, Brunei). Five urine specimens were collected from healthy adult volunteers. The urine samples used as blank and spiked samples were not pre-treated by any means except dilution of 10 times with Milli-Q water.An LCMS-8040 triple quadrupole coupled with a Nexera UHPLC system (Shimadzu Corporation) was used. The analytical column used was a Shim-pack XR-ODS III UHPLC column (1.6 µm) 50mm x 2mm. The mobile phases used
were water (A) and MeOH (B), both with 0.1% formic acid. A fast gradient elution program was developed for analysis of the ten compounds: 0-1.6min, B=2%->14%; 1.8-2.3min, B=70%; 2.4min, B=2%; end at 4min. The total �ow rate was 0.6 mL/min. Positive ESI ionization mode was applied with drying gas �ow of 15 L/min, nebulizing gas �ow of 3 L/min, heating block temperature of 400 ºC and DL temperature of 250 ºC. Various injection volumes from 0.1 uL to 5 uL were tested to develop a method with a lower injection volume to reduce contamination of untreated urine samples to the interface.
Results and Discussion
MRM optimization of the ten compounds (Table 1) was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions were selected for each compound, one for quantitation and second one for confirmation (Table 1). The ten compounds were separated and eluted in 0.75~2.2 minutes as sharp peaks as shown in Figure 1. In addition to analysis speed and detection sensitivity, this method development was also focused on evaluation of small to ultra-small injection volumes to develop a method suitable for direct injection of urine samples without any
pre-treatment while it should not cause significant contamination to the interface. The Nexera SIL-30A auto-sampler enables to inject as low as 0.10 uL of sample with excellent precision.Figure 1 shows a few selected results of direct injection of urine blank (a) and mixed standards spiked in urine with 1 uL (c and d) and 0.1 uL (b) injection. It can be seen that all compounds (12.5 ppb each in urine) could be detected with 0.1uL injection except MDA and Norpseudo-E. With 1uL injection, all of them were detected.
Method development of direct injection of amphetamines in urine
3
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
Figure 1: MRM chromatograms of urine blank (a) and spiked samples of amphetamines and related compounds in urine by LC/MS/MS method with 1uL and 0.1uL injection volumes.
Table 1: MRMs of amphetamines and related compounds
Compound Abbr. RT (min) MRM
Nor pseudo ephedrine
Ephedrine
Pseudo ephedrine
Amphetamine
Methampheta-mine
3,4-methylenedi oxyamphetamine
3,4-methylene dioxymeth amphetamine
3,4-methylene dioxy-N-ethyl amphetamine
Phentermine
Propyl amphetamine
Nor pseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
Cat.
B1
B2
B3
A1
A2
A3
A4
A5
B4
R
0.75
0.94
1.01
1.20
1.42
1.49
1.59
1.94
1.93
2.20
152>134
152>115
166>148
166>91
166>148
166>91
136>91
136>119
150>91
150>119
180>163
180>163
194>163
194>105
208>163
208>105
150>91
150>119
178>91
178>65
CE (V)
-13
-23
-14
-31
-14
-30
-20
-14
-20
-14
-12
-38
-13
-22
-12
-24
-20
-40
-22
-47
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x10,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x100,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
0.5
1.0
1.5
(x1,000,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
(a) Urine blank, 1 uL inj (b) 12.5ppb in urine, 0.1uL inj
(c) 12.5ppb, 1uL inj (d) 62.5ppb in urine, 1uL inj
0.0 0.5 1.0 1.5 2.0 2.5 min0.0
1.0
2.0
3.0
(x10,000)
4
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
Figure 2: Calibration Curves of amphetamines spiked in urine with 0.1uL injection
Linear calibration curves were established for the ten compounds spiked in urine with different injection volumes: 0.1, 0.2, 0.5, 1, 2 and 5 uL. Good linearity of calibration curves (R2>0.999) were obtained for all injection volumes including 0.1uL, an ultra-small injection
volume. The calibration curves with 0.1 uL injection volume are shown in Figure 2. The linearity (r2) of all compounds with 0.1 uL and 1 uL injection volumes are equivalently good as shown in Table 2.
Calibration curves with small and ultra-small injection volumes
Repeatability of peak area was evaluated with a same loading amount (6.25 pg) but with different injection volumes. The RSD shown in Table 2 were 1.6% ~ 7.9% and 1.6 ~ 7.8% for 0.1uL and 1uL injection, respectively. It is worth to note that the repeatability of every compounds with of 0.1uL injection is closed to that of 1uL injection as well as 5uL injection (data not shown).Matrix effect of the method was determined by comparison of peak areas of mixed standards in pure water and in urine matrix. The results of 62.5ppb with 1uL injection were at 102-115% except norpseudoephedrine (79%) as shown in Table 2.Accuracy and sensitivity of the method were evaluated with spiked samples of low concentrations. The results of
LOD and LOQ of the ten compounds in urine are shown in Table 3. Since the working samples (blank and spiked) were diluted for 10 times with water before injection, the concentrations and LOD/LOQ of the method described above for source urine samples have to multiply a factor of 10. Therefore, the LOQs of the method for urine specimens are at 2.1-17.1 ng/mL for AMPH, PAMP, MDMA and MDEA and 53 ng/mL for MDA. The LOQs for the potential interferences (Phentermine, Ephedrine, Pseudo-Ephedrine and Norpseudo-Ephedrine) are at 17-91 ng/mL, 2.4 ng/mL for the internal reference MAMP. The sensitivity of the direct injection LC/MS/MS method are significantly higher than the confirmation cutoff (250 ng/mL) required by the SAMHSA guidelines.
Performance validation
0 250 Conc.0.0
1.0
2.0
3.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
0 250 Conc.0.0
2.5
5.0
Area (x100,000)
0 250 Conc.0.0
2.5
5.0Area (x100,000)
0 250 Conc.0.0
0.5
1.0
1.5
Area (x1,000,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
0 250 Conc.0.00
0.25
0.50
0.75
1.00
1.25Area (x1,000,000)
0 250 Conc.0.0
2.5
5.0
7.5
Area (x100,000)
AMPH MAMP
Phent PAMP
MDA MDMA MDEA
Ephedrine Pseudo-ENor pseudo-E
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
5
Table 2: Method Performance with different inj. volumes
NameCalibration curve, R2
(0.1uL)
RSD% area (n=6)
(0.1uL)
M.E. %1
(1uL)
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
0.9992
0.9995
0.9994
0.9997
0.9998
0.9978
0.9993
0.9996
0.9998
0.9998
(1uL)
0.9996
0.9998
0.9986
0.9998
0.9999
0.9995
0.9998
0.9998
0.9998
0.9932
(ppb)2
1-500
2.5-500
1-500
1-500
1-500
2.5-500
1-500
1-500
2.5-500
1-500
4.5
3.2
3.7
3.5
1.6
7.9
1.8
3.5
4.1
2.9
(1uL)
5.7
2.9
3.3
2.4
2.3
7.8
4.5
2.9
1.6
2.0
79
115
113
102
110
103
115
115
106
102
The method operational stability with 1uL injection was tested with spiked samples of 25 ppb in five urine specimens, corresponding to 250 ng/mL in the source urine samples. Continuous injections of accumulated 120 times was carried out in about 10 hours. The purpose of the experiment was to evaluate the operational stability against the ESI source contamination by urine samples without pre-treatment. Figure 3 shows the first injection and the
120th injection of the same spiked sample (S1) as well as other spiked samples (S2, S3, S4 and S5) in between. Decrease in peak areas of the compounds occurred, but the degree of the decrease in average was about 17% from the first injection to the last injection. This result indicates that it is possible to carry out direct analysis of urine samples (10 times dilution with water) by the high sensitivity LC/MS/MS method with a very small injection volume.
Method operational stability
1: Measured with mixed stds of 62.5 ppb in clear solution and spiked in urine2: For 0.1uL injection, the lowest conc. is 2.5 or 12.5 ppb
Table 3: Method performance: sensitivity & accuracy (1uL)
NameMeas. S/N LOQ
Norpseudo-E
Ephe
Pseudo-E
AMPH
MAMP
MDA
MDMA
MDEA
Phent
PAMP
1.2
2.2
1.0
1.1
1.0
2.4
1.1
1.1
2.6
1.0
Accuracy
(%)
118.7
88.2
99.5
114.1
103.6
96.3
106.4
111.8
105.3
101.7
Conc. (ppb)
Prep.
1.0
2.5
1.0
1.0
1.0
2.5
1.0
1.0
2.5
1.0
2.3
2.7
5.9
6.7
21.8
4.5
51.9
28.5
2.9
42.2
Sensitivity (ppb)
LOD
1.53
2.41
0.50
0.51
0.14
1.60
0.06
0.12
2.73
0.07
5.09
8.04
1.67
1.71
0.47
5.34
0.21
0.39
9.10
0.24
Development and Validation of Direct Analysis Method for Screening and Quantitation of Amphetamines in Urine by LC/MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 3: Selected chromatograms of continuous injections of spiked samples (25 ppb) with 1 µL injection. Five urine specimens S1, S2, S3, S4 and S5 were used to prepare these spiked samples.
References1. Kudo K, Ishida T, Hara K, Kashimura S, Tsuji A, Ikeda N, J Chromatogr B, 2007, 855, 115-120. 2. Mandatory guidelines for Federal Workplace Drug Testing Program, 73 FR 71858-71907, Nov. 25, 2008. 3. Huei-Ru Lina, Ka-Ian Choia, Tzu-Chieh Linc, Anren Hu,, Journal of Chromatogr B, 2013, 929, 133–141.
ConclusionsIn this study, we developed a fast LC/MS/MS method for direct analysis of �ve amphetamines and related compounds in human urine for screening and quantitative con�rmation. Very small injection volumes of 0.1~1.0 uL were adopted to minimize ESI contamination and enhance
operational stability. The good performance results observed reveals that screening and con�rmation of amphetamines in human urine by direct injection to LC/MS/MS is possible and the method could be an alternative choice in forensic and toxicology analysis.
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
do
Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
do Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
7.5
(x100,000)
Phen
t
Nor
pseu
doPs
eudo
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
do Pseu
do
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
do Pseu
doEp
hedr
ine
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
0.0 1.0 2.0 min
0.0
2.5
5.0
(x100,000)
Phen
t
Nor
pseu
doPs
eudo
Ephe
drin
e
MD
EA
MD
MA
MD
A
PAM
P
MA
MP
AM
PH
S1 (1st inj)
S1 (110th inj)
S2 (11th inj) S3 (21st inj)
S4 (31st inj) S5 (41st inj)
PO-CON1482E
Next generation plasma collectiontechnology for the clinical analysis oftemozolomide by HILIC/MS/MS
ASMS 2014 WP641
Alan J. Barnes1, Carrie-Anne Mellor2,
Adam McMahon2, Neil Loftus1
1Shimadzu, Manchester, UK 22WMIC, University of Manchester, UK
2
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
IntroductionPlasma extraction technology is a novel technique achieved by applying a blood sample to a laminated membrane stack which allows plasma to �ow through the asymmetric �lter whilst retaining the cellular components of the blood sample.Plasma separation card technology was applied to the quantitative analysis of temozolomide (TMZ); an oral imidazotetrazine alkylating agent used for the treatment of Grade IV astrocytoma, an aggressive form of brain tumour.
Under physiological conditions TMZ is rapidly converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) which in-turn degrades by hydrolysis to 5-aminoimidazole-4-carboxamide (AIC). Storage of plasma has previously shown that both at -70C and 4C degradation still occurs. In these experiments, whole blood containing TMZ standard was applied to NoviPlex plasma separation cards (PSC). The aim was to develop a robust LC/MS/MS quantitative method for TMZ.
Materials and Methods
TMZ spiked human blood calibration standards (50uL) were applied to the PSC as described below in figure 1.
Plasma separation
1 3 42
A NoviPlex card is removed from foil packaging.
Approximately 50uL of whole blood is added to the test area.
After 3 minutes, the top layer is completely removed (peeled back).
The collection disc contains 2.5uL of plasma. Card is air dried for 15 minutes.
The collection disc is removed from the card and is ready for extraction for LC-MS/MS analysis.
Figure 1. Noviplex plasma separation card work�ow
3
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Control Spot:[Determines whether enough blood was placed on the card].
Filtration Layer[Filtration layer captures blood cells by a combination of �ltration and adsorption. The average linear vertical migration rate is approximately 1um/sec].
Collection Layer[Loads with a speci�c aliquot of plasma onto a 6.35mm disc]. Although �ow through the �ltration membrane is unlikely to be constant throughout the plasma extraction process, the average loading rate of the Collection Disc was 13 nL/sec. This corresponds to a volumetric �ow rate into the Collection Disc of 400 pL/mm2/sec.
Isolation Screen[Precludes lateral wicking along the card surface].
Spreading Layer[Lateral spreading layer rapidly spreads blood so it will enter the �ltration layer as a front while adding buffers and anticoagulants. The lateral spreading rate is 150um/sec].
Figure 1. Noviplex plasma separation card work�ow (Cont'd)
Figure 2. Applying a blood sample, either as a �nger prick or by accurately measuring the blood volume, to the laminated membrane stack retains red cells and allows a plasma sample to be collected. The red cells are retained by a combination of adsorption and �ltration whilst plasma advances through the membrane stack
by capillary action. After approximately three minutes the plasma Collection Disc was saturated with an aliquot of plasma and was ready for LC/MS/MS analysis.
TMZ was extracted from the dried plasma collection discs by adding 40uL acetonitrile + 0.1% formic acid, followed by centrifugation 16,000g for 5 min. 30uL supernatant was added directly to the LC/MS/MS sample vial for analysis.
As a control, conventional plasma samples were prepared by centrifuging the human blood calibration standards at 1000g for 10min. TMZ was extracted from 2.5uL of plasma using the same extraction protocol as applied for PSC.
Sample preparation
4
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Figure 3. HILIC LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99).HILIC was considered in response to previous published data and to minimize potential stability issues. However, to reduce sample cycle times a reverse
phase method was also developed.
Results
Temozolomide is known to be unstable under physiological conditions and is converted to 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC) by
a nonenzymatic, chemical degradation process. Previous studies have used HILIC to analyze the polar compound and to avoid degradation in aqueous solutions.
HILIC LC/MS/MS
LC/MS/MS analysis
Ionisation : Electrospray, positive mode
MRM 195.05 >138.05 CE -10
HPLC : HILIC
Nexera UHPLC system
Flow rate : 0.5mL/min (0-7min), 1.8mL/min (7.5min-17.5min)
Mobile phase : A= Water + 0.1% formic acid
B= Acetonitrile + 0.1% formic acid
Gradient : 95% B – 30%% B (6.5 min),
30% B (7.5 min), 95% B (18 min)
Analytical column : ZIC HILIC 150 x 4.6mm 5um 200ª
Column temperature : 40ºC
Injection volume : 10uL
Reverse Phase
Nexera UHPLC system
0.4mL/min
A= Water + 0.1% formic acid
B= methanol + 0.1% formic acid
5% B – 100%% B (3 min),
100% B (7 min), 5% B (10 min)
Phenomenex Kinetex XB C18 100 x 2.1mm 1.7um 100A
50ºC
2µL
Desolvation line : 300ºC
Drying/Nebulising gas : 10L/min, 2L/min
Heating block : 400ºC
Linear regression analysisy = 64578x + 18473
R² = 0.9988
0
100000
200000
300000
400000
500000
600000
700000
0 2 4 6 8 10 12
Peak Area
Blood Concentration (ug/mL)
Plasma separation cardHILIC analysisTMZ Single point calibration standardsCalibration curve 0.2-10ug/mL
0.0 2.5 5.0 min
0.0
1.0
2.0
3.0
4.0
5.0(x10,000)
Plasma separation cardHILIC analysisTMZ m/z 195.05 > 138.05
Q1 (V) -20 Collision energy -10 Q3 (V) -12
8.0ug/mL calibn std
0.5ug/mL calibn std
5
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
Figure 4. Reverse phase LC/MS/MS chromatograms for PSC TMZ analysis at 0.5 and 8ug/mL. The PSC calibration curve was linear between 0.2-10ug/mL (r2>0.99; replicate samples were included in the liner regression analysis at 0.5 and 8ug/mL; n=3).
Due to the relatively long cycle time (18 min), a faster reversed phase method was developed (10 min). Sample preparation was modified with PSC sample disk placed in 40uL methanol + 0.1% formic acid, followed by centrifugation 16,000g 5 min. 20uL supernatant was
added directly to the LC/MS sample vial plus 80uL water + 0.1% formic acid. In addition to reversed phase being faster, the sample injection volume was reduced to just 2uL as a result of higher sensitivity from narrower peak width (reversed phase,13 sec; HILIC, 42 sec).
Reversed Phase LC/MS/MS
Figure 5. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the con�rmatory ion transition 195.05>67.05 both the PSC and plasma sample are in broad agreement with regard to matrix ion signal distribution.
Comparison between PSC and plasma
Linear regression analysisy = 72219x - 355.54
R² = 0.9997
0
100,000
200,000
300,000
400,000
500,000
600,000
700,000
800,000
0 2 4 6 8 10 12
Peak Area
Blood Concentration (ug/mL)
Plasma separation cardRP analysisTMZ m/z 195.05 > 138.05
Q1 (V) -20Collision energy -10Q3 (V) -12
8.0ug/mL Calibration standard
0.5ug/mLCalibration standard
Plasma separation cardRP analysisTMZ calibration curveReplicate calibration points at 0.5ug/mL and 8ug/mL (n=3)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 min
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
(x10,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x1,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
(x100) Matrix blank comparisonMRM 195.05>67.05Plasma separation card matrix blank
Plasma matrix blank
500ng/mL comparisonMRM 195.05>67.05Plasma separation card 500ng/mL calibration standard
Plasma500ng/mL calibration standard
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Next generation plasma collection technology for the clinical analysis of temozolomide by HILIC/MS/MS
ConclusionsThis technology has the potential for a simplified clinical sample collection by the finger prick approach, with future work aimed to evaluate long term sample stability of PSC samples, stored at room temperature. Quantitation of drug metabolites MTIC and AIC also could help provide a measure of sample stability.
References• Andrasia, M., Bustosb, R., Gaspara,A., Gomezb, F.A. & Kleknerc, A. (2010) Analysis and stability study of
temozolomide using capillary electrophoresis. Journal of Chromatography B. Vol. 878, p1801-1808• Denny, B.J., Wheelhouse, R.T., Stevens, M.F.G., Tsang, L.L.H., Slack, J.A., (1994) NMR and molecular modeliing
investigation of the mechanism of activation of the antitumour drug temozolomide and its Interaction with DNA. Biochemistry, Vol. 33, p9045-9051
Figure 6. Human blood TMZ calibration standards were prepared using PSC and conventional plasma. Using the quantitation ion transition 195.05>138.05 both the PSC and plasma sample are in broad agreement in signal distribution and intensity including the presence of
a matrix peak at 2.4mins.
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x10,000)
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50
(x10,000) Matrix blank comparisonMRM 195.05>138.05Plasma separation card matrix blank
Plasma matrix blank
500ng/mL comparisonMRM 195.05>138.05Plasma separation card 500ng/mL calibration standard
Plasma500ng/mL calibration standard
TMZ
TMZRt
1.7mins
Matrix peak Matrix peak
PO-CON1475E
Application of a Sensitive Liquid Chromatography-Tandem Mass SpectrometricMethod to Pharmacokinetic Study of Telbivudine in Humans
ASMS 2014 WP 629
Bicui Chen1, Bin Wang1, Xiaojin Shi1, Yuling Song2,
Jinting Yao2, Taohong Huang2, Shin-ichi Kawano2,
Yuki Hashi2
1 Pharmacy Department, Huashan Hospital,
Fudan University,
2 Shimadzu Global COE, Shimadzu (China) Co., Ltd.
2
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
IntroductionTelbivudine is a synthetic L-nucleoside analogue, which is phosphorylated to its active metabolite, 5’-triphosphate, by cellular kinases. The telbivudine 5’-triphosphate inhibits HBV DNA polymerase (a reverse transcriptase) by competing with the natural substrate, dTTP. Incorporation
of 5’-triphosphorylated-telbivudine into viral DNA obligates DNA chain termination, resulting in inhibition of HBV replication. The objectives of the current studies were to develop a selective and sensitive LC-MS/MS method to determine of telbivudine in human plasma.
Method
(1) Add 100 μL of plasma into the polypropylene tube, add 40 μL of internal standard working solution (33 µg/mL, with thymidine phosphorylase) to all other tubes.
(2) Incubate the tubes for 1 h at 37 ºC in dark.(3) Add 200 μL of acetonitrile to all tubes, seal and vortex for 1 minutes.(4) Centrifuge the tubes for 5 minutes at 13000 rpm.(5) Transfer 200 μL supernatant to a clean glass bottle and inject into the HPLC-MS/MS system.
Sample Preparation
The analysis was performed on a Shimadzu Nexera UHPLC instrument (Kyoto, Japan) equipped with LC-30AD pumps, CTO-30A column oven, DGU-30A5 on-line egasser, and SIL-30AC autosampler. The separation was carried out on GL Sciences InertSustain C18 column (3.0 mmI.D. x 100
mmL.) with the column temperature at 40 ºC. A triple quadruple mass spectrometer (Shimadzu LCMS-8050, Kyoto, Japan) was connected to the UHPLC instrument via an ESI interface.
LC-MS/MS Analysis
Analytical Conditions
HPLC (Nexera UHPLC system)
Column : InertSustain (3.0 mmI.D. x 100 mmL., 2 μm, GL Sciences)
Mobile Phase A : water with 0.1% formic acid
Mobile Phase B : acetonitrile
Gradient Program : as shown in Table 1
Flow Rate : 0.4 mL/min
Column Temperature : 40 ºC
Injection Volume : 2 µL
Table 1 Time Program
Time (min) Module Command Value
0.00
4.00
4.10
6.00
Pumps
Pumps
Pumps
Controller
Pump B Conc.
Pump B Conc.
Pump B Conc.
Stop
5
80
5
3
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
Results and DiscussionHuman plasma samples containing telbivudine ranging from 1.0 to 10000 ng/mL were prepared and extracted by protein precipitation and the �nal extracts were analyzed by LC-MS/MS. MRM chromatograms of telbivudine (1 ng/mL) and deuterated internal standard are presented in Fig. 1 (blank) and Fig. 2 (spiked). The linear regression for telbivudine was found to be >0.9999. The calibration curve with human plasma as the matrix were shown in Fig. 3. Excellent precision and accuracy were maintained for four orders of magnitude, demonstrating a linear dynamic range suitable for real-world applications. LLOQ for telbivudine was 1.0 ng/mL, which met the criteria for bias (%) and precision within ±15% both within run and between run. The
intra-day and inter-day precision and accuracy of the assay were investigated by analyzing QC samples. Intra-day precision (%RSD) at three concentration levels (3, 30, and 8000 ng/mL) were below 2.5% and inter-day precision (%RSD) was below 2.5%. The recoveries of telbivudine were 100.6±2.5 %, 104.5±1.5% and 104.3±1.6% at three concentration levels, respectively. The stability data showed that the processed samples were stable at the room temperature for 8 h, and there was no signi�cant degradation during the three freeze/thaw cycles at -20 ºC. The reinjection reproducibility results indicated that the extracted samples could be stable for 72 h at 10 ºC.
MS (LCMS-8050 triple quadrupole mass spectrometer)
Ionization : ESI
Polarity : Positive
Ionization Voltage : +0.5 kV (ESI-Positive mode)
Nebulizing Gas Flow : 3.0 L/min
Heating Gas Flow : 8.0 L/min
Drying Gas Flow : 12.0 L/min
Interface Temperature : 250 ºC
Heat Block Temperature : 300 ºC
DL Temperature : 350 ºC
Mode : MRM
Table 2 MRM Parameters
CompoundPrecursor
m/z
243.10
246.10
Productm/z
127.10
130.10
Dwell Time(ms)
100
100
Q1 Pre Bias(V)
-26
-16
Q3 Pre Bias(V)
-13
-25
CE (V)
-10
-9
Telbivudine
Telbivudine-D3
4
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
Figure 1 Representative MRM chromatograms of blank human plasma(left: transition for telbivudine, right: transition for internal standard)
Figure 2 Representative MRM chromatograms of telbivudine (left, 1 ng/mL) and internal standard (right) in human plasma
Figure 3 Calibration curve of telbivudine in human plasma
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
4.0(x100)
1:Telbivudine 243.10>127.10(+) CE: -10.0
1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
4.0
(x1,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
2.5
5.0
7.5
(x100)1:Telbivudine 243.10>127.10(+) CE: -10.0
Telb
ivud
ine
1.0 2.0 3.0 4.0 5.0 min
0.00
0.25
0.50
0.75
1.00
1.25
1.50(x1,000,000)
2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
Telb
ivud
ine-
D3
0 2500 5000 7500 Conc. Ratio0.0
0.5
1.0
1.5
2.0
2.5
Area Ratio
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
5
Figure 4 Representative MRM chromatograms of real-world sample
CompoundCalibration
Curve
Y = (2.77×10-4)X + (3.39×10-5)
Linear Range(ng/mL)
1~10000
Accuracy(%)
93.1~116.6%
r
0.9998Telbivudine
Table 3 Accuracy and precision for the analysis of amlodipine in human plasma(in pre-study validation, n=3 days, six replicates per day)
Added Conc.(ng/mL)
3
400
8000
Intra-day Precision(%RSD)
2.18
1.52
1.76
Inter-day Precision(%RSD)
2.11
1.58
1.68
Accuracy(%)
107.7~114.4
91.6~95.9
95.4~101.3
Table 5 Matrix effect for QC samples (n=6)
QC Level
LQC
MQC
HQC
Added Conc.(ng/mL)
3
400
8000
Matrix Factor
82.3%
81.7%
90.8%
IS-NormalizedMatrix Factor
99.0%
101.0%
101.5%
Table 4 Recovery for QC samples (n=6)
QC Level
LQC
MQC
HQC
Concentartion(ng/mL)
3
400
8000
Recovery(%)
100.6
104.5
104.3
0.0 1.0 2.0 3.0 4.0 5.0 min
0.0
1.0
2.0
3.0
(x10,000)1:Telbivudine 243.10>127.10(+) CE: -10.0
1.0 2.0 3.0 4.0 5.0 min
0.00
0.25
0.50
0.75
1.00
(x1,000,000)2:Telbivudine-D3 246.10>130.10(+) CE: -9.0
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Application of a Sensitive Liquid Chromatography-Tandem Mass Spectrometric Method to Pharmacokinetic Study of Telbivudine in Humans
ConclusionResults of parameters for method validation such as dynamic range, linearity, LLOQ, intra-day precision, inter-day precision, recoveries, and matrix effect factors were excellent. The sensitive LC-MS/MS technique provides a powerful tool for the high-throughput and highly selective analysis of telbivudine in clinical trial study.
PO-CON1449E
Accelerated and robust monitoringfor immunosuppressants using triplequadrupole mass spectrometry
ASMS 2014 WP628
Natsuyo Asano1, Tairo Ogura1, Kiyomi Arakawa1
1 Shimadzu Corporation. 1, Nishinokyo Kuwabara-cho,
Nakagyo-ku, Kyoto 604–8511, Japan
2
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
IntroductionImmunosuppressants are drugs which lower or suppress activity of the immune system. They are used to prevent the rejection after transplantation or treat autoimmune disease. To avoid immunode�ciency as adverse effect, it is recommended to monitor blood level of therapeutic drug with high throughput and high reliability. There are several analytical technique to monitor drugs, LC/MS is superior in terms of cross-reactivity at low level and throughput of
analysis. Therefore, it is important to analyze these drugs in blood by using ultra-fast mass spectrometer to accelerate monitoring with high quantitativity. We have developed analytical method for four immunosuppressants (Tacrolimus, Rapamycin, Everolimus and Cyclosporin A) with two internal standards (Ascomycin and Cyclosporin D) using ultra-fast mass spectrometer.
Figure 1 Structure of immunosuppressants and internal standards (IS)
O
HO
O
O OH
ON
OO
OHO
O
H O
HOO
O
O O OH
OOO
N
OO
O
HO
O
HO
O
O O OH
OOO
N
OO
O
HO
O
TacrolimusMW: 804.02
EverolimusMW: 958.22
RapamycinMW: 914.17
N
O
N O
NH
OHN
O
N
OHN
O
N
N
O
O
N
HO
HN
O
O
N
O
H
O
O
HO HO
N
O
OO
O
O
H
OH
O
HO
N N
OO
HN
O
N
O
N O
N
ON
OH
O
NH
OHN
O
NO
HNO
Cyclosporin AMW: 1202.61
Ascomycin (IS)MW: 792.01
Cyclosporin D (IS)MW: 1216.64
3
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
Methods and MaterialsStandard samples of each compound were analyzed to optimize conditions of liquid chromatograph and mass spectrometer. Whole blood extract was prepared based on liquid-liquid extraction described bellow.
2.7 mL of Whole blood and 20.8 mL of Water ↓Vortex for 15 seconds ↓Add 36 mL of MTBE/Cyclohexane (1:3) ↓Vortex for 15 seconds and Centrifuge with 3000 rpm at 20 ºC for 10 minutes ↓Extract an Organic phase ↓Evaporate and Dry under a Nitrogen gas stream ↓Redissolve in 1.8 mL of 80 % Methanol solution with 1 mmol/L Ammonium acetate ↓Vortex for 1 minute and Centrifuge with 3000 rpm at 4 ºC for 5 minutes ↓Filtrate and Transfer into 1 mL glass vial
Table 1 Analytical conditions
UHPLC
Liquid Chromatograph : Nexera (Shimadzu, Japan)
Analysis Column : YMC-Triart C18 (30 mmL. × 2 mmI.D.,1.9 μm)
Mobile Phase A : 1 mmol/L Ammonium acetate - Water
Mobile Phase B : 1 mmol/L Ammonium acetate - Methanol
Gradient Program : 60 % B. (0 min) – 75 % B. (0.10 min) – 95 % B. (0.70 – 0.90 min) –
60 % B. (0.91 – 1.80 min)
Flow Rate : 0.45 mL/min
Column Temperature : 65 ºC
Injection Volume : 1.5 µL
MS
MS Spectrometer : LCMS-8050 (Shimadzu, Japan)
Ionization : ESI (negative)
Probe Voltage : -4.5 ~ -3 kV
Nebulizing Gas Flow : 3.0 L/min
Drying Gas Flow : 5.0 L/min
Heating Gas Flow : 15.0 L/min
Interface Temperature : 400 ºC
DL Temperature : 150 ºC
HB Temperature : 390 ºC
4
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
ResultImmunosuppressants, which we have developed a method for monitoring of, has been often observed as ammonium or sodium adduct ion by using positive ionization. In general, protonated molecule (for positive) or deprotonated molecule (for negative) is more preferable for reliable quantitation than adduct ions such as ammonium, sodium, and potassium adduct. In this study,
each compound was detected as deprotonated molecule in negative mode by using heated ESI source of LCMS-8050 (Table 2).The separation of all compounds was achieved within 1.8 min, with a YMC-Triart C18 column (30 mmL. × 2 mmI.D.,1.9 μm) and at 65 ºC of column oven temperature.
Figure 2 MRM chromatograms of immnosuppresants in human whole blood (50 ng/mL)
Peak No.
1
2
3
4
5
6
Compound
Ascomysin (IS)
Tacrolimus
Rapamycin
Everolimus
Cyclosporin A
Cyclosporin D (IS)
Porality
neg
neg
neg
neg
neg
neg
Precursor ion (m/z)
790.40
802.70
912.70
956.80
1200.90
1215.10
Product ion (m/z)
548.20
560.50
321.20
365.35
1088.70
1102.60
Table 2 MRM transitions
0.75 1.00 1.25 min
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
(x100,000)
2
4
3
1
5
6
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
5
Figure 3 MRM chromatograms at LLOQ and ISTD (left), and calibration curves (right) for four immnosuppresants in human whole blood
a) Tacrolimus
0.5 – 1000 ng/mL
0.5 ng/mL
Ascomycin40 ng/mL
c) Everolimus
0.5 ng/mL
Ascomycin40 ng/mL 0.5 – 100 ng/mL
b) Rapamycin
0.5 ng/mL
Ascomycin40 ng/mL 0.5 – 500 ng/mL
d) Cyclosporin A
Cyclosporin D100 ng/mL
0.5 ng/mL
0.5 – 1000 ng/mL
Accelerated and robust monitoring for immunosuppressants using triple quadrupole mass spectrometry
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Figure 3 illustrates both a calibration curve and chromatogram at the lowest calibration level for all immunosuppressants analyzed. Table 3 lists both the reproducibility and accuracy for each immunosuppressant that has been simultaneously measured in 1.8 minutes.
In high speed measurement condition, we have achieved high sensitivity and wide dynamic range for all analytes. Additionally, the accuracy of each analyte ranged from 88 to 110 % and area reproducibility at the lowest calibration level of each analyte was less than 20%.
Conclusions• Monitoring with negative mode ionization permitted more sensitive, robust and reliable quantitation for four
immunosuppressants.• A total of six compounds were measured in 1.8 minutes. The combination of Nexera and LCMS-8050 provided a faster
run time without sacri�cing the quality of results.• Even with a low injection volume of 1.5 μL, the lower limit of quantitation (LLOQ) for all compounds was 0.5 ng/mL. • In this study, it is demonstrated that LCMS-8050 is useful for the rugged and rapid quantitation for immunosuppressants
in whole blood.
AcknowledgementWe appreciate suggestions from Prof. Kazuo Matsubara and Assoc. Prof. Ikuko Yano from the department of pharmacy, Kyoto University Hospital, and Prof. Satohiro Masuda from the department of pharmacy, Kyusyu University Hospital.
Table 3 Reproducibility and Accuracy
Compound
Tacrolimus
Concentration
Low (0.5 ng/mL) Low-Mid (2 ng/mL)High (1000 ng/mL)
CV % (n = 6)
18.013.02.87
Accuracy %
99.499.588.7
RapamycinLow (0.5 ng/mL)
Low-Mid (5 ng/mL)High (500 ng/mL)
6.872.883.41
95.6109.390.0
EverolimusLow (0.5 ng/mL)
Low-Mid (5 ng/mL)High (100 ng/mL)
10.45.112.26
95.3104.493.3
Cyclosporin ALow (0.5 ng/mL)
Low-Mid (10 ng/mL)High (1000 ng/mL)
7.312.362.67
95.199.994.9
PO-CON1468E
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazidefrom plasma using LC/MS/MS
ASMS 2014 TP497
Shailendra Rane, Rashi Kochhar, Deepti Bhandarkar,
Shruti Raju, Shailesh Damale, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
IntroductionFelodipine is a calcium antagonist (calcium channel blocker), used as a drug to control hypertension[1]. Hydrochlorothiazide is a diuretic drug of the thiazide class that acts by inhibiting the kidney’s ability to retain water. It is, therefore, frequently used for the treatment of hypertension, congestive heart failure, symptomatic edema, diabetes insipidus, renal tubular acidosis and the prevention of kidney stones[2].Efforts have been made here to develop high sensitive
methods of quantitation for these two drugs using LCMS-8050 system from Shimadzu Corporation, Japan.Presence of heated Electro Spray Ionization (ESI) probe in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development quantitation method at low ppt level for both Felodipine and Hydrochlorthiazide.
Method of Analysis
To 100 µL of plasma, 500 µL of cold acetonitrile was added for protein precipitation then put in rotary shaker at 20 rpm for 15 minutes for uniform mixing. It was centrifuged
at 12000 rpm for 15 minutes. Supernatant was collected and evaporated to dryness at 70 ºC and finally reconstituted in 200 µL Methanol.
Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile
Figure 2. Structure of Hydrochlorothiazide
HydrochlorothiazideHydrochlorothiazide, abbreviated HCTZ (or HCT, HZT), is a diuretic drug of the thiazide class that acts by inhibiting the kidney‘s ability to retain water. Hydrochlorothiazide is 6-chloro-1,1-dioxo-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide.Its empirical formula is C7H8ClN3O4S2 and its structure is shown in Figure 2.
Figure 1. Structure of Felodipine
FelodipineFelodipine is a calcium antagonist (calcium channel blocker). Felodipine is a dihydropyridine derivative that is chemically described as ± ethyl methyl 4-(2,3-dichlorophenyl)1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Its empirical formula is C18H19Cl2NO4 and its structure is shown in Figure 1.
3
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
LC/MS/MS analysisCompounds were analyzed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8050 triple quadrupole system (Shimadzu
Corporation, Japan), The details of analytical conditions are given in Table 1 and Table 2.
• Felodipine Calibration Std : 5 ppt, 10 ppt, 50 ppt, 100 ppt, 500 ppt, 1 ppb and 10 ppb• HCTZ Calibration Std : 2 ppt, 5 ppt, 10 ppt, 50 ppt, 100 ppt, and 500 ppt
To 500 µL plasma, 100 µL sodium carbonate (1.00 mol/L) and 5 mL of diethyl ether : hexane (1:1 v/v) was added. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing and centrifuged at 12000 rpm for 15
minutes. Supernatant was collected and evaporated to dryness at 60 ºC. It was finally reconstitute in 1000 µL Methanol.
Preparation of matrix matched plasma by liquid-liquid extraction method using diethyl ether and hexane mixture (1:1 v/v)
Response of Felodipine and Hydrochlorothiazide were checked in both above mentioned matrices. It was found that cold acetonitrile treated plasma and diethyl ether: hexane (1:1 v/v) treated plasma were suitable for
Felodipine and Hydrochlorothiazide molecules respectively. Calibration standards were thus prepared in respective matrix matched plasma.
Preparation of calibration standards in matrix matched plasma
Figure 3. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 4. Heated ESI probe
LCMS-8050 triple quadrupole mass spectrometer by Shimadzu (shown in Figure 3), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity), Ultra fast scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.
In order to improve ionization efficiency, the newly developed heated ESI probe (shown in Figure 4) combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide range of target compounds with considerable reduction in background.
4
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
Results
LC/MS/MS method for Felodipine was developed on ESI positive ionization mode and 383.90>338.25 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest concentrations (5 ppt) as seen in Figure 5 and Figure 6
respectively. Calibration curves as mentioned with R2 = 0.998 were plotted (shown in Figure 7). Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Also, LOD as 2 ppt and LOQ as 5 ppt was obtained.
LC/MS/MS analysis results of Felodipine
• Column : Shim-pack XR-ODS (75 mm L x 3 mm I.D.; 2.2 µm)
• Flow rate : 0.3 mL/min
• Oven temperature : 40 ºC
• Mobile phase : A: 10 mM ammonium acetate in water
B: methanol
• Gradient program (%B) : 0.0 – 3.0 min → 90 (%); 3.0 – 3.1 min → 90 – 100 (%);
3.1 – 4.0 min → 100 (%); 4.0– 4.1 min → 100 – 90 (%)
4.1 – 6.5 min → 90 (%)
• Injection volume : 10 µL
• MS interface : ESI
• Nitrogen gas �ow : Nebulizing gas 1.5 L/min; Drying gas 10 L/min;
• Zero air �ow : Heating gas 10 L/min
• MS temperature : Desolvation line 200 ºC; Heating block 400 ºC
Interface 200 ºC
Table 1. LC/MS/MS conditions for Felodipine
• Column : Shim-pack XR-ODS (100 mm L x 3 mm I.D.; 2.2 µm)
• Flow rate : 0.2 mL/min
• Oven temperature : 40 ºC
• Mobile phase : A: 0.1% formic acid in water
B: acetonitrile
• Gradient program (%B) : 0.0 – 1.0 min → 80 (%); 1.0 – 3.5 min → 40 – 100 (%);
3.5 – 4.5 min → 100 (%); 4.5– 4.51min → 100 – 80 (%)
4.51 – 8.0 min → 90 (%)
• Injection volume : 25 µL
• MS interface : ESI
• Nitrogen gas �ow : Nebulizing gas 2.0 L/min; Drying gas 10 L/min;
• Zero air �ow : Heating gas 15 L/min
• MS temperature : Desolvation line 250 ºC; Heating block 500 ºC
Interface 300 ºC
Table 2. LC/MS/MS conditions for Hydrochlorothiazide
5
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
Figure 5. Felodipine at 10 ppb in matrix matched plasma Figure 6. Felodipine at 5 ppt in matrix matched plasma
Figure 7. Calibration curve of Felodipine
LC/MS/MS method for Hydrochlorothiazide was developed on ESI negative ionization mode and 296.10>204.90 MRM transition was optimized for it. Checked matrix matched plasma standards for highest (500 ppt) as well as lowest (2 ppt) concentrations as seen in Figures 8 and 9 respectively.
Calibration curves as mentioned with R2=0.998 were plotted (shown in Figure 10). Also as seen in Table 4, % Accuracy was studied to confirm the reliability of method. Also, LOD as 1 ppt and LOQ as 2 ppt were obtained.
LC/MS/MS analysis results of Hydrochlorothiazide
Table 3: Results of Felodipine calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.005
0.01
0.05
0.1
0.5
1
10
Standard
STD-FEL-01
STD-FEL-02
STD-FEL-03
STD-FEL-04
STD-FEL-05
STD-FEL-06
STD-FEL-07
Sr. No.
1
2
3
4
5
6
7
0.005
0.010
0.053
0.103
0.469
0.977
10.023
97.43
103.80
104.47
103.13
94.88
97.33
100.90
9.87
8.76
2.24
1.23
1.33
0.95
0.60
0.0 2.5 5.0
0.0
2.5
5.0(x100,000)383.90>338.25(+)
FELO
DIP
INE
0.0 2.5 5.0
0.0
0.5
1.0
1.5
2.0
(x1,000)383.90>338.25(+)
FELO
DIP
INE
0.0 2.5 5.0 7.5 Conc.0.0
0.5
1.0
1.5
2.0Area (x1,000,000)
1 2 3 4 5
6
7
0.05 0.10 Conc.0.0
0.5
1.0
1.5
2.0
2.5
3.0Area (x10,000)
1 2
3
4
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
6
Figure 8. Hydrochlorothiazide at 500 ppt in matrix matched plasma Figure 9. Hydrochlorothiazide at 2 ppt in matrix matched plasma
Figure 10. Calibration curve of Hydrochlorothiazide
Conclusion• Highly sensitive LC/MS/MS method for Felodipine and Hydrochlorothiazide was developed on LCMS-8050 system.• LOD of 2 ppt and LOQ of 5 ppt was achieved for Felodipine and LOD of 1 ppt and LOQ of 2 ppt was achieved for
Hydrochlorothiazide by matrix matched methods.• Heated ESI probe of LCMS-8050 system enables drastic augment in sensitivity with considerable reduction in
background. Hence, LCMS-8050 system from Shimadzu is an all rounder solution for bioanalysis.
Table 4. Results of Hydrochlorothiazide calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.002
0.005
0.01
0.05
0.1
0.5
Standard
STD-HCTZ-01
STD-HCTZ-02
STD-HCTZ-03
STD-HCTZ-04
STD-HCTZ-05
STD-HCTZ-06
Sr. No.
1
2
3
4
5
6
0.0020
0.0048
0.0099
0.0512
0.1019
0.4944
102.03
95.50
100.07
102.67
102.11
102.13
6.65
3.53
3.80
1.60
3.58
1.68
0.0 2.5 5.0 7.5
0.0
0.5
1.0
1.5
(x10,000)296.10>204.90(-)
HC
TZ
0.0 2.5 5.0 7.5
0.0
0.5
1.0
1.5
2.0
2.5(x100)
296.10>204.90(-)
HC
TZ
0.0 0.1 0.2 0.3 0.4 Conc.0.00
0.25
0.50
0.75
1.00Area (x100,000)
1 2 3
4
5
6
0.000 0.025 0.050 Conc.0.0
0.5
1.0
1.5
Area (x10,000)
1 2 3
4
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Highly sensitive quantitative analysis of Felodipine and Hydrochlorothiazide from plasma using LC/MS/MS
References[1] YU Peng; CHENG Hang, Chinese Journal of Pharmaceutical Analysis, Volume 32, Number 1, (2012), 35-39(5).[2] Hiten Janardan Shah, Naresh B. Kataria, Chromatographia, Volume 69, Issue 9-10, (2009), 1055-1060.
PO-CON1467E
Highly sensitive quantitative estimationof genotoxic impurities from API and drug formulation using LC/MS/MS
ASMS 2014 TP496
Shruti Raju, Deepti Bhandarkar, Rashi Kochhar,
Shailesh Damale, Shailendra Rane, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd.,
1 A/B Rushabh Chambers, Makwana Road, Marol,
Andheri (E), Mumbai-400059, Maharashtra, India.
2
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
IntroductionThe toxicological assessment of Genotoxic Impurities (GTI) and the determination of acceptable limits for such impurities in Active Pharmaceutical Ingredients (API) is a dif�cult issue. As per European Medicines Agency (EMEA) guidance, a Threshold of Toxicological Concern (TTC) value of 1.5 µg/day intake of a genotoxic impurity is considered to be acceptable for most pharmaceuticals[1]. Dronedarone is a drug mainly used for indications of cardiac arrhythmias. GTI of this drug has been quantitated here. Method has been optimized for simultaneous analysis of DRN-IA {2-n-butyl-3-[4-(3-di-n-butylamino-propoxy)benzoyl]-5-nitro
benzofuran}, DRN-IB {5-amino-3-[4-(3-di-n-butylamino-propoxy)benzoyl}-2-n-butyl benzofuran} and BHBNB {2-n-butyl-3-(4-hydroxy benzoyl)-5-nitro benzofuran}. Structures of Dronedarone and its GTI are shown in Figure 1.As literature references available on GTI analysis are minimal, the feature of automatic MRM optimisation in LCMS-8040 makes method development process less tedious. In addition, the lowest dwell time and pause time and ultrafast polarity switching of LCMS-8040 ensures uncompromised and high sensitive quantitation.
Figure 1. Structures of Dronedarone and its GTI
O
OOH
NO2
C4H9
O
O O
C4H9
NH2
N
C4H9
C4H9
O
O O
C4H9
NO2
N
C4H9
C4H9
DRN-IA
O
O O
C4H9
NHSO2Me
N
C4H9
C4H9
Dronedarone
DRN-IB BHBNB
3
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
LC/MS/MS Analytical ConditionsAnalysis was performed using Ultra High Performance Liquid Chromatography (UHPLC) Nexera coupled with LCMS-8040 triple quadrupole system (Shimadzu Corporation, Japan), shown in Figure 2. Limit of GTI for Dronedarone is 2 mg/kg. However, general dosage of Dronedarone is 400 mg, hence, limit for GTI is 0.8 µg/400 mg. This when reconstituted in 20 mL system, gives an
effective concentration of 40 ppb. For analytical method development it is desirable to have LOQ at least 30 % of limit value, which in this case corresponds to 12 ppb. The developed method gives provision for measuring GTI at much lower level. However, recovery studies have been done at 12 ppb level.
Figure 2. Nexera with LCMS-8040 triple quadrupole system by Shimadzu
Method of Analysis
• Preparation of DRN-IA and DRN-IB and BHBNB stock solutions 20 mg of each impurity standard was weighed separately and dissolved in 20 mL of methanol to prepare stock solutions
of each standard.
• Preparation of calibration levels GTI mix standards (DRN-IA, DRN-IB and BHBNB) at concentration levels of 0.5 ppb, 1 ppb, 5 ppb, 10 ppb, 40 ppb, 50
ppb and 100 ppb were prepared in methanol using stock solutions of all the three standards.
• Preparation of blank sample 400 mg of Dronedarone powder sample was weighed and mixed with 20 mL of methanol. Mixture was sonicated to
dissolve sample completely.
• Preparation of spiked (at 12 ppb level) sample 400 mg of sample was weighed and spiked with 60 µL of 1 ppm stock solution. Solution was then mixed with 20 mL of
methanol. Mixture was sonicated to dissolve sample completely.
Sample Preparation
4
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Table 1. LC/MS/MS analytical conditions
Results
LC/MS/MS method was developed for simultaneous quantitation of GTI mix standards. MRM transitions used for all GTI are given in Table 2. No peak was seen in diluent (methanol) at the retention times of GTI for selected MRM transitions which confirms the absence of any interference from diluent (shown in Figure 3). MRM chromatogram of GTI mix standard at 5 ppb level is shown in Figure 4. Linearity studies were carried out using external standard
calibration method. Calibration graphs of each GTI are shown in Figure 5. LOQ was determined for each GTI based on the following criteria – (1) % RSD for area < 15 %, (2) % Accuracy between 80-120 % and (3) Signal to noise ratio (S/N) > 10. LOQ of 0.5 ppb was achieved for DRN-IB and BHBNB whereas 1 ppb was achieved for DRN-IA. Results of accuracy and repeatability for all GTI are given in Table 3.
LC/MS/MS analysis
• Column : Shim-pack XR-ODS II (75 mm L x 3 mm I.D.; 2.2 µm)
• Mobile phase : A: 0.1% formic acid in water
B: acetonitrile
• Flow rate : 0.3 mL/min
• Oven temperature : 40 ºC
• Gradient program (B%) : 0.0–2.0 min → 35 (%); 2.0–2.1 min → 35-40 (%);
2.1–7.0 min → 40-60 (%); 7.0–8.0 min → 60-100 (%);
8.0–10.0 min → 100 (%); 10.0–10.01 min → 100-35 (%);
10.01–13.0 min → 35 (%)
• Injection volume : 1 µL
• MS interface : Electro Spray Ionization (ESI)
• MS analysis mode : MRM
• Polarity : Positive and negative
• MS gas �ow : Nebulizing gas 2 L/min; Drying gas 15 L/min
• MS temperature : Desolvation line 250 ºC; Heat block 400 ºC
Note: Flow Control Valve (FCV) was used for the analysis to divert HPLC �ow towards waste during elution of Dronedarone so as to prevent contamination of Mass Spectrometer.
Table 2: MRM transitions selected for all GTI
Name of GTI MRM transition Retention time (min) Mode of ionization
DRN-IB
DRN-IA
BHBNB
479.15>170.15
509.10>114.10
338.20>244.05
1.83
5.85
8.77
Positive ESI
Positive ESI
Negative ESI
Below mentioned table shows the analytical conditions used for analysis of GTI.
5
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Figure 4. MRM chromatogram of GTI mix standard at 5 ppb level
Figure 5. Calibration graphs for GTI
Figure 3. MRM chromatogram of diluent (methanol)
0.0 2.5 5.0 7.5 10.0 min
0
5000
10000
15000
20000
25000
30000
35000
40000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
0.0 2.5 5.0 7.5 10.0 min
0
250
500
750
1000
3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
0.0 25.0 50.0 75.0 Conc.0
250000
500000
750000Area
DRN-IB R2-0.9989
0.0 25.0 50.0 75.0 Conc.0
250000
500000
750000
1000000
1250000
Area
DRN-IA R2-0.9943
0.0 25.0 50.0 75.0 Conc.0
50000
100000
150000
Area
BHBNB R2-0.9922
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
6
Figure 6. MRM chromatogram of blank sample
Table 3: Results of accuracy and repeatability for all GTI
Standard concentration (ppb)
Calculated concentration from calibration graph
(ppb) (n=6)
% Accuracy (n=6)
% RSD for area counts (n=6)
0.5
1
5
12
40
50
100
1
5
12
40
50
100
0.5
1
5
12
40
50
100
Name of GTI
DRN-IB
DRN-IA
BHBNB
Sr. No.
1
2
3
0.492
1.044
4.961
12.014
38.360
49.913
103.071
0.994
4.916
11.596
37.631
48.605
100.138
0.486
1.062
4.912
11.907
37.378
48.518
96.747
98.40
104.40
99.22
100.12
95.90
99.83
103.07
99.40
98.32
96.63
94.08
97.21
100.14
97.20
106.20
98.24
99.23
93.45
97.04
96.75
9.50
6.62
3.10
2.97
1.17
1.08
0.86
5.02
2.82
2.43
1.27
1.40
0.99
4.88
6.97
2.16
1.31
0.37
0.43
0.91
Recovery studiesFor recovery studies, samples were prepared as described previously. MRM chromatogram of blank and spiked samples are shown in Figures 6 and 7 respectively. Results
of recovery studies have been shown in Table 4. Recovery could not be calculated for DRN-IB as blank sample showed higher concentration than spiked concentration.
0.0 2.5 5.0 7.5 10.0 min
0
50000
100000
150000
200000
250000
300000
350000
4000003:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Highly sensitive quantitative estimation of genotoxic impurities from API and drug formulation using LC/MS/MS
Figure 7. MRM chromatogram of spiked sample
Conclusion• A highly sensitive method was developed for analysis of GTI of Dronedarone.• Ultra high sensitivity, ultra fast polarity switching (UFswitching) enabled sensitive, selective, accurate and reproducible
analysis of GTI from Dronedarone powder sample.
References[1] Guideline on The Limits of Genotoxic Impurities, (2006), European Medicines Agency (EMEA).
Table 4. Results of the recovery studies
Concentration of GTI mix standard spiked
in blank sample (ppb)
Average concentration obtained from calibration graph for blank sample (ppb) (A) (n=3)
Average concentration obtained from calibration graph
for spiked sample (ppb) (B) (n=3)
% Recovery = (B-A)/ 12 * 100
12
12
12
Name of Impurity
DRN-IB
DRN-IA
BHBNB
94.210
3.279
1.241
NA
12.840
12.723
NA
79.678
95.689
0.0 2.5 5.0 7.5 10.0 min
0
25000
50000
75000
100000
125000 3:BHBNB 338.20>244.05(-) CE: 20.02:DRA-IA 509.10>114.10(+) CE: -41.01:DRA-IB 479.15>170.15(+) CE: -29.0
BHBN
B 33
8.20
>24
4.05
DRN
-IA 5
09.1
0>11
4.10
DRN
-IB 4
79.1
5>17
0.15
PO-CON1470E
Development of 2D-LC/MS/MS Method for Quantitative Analysis of1α,25-Dihydroxylvitamin D3 in Human Serum
ASMS 2014 WP449
Daryl Kim Hor Hee1, Lawrence Soon-U Lee1,
Zhi Wei Edwin Ting2, Jie Xing2, Sandhya Nargund2,
Miho Kawashima3 & Zhaoqi Zhan2
1 Department of Medicine Research Laboratories,
National University of Singapore, 6 Science Drive 2,
Singapore 1175462 Customer Support Centre, Shimadzu (Asia Paci�c) Pte
Ltd, 79 Science Park Drive, #02-01/08, Singapore 1182643 Global Application Development Centre, Shimadzu
Corporation, 1-3 Kanda Nishihiki-cho, Chiyoda-ku,
Tokyo 101-8448, Japan
2
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
IntroductionDevelopments of LC/MS/MS methods for accurate quantitation of low pg/mL levels of 1α,25-dihydroxy vitamin D2/D3 in serum were reported in recent years, because their levels in serum were found to be important indications of several diseases associated with vitamin D metabolic disorder in clinical research and diagnosis [1]. However, it has been a challenge to achieve the required sensitivity directly, due to the intrinsic dif�culty of ionization of the compounds and interference from matrix [2,3]. Sample extraction and clean-up with SPE and immunoaf�nity methods were applied to remove the interferences [4] prior to LC/MS/MS analysis. However, the
amount of serum required was normally rather high from 0.5mL to 2mL, which is not favourite in the clinical applications. Direct analysis methods with using smaller amount of serum are in demand. Research efforts have been reported in literatures to enhance ionization ef�ciency by using different interfaces such as ESI, APCI or APPI and ionization reagents to form purposely NH3 adduct or lithium adduct [4,5]. Here, we present a novel 2D-LC/MS/MS method with APCI interface for direct analysis of 1α,25-diOH-VD3 in serum. The method achieved a detection limit of 3.1 pg/mL in spiked serum samples with 100 uL injection.
ExperimentalHigh purity 1α,25-dihydroxyl Vitamin D3 and deuterated 1α,25-dihydroxyl-d6 Vitamin D3 (as internal standard) were obtained from Toronto Research Chemicals. Charcoal-stripped pooled human serum obtained from Bioworld was used as blank and matrix to prepare spiked samples in this study. A 2D-LC/MS/MS system was set up on LCMS-8050 (Shimadzu Corporation) with a column switching valve installed in the column oven and controlled by LabSolutions workstation. The details of columns, mobile phases and gradient programs of 1st-D and 2nd-D LC
separations and MS conditions are compiled into Table 1. The procedure of sample preparation of spiked serum samples is shown in Figure 1. It includes protein precipitation by adding ACN-MeOH solvent into the serum in 3 to 1 ratio followed by vortex and centrifuge at high speed. The supernatant collected was �ltered before standards with IS were added (post-addition). The clear samples obtained were then injected into the 2-D LC/MS/MS system.
Table 1: 2D-LC/MS/MS analytical conditions
LC condition
1st D: FC-ODS (2.0mml.D. x 75mm L, 3μm)2nd D: VP-ODS (2.0mmI.D. x 150mm L, 4.6μm)
A: Water with 0.1% formic acidB: Acetontrile
C: Water with 0.1% formic acidD: MeOH with 0.1% formic acid
B: 40% (0 to 0.1min) → 90% (5 to 7.5min) → 15% (11 to 12min) → 40% (14 to 25min); Total �ow rate: 0.5mL/min
D: 15% (0min) → 80% (20 to 22.5min) → 15% (23 to 25min); Peak cutting: 3.15 to 3.40; Total �ow rate: 0.5 mL/min
45ºC
100 uL
Column
Mobile Phase of 1st D
Mobile Phase of 2nd D
1st D gradient pro-gram & �ow rate
2nd D gradient pro-gram & �ow rate
Oven Temp.
Injection Vol.
MS Interface condition
APCI, 400ºC
Positive, MRM
300ºC & 200ºC
Ar (270kPa)
N2, 2.5 L/min
N2, 7.0 L/min
Interface
MS mode
Heat Block & DL Temp.
CID Gas
Nebulizing Gas Flow
Drying Gas Flow
3
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
Figure 1: Flow chart of serum sample pre-treatment method
150µL of serum 450µL of ACN/MeOH (1:1)
Shake and Vortex 10mins
Centrifuge for 10 minutes at 13000rpm
480µL of Supernatant
0.2µm nylon �lter
400µL of �ltered protein precipitated Serum
500µL of calibrate50µL of of Std stock
50µL of IS stock
Results and Discussion
An APCI interference was employed for effective ionization of 1α,25-diOH-VitD3 (C27H44O3, MW 416.7). A MRM quantitation method for 1α,25-diOH-VitD3 with its deuterated form as internal standard (IS) was developed. MRM optimization was performed using an automated MRM optimization program with LabSolutions workstation. Two MRM transitions for each compound were selected
(Table 2), the first one for quantitation and the second one for confirmation. The parent ion of 1α,25-diOH-VitD3 was the dehydrated ion, as it underwent neutral lost easily in ionization with ESI and APCI [2,3]. The MRM used for quantitation (399.3>381.3) was dehydration of the second OH group in the molecule.
Development of 2D-LC/MS/MS method
Table 2: MRM transitions and CID parameters of 1α,25-diOH-VitD3 and deuterated IS
Q1 Pre Bias Q3 Pre BiasName
1α,25-dihydroxyl Vitamin D3
1α,25-dihydroxyl-d6 Vitamin D3 (IS)
RT1 (min)
22.74
22.71
Transition (m/z)
399.3 > 381.3
399.3 > 157.0
402.3 > 366.3
402.3 > 383.3
-20
-20
-20
-20
CID Voltage (V)
CE
-13
-29
-12
-15
-14
-17
-18
-27
1, Retention time by 2D-LC/MS/MS method
4
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
Figure 2: 1D-LC/MS/MS chromatograms of 22.7 pg/mL 1α,25-diOH-VitD3 (top) and 182 pg/mL internal
standard (bottom) in serum (injection volume: 50uL)
0.0 2.5 5.0 7.5 10.0 min0
1000
2000
3000
4000
5000 1:OH2D3 399.30>105.00(+) CE: -44.01:OH2D3 399.30>157.00(+) CE: -29.01:OH2D3 399.30>381.30(+) CE: -13.0
OH
2-V
D3
2.5 5.0 7.5 10.0 min0
100
200
300
400
500
600
700 2:OH2D3-D6 402.30>366.30(+) CE: -12.02:OH2D3-D6 402.30>383.30(+) CE: -15.0
OH
2-V
D3-
D3
Peak cutting (125 uL) in 1st D separationand transferred to 2nd D LC
The reason to develop a 2-D LC separation for a LC/MS/MS method was the high background and interferences occurred with 1D LC/MS/MS observed in this study and also reported in literatures. Figure 2 shows the MRM chromatograms of 1D-LC/MS/MS of spiked serum sample. It can be seen that the baseline of the quantitation MRM (399.3>381.3) rose to a rather high level and interference peaks also appeared at the same retention time. The 2-D LC/MS/MS method developed in this study involves “cutting the targeted peak” in the 1st-D separation precisely (3.1~3.4 min) and the portion retained in a stainless steel sample loop (200 uL) was transferred into the 2nd-D column for further separation. The operation was accomplished by switching the 6-way valve in and out by a time program. Both 1st-D and 2nd-D separations were carried out in gradient elution mode. The organic mobile phase of 2nd-D (MeOH with 0.1% formic acid) was different from that of 1st-D (pure ACN). The interference peaks co-eluted with the analyte in 1st-D were separated from the analyte peak (22.6 min) as shown in Figure 3.
Two sets of standard samples were prepared in serum and in clear solution (diluent). Each set included seven levels of 1α,25-diOH-VitD3 from 3.13 pg/mL to 200 pg/mL, each added with 200 pg/mL of IS (See Table 3). The chromatograms of the seven spiked standard samples in serum are shown in Figure 3. A linear IS calibration curve (R2 > 0.996) was established from these 2D-LC/MS/MS analysis results, which is shown in Figure 4. It is worth to
note that the calibration curve has a non-zero Y-intercept, indicating that the blank (serum) contains either residual 1α,25-diOH-VitD3 or other interference which must be deducted in the quantitation method. The peak area ratios shown in Table 3 are the results after deduction of the background peaks. The accuracy of the method after this correction is between 92% and 117%.
Calibration curve (IS), linearity and accuracy
Figure 3: Overlay of 2nd-D chromatograms of 7 levels from 3.13 pg/mL to 200 pg/mL spiked in serum.
Figure 4: Calibration curves of1α,25-diOH VD3 in serum by IS method.
0 10 20 min
0
1000
2000
3000
4000
22.0 23.0 min
1000
2000
3000
4000
1α,25-diOH-VitD3
0.00 0.25 0.50 0.75 Conc. Ratio0.0
1.0
2.0
3.0
4.0
5.0
Area Ratio
R2 = 0.9967
Non-zero intercept
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
5
Table 3: Seven levels of standard samples for calibration curve and performance evaluation
Figure 5: MRM peaks of 1α,25-diOH-VitD3 spiked in pure diluent (top) and in serum (bottom) of L1, L3, L5 and L7 (spiked conc. refer to Table 3)
Matrix effect of the 2D-LC/MS/MS method was determined by comparison of peak area ratios of standard samples in diluent and in serum at the seven levels. The results are compiled into Table 3. The matrix effect of the method are between 58% and 95%. It seems that the matrix effect is stronger at lower concentrations than at higher concentrations. Repeatability of peak area of the method was evaluated with L2 and L3 spiked serum samples for both target and IS. The Results of RSD (n=6) are displayed in Table 4. The MRM peaks of 1α,25-diOH VD3 in clear solution and in serum are displayed in pairs (top and bottom) in Figure 5. It can be seen from the first pair (diluent and serum blank) that a peak appeared at the same retention of 1α,25-diOH VD3 in the blank serum. As pointed out above, this peak is
from either the residue of 1α,25-diOH VD3 or other interference present in the serum. Due to this background peak, the actual S/N ratio could not be calculated. Therefore, it is difficult to determine the LOD and LOQ based on the S/N method. Tentatively, we propose a reference LOD and LOQ of the method for 1α,25-diOH VD3 to be 3.1 pg/mL and 10 pg/mL, respectively. The specificity of the method relies on several criteria: two MRMs (399>381 and 399>157), their ratio and RT in 2nd-D chromatogram. The MRM chromatograms shown in Figure 5 demonstrate the specificity of the method from L1 (3.1 pg/mL) to L7 (200 pg/mL). It can be seen that the results of spiked serum samples (bottom) meet the criteria if compared with the results of samples in the diluent (top).
Matrix effect, repeatability, LOD/LOQ and speci�city
Conc. Level of Std.
L1
L2
L3
L4
L5
L6
L7
1α,25-diOH VD3 (pg/mL)
3.13
6.25
12.5
25.0
50.0
100.0
200.0
Conc. Ratio1 (Target/IS)
0.0156
0.0313
0.0625
0.1250
0.2500
0.5000
1.0000
Area Ratio2
(in serum)
0.243
0.321
0.456
0.757
1.188
2.168
4.531
Area Ratio2
(in clear solu)
0.414
0.481
0.603
0.914
1.354
2.580
4.740
Accuracy3
(%)
103.8
100.0
117.3
115.9
95.5
92.15
102.0
Matrix Effect (%)
58.7
66.8
75.6
82.9
87.7
84.0
95.6
1, Target = 1α,25-diOH VD3; 2, Area ratio = area of target / area of IS; 3, Based on the data of spiked serum samples
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.565
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.565
22.5 24.7
0
500
1000
1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.573
22.5 24.7
0
1000
2000
3000
4000 1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.598
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.595
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
22.5 24.7
0
250
500
750
10001:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.619
22.5 24.7
0
1000
2000
3000
4000 1:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.630
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.622
22.5 24.7
0
250
500
7501:399.30>157.00(+)1:399.30>381.30(+)
OH
2VD
3/22
.602L1 L3 L5 L7 Diluent
L1 L3 L5 L7 Serum blank
Development of 2D-LC/MS/MS Method for Quantitative Analysis of 1α,25-Dihydroxylvitamin D3 in Human Serum
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
ConclusionsA 2D-LC/MS/MS method with APCI interface has been developed for quantitative analysis of 1α,25-dihydroxylvitamin D3 in human serum without of�ine extraction and cleanup. The detection and quantitation range of the method is from 3.1 pg/mL to 200 pg/mL, which meets the diagnosis requirements in clinical applications. The performance of the method was evaluated thoroughly, including linearity, accuracy,
repeatability, matrix effect, LOD/LOQ and speci�city. The results indicate that the 2D-LC/MS/MS method is sensitive and reliable in detection and quantitation of trace 1α,25-dihydroxylvitamin D3 in serum. Further studies to enable the method for simultaneous analysis of both 1α,25-dihydroxylvitamin D3 and 1α,25-dihydroxylvitamin D2 are needed.
References1. S. Wang. Nutr. Res. Rev. 22, 188 (2009).2. T. Higashi, K. Shimada, T. Toyo’oka. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2010) 878, 1654.3. J. M. El‐Khoury, E. Z. Reineks, S. Wang. Clin. Biochem. 2010. DOI: 10.1002/jssc.20200911.4. Chao Yuan, Justin Kosewick, Xiang He, Marta Kozak and Sihe Wang, Rapid Commun. Mass Spectrom. 2011, 25,
1241–12495. Casetta, I. Jans, J. Billen, D. Vanderschueren, R. Bouillon. Eur. J. Mass Spectrom. 2010, 16, 81.
For Research Use Only. Not for use in diagnostic procedures.
Table 4: Repeatability Test Results (n=6)
Sample
L2
L3
Compound
1α,25-diOH VD3
IS
1α,25-diOH VD3
IS
Spiked Conc. (pg/mL)
6.25
200
12.5
200
%RSD
10.10
7.66
9.33
6.28
PO-CON1450E
Analysis of polysorbates in biotherapeuticproducts using two-dimensional HPLC coupled with mass spectrometer
ASMS 2014 WP 182
William Hedgepeth, Kenichiro Tanaka Shimadzu Scienti�c Instruments, Inc., Columbia MD
2
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
IntroductionPolysorbate 80 is commonly used for biotherapeutic products to prevent aggregation and surface adsorption, as well as to increase the solubility of biotherapeutic compounds. A reliable method to quantitate and characterize polysorbates is required to evaluate the quality and stability of biotherapeutic products. Several methods for polysorbate analysis have been reported, but most of
them require time-consuming sample pretreatment such as derivatization and alkaline hydrolysis because polysorbates do not have suf�cient chromophores. Those methods also require an additional step to remove biotherapeutic compounds. Here we report a simple and reliable method for quantitation and characterization of polysorbate 80 in biotherapeutic products using two-dimensional HPLC.
Fig.1 Typical structure of polysorbate 80
Materials
Reagents: Tween® 80 (Polysorbate 80), IgG from human serum, potassium phosphate monobasic, potassium phosphate dibasic, and ammnonium formate were purchased from Sigma-Aldrich. Water was made in house using a Millipore Milli-Q Advantage A10 Ultrapure Water Purification System. Isopropanol was purchased from Honeywell. Standard solutions: 10 mmol/L phosphate buffer (pH 6.8) was prepared by dissolving 680 mg of potassium
phosphate monobasic and 871 mg of potassium phosphate dibasic in 1 L of water. Polysorbate 80 was diluted with 10 mmol/L phosphate buffer (pH 6.8) to 200, 100, 50, 20, 10 mg/L and transferred to 1.5 mL vials for analysis.Sample solutions: A model sample was prepared by dissolving 2 mg of IgG in 0.1 mL of a 100 mg/L polysorbate 80 standard solution. The sample was centrifuged and transferred to a 1.5 mL vial for analysis.
Reagents and standards
w+x+y+z=approx. 20
OO
OH
OOH
O
OOH
O
O
CH3
yz
x
w
3
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.2 Flow diagram of Co-Sense for BA
The standard and sample solutions were injected into a Shimadzu Co-Sense for BA system consisting of two LC-20AD pumps and a LC-20AD pump equipped with a solvent switching valve, DGU-20A5R degassing unit, SIL-20AC autosampler, CTO-20AC column oven equipped with a 6-port 2-position valve, and a CBM-20A system controller. Polysorbate 80 was detected by a LCMS-2020 single quadrupole mass spectrometer or a LCMS-8050 triple quadrupole mass spectrometer because polysorbates do not have any chromophores and are present at low concentrations in antibody drugs. A SPD-20AV UV-VIS
detector was used to check protein removal.Fig. 2 shows the flow diagram of the Co-Sense for BA system. In step 1, a sample pretreatment column “Shim-pack MAYI-ODS” traps polysorbate 80 in the sample. Proteins (antibody) cannot enter the pore interior that is blocked by a hydrophilic polymer bound on the outer surface. Other additives and excipients such as sugars, salts, and amino acids cannot be retained by the ODS phase of the inner surface due to their polarity. In step 2, the trapped polysorbate 80 is introduced to the analytical column by valve switching.
System
Step 1 : Protein removal
Step 2 : Analyzing the trapped polysorbate
Autosampler
Valve(Position 0)
Pump 1
Pump 2
Sample pretreatment column
Analytical column
Mass spectrometer
UV-VIS detector
Mobile phase C
Mobile phase D
Mobile phase A(solution for sample injection)
Mobile phase B(solution for rinse)
Protein,Salts,
Amino acids,Sugars
Polysorbate80
Autosampler
Valve(Position 1)
Pump 1
Pump 2
Sample pretreatment column
Analytical column
Mass spectrometer
UV-VIS detector
Mobile phase C
Mobile phase D
Mobile phase B(solution for rinse)
Mobile phase A(solution for sample injection)
Polysorbate80
4
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Results
A fast analysis for quantitation will be shown here. Table 1 shows the analytical conditions and Fig. 3 shows the TIC chromatogram of a 100 mg/L polysorbate 80 standard solution and the mass spectrum of the peak at 4.4 min. Polysorbates contain many by-products, so several peaks appeared on the TIC chromatogram. The peak at 4.4 min was identified as polyoxyethylene sorbitan monooleate (typical structure of polysorbate 80) based on E. Hvattum et al 2011. The ion at 783 was used as a marker for detection in selected ion mode (SIM). This ion is attributable to the 2NH4
+ adduct of polyoxyethylene sorbitan monooleate containing 25 polyoxyethylene groups. Fig. 4 shows the SIM chromatogram of the model sample (20 g/L of IgG, 100 mg/L of polysorbate 80 in 10
mmol/L phosphate buffer pH6.8). Polysorbate 80 in the model sample was successfully analyzed. The peak at 4.4 min was used for quantitation.Six replicate injections for the model sample were made to evaluate the reproducibility. The relative standard deviations of retention time and peak area were 0.034 % and 1.11 %, respectively. The recovery ratio was obtained by comparing the peak area of the model sample and a 100 mg/L polysorbate 80 standard solution and was 99 %. Five different levels of polysorbate 80 standard solutions ranging from 10 to 200 mg/L were used for the linearity evaluation. The correlation coefficient (R2) of determination was higher than 0.999.
Quantitation method
Table 1 Analytical Conditions
System : Co-Sense for BA equipped with LCMS-2020
[Sample Injection]
Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
Flow Rate : 0.6 mL/min
Valve Position : 0 (0-1 min, 7-9 min), 1 (1-7 min)
Injection Volume : 1 µL
[Separation]
Column : Kinetex 5u C18 100A (50 mm L. x 2.1 mm I.D., 5 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Time Program : B. Conc 5 % (0-1 min) - 100 % (6-7 min) -5 % (7.01-9 min)
Flow Rate : 0.3 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection : 280 nm
Flow Cell : Semi-micro cell
[MS Detection]
Ionization Mode : ESI Positive
Applied Voltage : 4.5 kV
Nebulizer Gas Flow : 1.5 mL/min
DL Temperature : 250 ºC
Block Heater Temp. : 400 ºC
Scan : m/z 300-2000
SIM : m/z 783
5
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.4 SIM chromatogram of the model sample
An analysis for characterization will be shown here. Table 2 shows the analytical conditions and Fig. 5 shows the TIC chromatogram of the model sample and mass spectra of the peaks from 10 to 30 min. A longer column and gradient were applied to obtain better resolution. Polysorbate 80 consists of not only monooleate (typical structure of polysorbate 80), but also many by-products such as polyoxyethylene, polyoxyethylene sorbitan, polyoxyethylene isosorbide, dioleate, trioleate, tetraoleate
and others. The peaks on the TIC chromatogram are assumed to correspond to those by-products. For example, the peaks from 10 to 22 min correspond to polyoxyethylene and polyoxyethylene isosorbide and the peaks from 22 to 30 min correspond to polyoxyethylene sorbitan. This method is helpful for characterization as well as checking degradation such as auto-oxidation and hydrolysis.
Characterization method
Fig.3 TIC Chromatogram of 100 mg/L polysorbate 80 standard solution and mass spectrum of the peak at 4.4 min
500 550 600 650 700 750 800 850 900 950 m/z0.0
0.5
1.0
1.5
Inten.(x100,000)
601587 616631
572645
660557
783675 805 827543849761689 739528 871 893704 915717
Doubly charged ions
Triply charged ions
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
1000000
2000000
3000000
4000000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min0
25000
50000
75000
100000
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
6
Fig.5 TIC chromatogram of the model sample
Table 2 Analytical Conditions
System : Co-Sense for BA equipped with LCMS-8050
[Sample Injection]
Column : Shim-pack MAYI-ODS (5 mm L. x 2.0 mm I.D., 50 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Solvent Switching : A (0-1.5 min), B (1.5-3.5 min), A (3.5-9 min)
Flow Rate : 0.6 mL/min (0-10 min, 95.01-110 min), 0.1 mL/min (10.01-95 min)
Valve Position : 0 (0-3 min, 100-110 min), 1 (3-100 min)
Injection Volume : 5 µL
[Separation]
Column : Kinetex 5u C18 100A (100 mm L. x 2.1 mm I.D., 5 μm)
Mobile Phase : A: 10 mmol/L ammonium formate in water
B: Isopropanol
Time Program : B. Conc 5 % % (0-3min) – 35% (15min) – 100% (100min) – 5% (100.01-110min)
Flow Rate : 0.2 mL/min
Column Temperature : 40 ºC
[UV Detection]
Detection : 280 nm
Flow Cell : Semi-micro cell
[MS Detection]
Ionization Mode : ESI Positive
Applied Voltage : 4.5 kV
Nebulizer Gas Flow : 2 mL/min
Drying Gas Flow : 10 mL/min
Heating Gas Flow : 10 mL/min
Interface Temperature : 300 ºC
DL Temperature : 250 ºC
Block Heater Temp. : 400 ºC
Q1 Scan : m/z 300-2000
0 10 20 30 40 50 60 70 80 90 100 min
0.0
1.0
2.0
3.0
4.0
(x100,000,000)1:TIC(+)
10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min
0.0
2.5
5.0
7.5
(x10,000,000)1:TIC(+)
Polyoxyethylene sorbitan
Polyoxyethylene isosorbide
Polyoxyethylene
400 500 600 700 800 m/z0.0
1.0
2.0
3.0
4.0
5.0
6.0Inten.(x100,000)
513.6528.3498.9 543.0
484.2557.6
469.5651.0673.0628.9 695.0572.3
717.1454.8 606.9 739.0587.0761.1
440.2 783.1805.1425.4 827.1
300 400 500 600 700 800 900 m/z0.0
1.0
2.0
3.0
Inten.(x100,000)
692.8648.8
736.8604.7
560.7 780.9421.7443.8399.7 465.8 564.7 608.8 652.8520.7377.6 824.9516.6
696.9445.4 740.9355.6 423.5401.6 869.0379.5
784.9913.0
O
O OOH
OOH
y
z
OHO
H
x
OO
OH
OOH
O
OOH
OH
yz
x
w
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Analysis of polysorbates in biotherapeutic products using two-dimensional HPLC coupled with mass spectrometer
Fig.6 Chromatogram of elution from the sample pretreatment column
Fig. 6 shows the chromatogram of elution from the sample pretreatment column. Protein (IgG) was successfully removed from the sample by using the MAYI-ODS column.
Con�rmation of protein removal
E. Hvattum, W.L. Yip, D. Grace, K. Dyrstad, Characterization of polysorbate 80 with liquid chromatography mass spectrometry and nuclear magnetic resonance spectroscopy: Specific determination of oxidation products of thermally oxidized polysorbate 80, J Pharm Biomed Anal 62, (2012) 7-16
Reference
Conclusions1. Co-Sense for BA system automatically removed protein from the sample and enabled quantitation and characterization
of polysorbate 80 in a protein formulation.2. The quantitation method was successfully applied to the model sample with excellent reproducibility and recovery.3. The high-resolution chromatogram was obtained by the characterization method. This method is helpful for
characterization as well as checking degradation such as auto-oxidation and hydrolysis.
5uL injection of model sample
1uL injection of model sample
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
0
250000
500000
750000
1000000
1250000
uV
PO-CON1457E
A Rapid and Reproducible Immuno-MSPlatform from Sample Collection to Quantitation of IgG
ASMS 2014 WP161
Rachel Lieberman1, David Colquhoun1, Jeremy Post1,
Brian Feild1, Scott Kuzdzal1, Fred Regnier2, 1Shimadzu Scienti�c Instruments, Columbia, MD, USA 2Novilytic L.L.C, North Webster, IN, USA
2
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Sample Work�ow
Using rapid, automated processing, coupled to the speed and sensitivity of the LCMS-8050 allows for improved analysis of Immunoglobulin G.
Introduction
Novel Aspect
Dried blood spot analysis (DBS) has provided clinical laboratories a simple method to collect, store and transport samples for a wide variety of analyses. However, sample stability, hematocrit effects and inconsistent spotting techniques have limited the ability for wide spread adoption in clinical applications. Dried plasma spots (DPS) offer new opportunities by providing stable samples that
avoid variability caused by the hematocrit. This presentation focuses on an ultra-fast-immuno-MS platform that combines next generation plasma separator cards (Novilytic L.L.C., North Webster, IN) with fully automated immuno-af�nity enrichment and rapid digestion as an upfront sample preparation strategy for mass spectrometric analysis of immunoglobulins.
LC/MS/MSAffinitySelection
EnzymeDigestion Desalting
Automates and integrates key proteomic workflow steps: - Affinity Selection (15 min) - Trypsin digestion (1-8 min) - Online Desalting - Reversed phase LCExceptional reproducibility (CV less than 10%)
Rapid plasma extraction technology from whole blood (~ 18 minutes) - 2.5 uL of plasma collected (3 min) - Air dry for 15 minutes - Extract plamsa disc for analysis
- Ultrafast MRM methods - Up to 555 MRM transitions per run - Heated electrospray source - Scan speeds up to 30,000 u/sec - Polarity switching 5 msec
Perfinity WorkstationNoviplexTM Card LCMS-8050 Triple Quadrupole MS
BufferExchange
PlasmaGeneration
3
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
MethodsIgG was weighed out and then diluted in 500 μL of 0.5% BSA solution. Approximately15 uL of IgG standard was spiked into mouse whole blood and processed using the Noviplex card. The resulting plasma collection disc was extracted with 30 uL of buffer and each sample was
reduced and alkylated to yield a total sample volume of 100 uL. IgG standards and QC samples were directly injected onto the Per�nity-LCMS-8050 platform for af�nity pulldown with a Protein G column followed by trypsin digestion and LC/MS/MS analysis.
Noviplex Cards
Approximately 50 uL of the spiked whole blood was pipetted onto the Noviplex card test area (1). The spot was allowed to dry for 3 minutes (2). The top layer of the card was then peeled back (3) to reveal the plamsa collection
disc. The plasma collection disc was allowed to dry for an additional 15 minutes. Once the disc was dry (4), it was placed into an eppendorf tube for solvent extraction.
IgG concentrations for calibration levels. LCMS gradient conditions.
0
20
40
60
80
100
0 2 4 6 8 10 12 14 16
%B
Time (minutes)
MRM transitions on LCMS-8050 for two IgG peptides monitored.
Compound Name
TTPPVLDSDGSFFLYSK
VVSVLTVLHQDWLNGK
Transitions
937.70>836.25
937.70>723.95
603.70>805.7
+/-
+
+
+
Q1 Rod Bias(V)
-27
-27
-22
CE (V)
-28
-30
-16
Q3 Rod Bias(V)
-26
-22
-13
Level
1
2
3
4
5
6
7
Conc.(μg/mL)
465
315
142.5
127.5
102
60
22.5
Amount oncolumn (μg)
34.88
23.63
10.69
9.56
7.65
4.50
1.69
Time (min)
0
0.2
8
9.5
10
12.5
12.51
16
%B
2
2
50
50
90
90
2
2
Amount oncolumn (pmol)
581.25
393.75
178.13
159.37
127.50
75.00
28.12
(1)
(2) (3) (4)
4
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Results - Chromatograms
Total Ion Chromatogram for IgG
Optimization of Collision Energies for peptides of interest
MRM Chromatogram for Level 4 standard of spiked IgG in whole blood.
VVSVLTVLHQDWLNGKTTPPVLDSDGSFFLYSK
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min
0
25000000
50000000
75000000
100000000
125000000
150000000
175000000
200000000
225000000
250000000
275000000
300000000
6.200 6.225 6.250 6.275 6.300 6.325 6.350 6.375 6.400 6.425 6.450 6.475 6.500 6.525 6.550 6.575 6.600 6.625 6.650 6.675 min
0
250000
500000
750000
1000000
1250000
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00Inten.
938
836915510
397
938
937
836
836
724283
891379
397
836 1046640591283
809443
352295 524
723407 851
407337 724466 756658
837
1163561397369
449
Range CE: -50 to -10 VTTPPVLDSDGFFLYSK
[M+2H]+2
[P1+2H]+2
[P2+2H]+2
Carryover Assessment
Blank InjectionControl - Mouse blood
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
100
200
300
400
500
600
700
800
900
1000
1100
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 min
0
10
20
30
40
50
60
70
80
90
5
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
Results - Calibration Curves
VVSVLTVLHQDWLNGK
Sample
QC 1
QC 2
QC 3
QC 4
Ret. Time
6.49
6.516
6.514
6.492
Area
32,492
11,726
8,507
2,727
Calc. Conc.
502.804
167.189
115.155
21.745
Std. Conc.
465
142.5
102
22.5
% Accuracy
108.1
117.3
112.9
96.6
TTPPVLDSDGSFFLYSK
Sample
QC 1
QC 2
QC 3
QC 4
Ret. Time
6.029
6.052
6.047
6.029
Area
61,525
25,355
16,900
6,502
Calc. Conc.
416.447
155.568
94.58
19.587
Std. Conc.
465
142.5
102
22.5
% Accuracy
89.6
109.2
92.7
87.1
0 100 200 300 400 Conc.0
25000
50000
Area
r2 = 0.979
TTPPVLDSDGSFFLYSK VVSVLTVLHQDWLNGK
r2 = 0.989
0 100 200 300 400 Conc.0
5000
10000
15000
20000
25000
30000
Area
Level 7
5.50 5.75 6.00 6.25 6.50
0
500
1000
1500
2000 937.70>723.95(+)937.70>836.25(+)Level 1
5.50 5.75 6.00 6.25 6.50
0
5000
10000
15000
20000
25000937.70>723.95(+)937.70>836.25(+) Level 7
6.00 6.25 6.50 6.75
0
100
200
300
400
500
600603.70>805.70(+)Level 1
6.00 6.25 6.50 6.75
0
2500
5000
7500
10000603.70>805.70(+)
Calibration Curve and MS Chromatograms
Results - Tables and Replicates
QC data and Calculations for IgG Peptides
A Rapid and Reproducible Immuno-MS Platform from Sample Collection to Quantitation of IgG
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
VVSVLTVLHQDWLNGK TTPPVLDSDGSFFLYSK
Skyline Data - Retention Time Replicates
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
4
Replicate
5.90
5.95
6.00
6.05
6.10
6.15
6.20
Ret
enti
on
Tim
e
y15 - 836.4169++
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
46.35
6.40
6.45
6.50
6.55
6.60
6.65y14 - 805.4385++
839
AM
_226
2014
...L1
...00
5
839
AM
_226
2014
...L2
...00
4
839
AM
_226
2014
...L3
...00
3
839
AM
_226
2014
...L4
...00
2
1433
PM
_225
2014
...L5
...00
8
1433
PM
_225
2014
...L6
...00
6
1433
PM
_225
2014
...L7
...00
4
Replicate
6.35
6.40
6.45
6.50
6.55
6.60
6.65
Ret
enti
on
Tim
e
y14 - 805.4385++
Integration of Skyline Software into LabSolutions allows for further interrogation of data. Here are representative �gures showing the retention time reproducibility for each peptide monitored during the analysis.
ConclusionsCombining the sample collection technique of next generation plasma separator Noviplex cards for quick plamsa collection from whole blood, with the automated af�nity selection and trypsin digestion of the Per�nity workstation coupled to LCMS-8050, provides a very rapid and reproducible Immuno-MS platform for quantitation of IgG peptides. Furthermore, this rapid immuno-MS platform can be applied to many other peptide/protein applications.
PO-CON1473E
Simultaneous Determinations of 20 kindsof common drugs and pesticides in human blood by GPC-GC-MS/MS
ASMS 2014 TP 757
Qian Sun, Jun Fan, Taohong Huang,
Shin-ichi Kawano, Yuki Hashi,
Shimadzu Global COE, Shanghai, China
2
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
IntroductionOn-line gel permeation chromatography-gas chromatography/mass spectrometry (GPC-GC-MS) is a unique technique to cleanup sample that reduce the time of sample preparation. GPC can ef�ciently separates fats, protein and pigments from samples, due to this advantage, on-line GPC is widely used for pesticide analysis. Meanwhile, compared to widely used GC-MS, GC-MS/MS
techniques provide much better selectivity thus signi�cantly lower detection limits. In this work, a new method was developed for rapid determination of 20 common drugs and pesticides in human blood by GPC-GC-MS/MS. The modi�ed QuEChERS method was used for sample preparation.
ExperimentalThe human blood samples were extracted with acetonitrile, then was puri�ed by PSA, C18 and MgSO4 to remove most of the fats, protein and pigments in samples, then after on-line GPC-GC-MS/MS analysis which further removed
macromolecular interference material, such as protein and cholesterol, the background interference brought about by the complex matrix in samples was effectively reduced.
Figure 1 Schematic �ow diagram of the sample preparation
Sample pretreament
PSA/C18/MgSO4
vortex
centrifuge
CH3CN
vortex
human blood 2 mL
evaporate
GPC-GC-MS/MS
supernatant
set volume using moblie phase
3
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
ResultsFor all of analytes, recoveries in the acceptable range of 70~120% and repeatability (relative standard deviations, RSD)≤5% (n=3) were achieved for matrices at spiking levels of 0.01 µg/mL. The limitis of detection were 0.03~4.4 µg/L.
The method is simple, rapid and characterized with acceptable sensitivity and accuracy to meet the requirements for the analysis of common drugs and pesticides in the human blood.
Figure 2 MRM chromatograms of standard mixture
Instrument
GPC
Mobile phase : acetone/cyclohexane (3/7, v/v)
Flow rate : 0.1mL/min
Column : Shodex CLNpak EV-200 (2 mmI.D. x 150 mmL.)
Oven temperature : 40 ºC
Injection volume : 10 μL
GCMS-TQ8030
Column : deactivated silica tubing [0.53 mm(ID) x 5 m(L)]
+pre-column Rtx-5ms [0.25 mm(ID) x 5 m(L)]
Rtx-5ms [0.25mm(ID) x 30 m(L), Thickness: 0.25 μm]
Injector : PTV
Injector time program : 120 ºC(4.5min)-(80 ºC/min)-280 ºC(33.7 min)
Oven temperature program : 82 ºC(5min)-(8 ºC/min)-300 ºC(7.75 min)
Linear velocity : 48.8 cm/sec
Ion Source temperature : 210 ºC
Interface temperature : 300 ºC
15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5
0.00
0.25
0.50
0.75
1.00
(x10,000,000)
Simultaneous Determinations of 20 kinds of common drugs and pesticides in human blood by GPC-GC-MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
Table 1 Results of method validation for drugs and pesticides(Concentration range: 5-100 μg/L, LODs: S/N≥3, LOQs: S/N≥10, RSDs: n=3)
ConclusionA very quick, easy, effective, reliable method in human blood based on modi�ed QuEChERS method was developed using GPC-GCMS-TQ8030. The performance of the method was very satisfactory with results meeting
validation criteria. The method has been successfully applied for determination of human blood samples and ostensibly has further application opportunities, e.g. biological samples.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Compound Name
Dichlorvos
Methamidophos
Barbital
Sulfotep
Dimethoate
Malathion
Chlorpyrifos
Phenobarbital
Parathion
Triazophos
Zopiclone deg.
Diazepam
Midazolam
Zolpidem
Clonazepam
Estazolam
Clozapine
Alprazolam
Zolpidem
Triazolam
10.795
11.800
15.210
17.580
18.310
21.555
21.715
22.000
22.180
25.675
26.025
27.635
29.250
31.225
31.795
32.335
32.400
32.730
33.095
33.700
tR
(min)
0.9993
0.9994
0.9994
0.9995
0.9993
0.9997
0.9996
0.9995
0.9993
0.9994
0.9993
0.9992
0.9994
0.9993
0.9995
0.9994
0.9991
0.9993
0.9995
0.9992
CorrelationCoef�cient*
0.103
0.023
0.018
0.011
0.400
0.005
0.010
0.353
0.003
0.046
0.189
0.007
0.048
1.298
0.432
0.092
0.050
0.028
1.027
0.027
LOD(µg/L)
0.345
0.076
0.058
0.037
1.333
0.016
0.033
1.177
0.009
0.155
0.631
0.022
0.160
4.325
1.440
0.305
0.167
0.095
3.425
0.091
LOQ(µg/L) Recovery (%)
72.9
85.3
72.4
110.7
103.7
82.7
85.7
79.6
92.3
87.7
83.5
98.3
87.1
99.3
110.0
103.7
100.6
103.3
87.3
81.3
RSD (%)
2.99
3.58
1.72
2.27
3.10
2.52
3.57
3.25
3.17
1.32
1.28
1.55
2.01
1.01
1.57
1.37
3.12
1.48
1.75
2.56
0.01 µg/mL
PO-CON1466E
Low level quantitation of Loratadinefrom plasma using LC/MS/MS
ASMS 2014 TP498
Shailesh Damale, Deepti Bhandarkar, Shruti Raju,
Rashi Kochhar, Shailendra Rane, Ajit Datar,
Pratap Rasam, Jitendra Kelkar
Shimadzu Analytical (India) Pvt. Ltd., 1 A/B Rushabh
Chambers, Makwana Road, Marol, Andheri (E),
Mumbai-400059, Maharashtra, India.
2
Low level quantitation of Loratadine from plasma using LC/MS/MS
IntroductionLoratadine is a histamine antagonist drug used for the treatment of itching, runny nose, hay fever and such other allergies. Here, an LC/MS/MS method has been developed for high sensitive quantitation of this molecule from plasma using LCMS-8050, a triple quadrupole mass spectrometer from Shimadzu Corporation, Japan. Presence
of heated Electro Spray Ionization (ESI) interface in LCMS-8050 ensured good quantitation and repeatability even in the presence of a complex matrix like plasma. Ultra high sensitivity of LCMS-8050 enabled development of a low ppt level quantitation method for Loratadine.
Method of AnalysisThis bioanalytical method was developed for measuring Loratadine in therapeutic concentration range for the analysis of routine samples. It was important to develop a
simple and accurate method for estimation of Loratadine in human plasma.
To 100 µL of plasma 500 µL cold acetonitrile was added for protein precipitation. It was placed in rotary shaker at 20 rpm for 15 minutes for uniform mixing. This solution
was centrifuged at 12000 rpm for 15 minutes. Supernatant was taken and evaporated to dryness at 70 ºC . The residue was reconstituted in 200 µL Methanol.
Preparation of matrix matched plasma by protein precipitation method using cold acetonitrile
1 ppt, 5 ppt, 50 ppt, 100ppt, 500 ppt, 1 ppb, 5 ppb and 10 ppb of Loratadine calibration standards were prepared
in cold acetonitrile treated matrix matched plasma.
Preparation of calibration standards in matrix matched plasma
Figure 1. Structure of Loratadine
LoratadineLoratadine, a piperidine derivative, is a potent long-acting, non-sedating tricyclic antihistamine with selective peripheral H1-receptor antagonist activity. It is used for relief of nasal and non-nasal symptoms of seasonal allergies and skin rashes[1,2,3]. Due to partial distribution in central nervous system, it has less sedating power compared to traditional H1 blockers. Loratadine is given orally, is well absorbed from the gastrointestinal tract, and has rapid �rst-pass hepatic metabolism; it is metabolized by isoenzymes of the cytochrome P450 system, including CYP3A4, CYP2D6, and, to a lesser extent, several others. Loratadine is almost totally (97–99 %) bound to plasma proteins and reaches peak plasma concentration (Tmax) in ~ 1–2 h[4,5].
Ethyl 4- (8-chloro-5, 6-dihydro-11H-benzo [5, 6] cyclohepta [1, 2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
3
Low level quantitation of Loratadine from plasma using LC/MS/MS
LC/MS/MS analysisLCMS-8050 triple quadrupole mass spectrometer by Shimadzu Corporation, Japan (shown in Figure 2A), sets a new benchmark in triple quadrupole technology with an unsurpassed sensitivity (UFsensitivity) with Scanning speed of 30,000 u/sec (UFscanning) and polarity switching speed of 5 msecs (UFswitching). This system ensures highest quality of data, with very high degree of reliability.In order to improve ionization ef�ciency, the newly developed heated ESI probe combines high-temperature gas with the nebulizer spray, assisting in the desolvation of large droplets and enhancing ionization. This development allows high-sensitivity analysis of a wide
range of target compounds with considerable reduction in background.Presence of heated Electro spray interface in LCMS-8050 (shown in Figure 2B) ensured good quantitative sensitivity even in presence of a complex matrix like plasma.The parent m/z of 382.90 giving the daughter m/z of 337.10 in the positive mode was the MRM transition used for quantitation of Loratadine. MS voltages and collision energy were optimized to achieve maximum transmission of mentioned precursor and product ion. Gas �ow rates, source temperature conditions and collision gas were optimized, and linearity graph was plotted for 4 orders of magnitude.
Figure 2A. LCMS-8050 triple quadrupole mass spectrometer by Shimadzu Figure 2B. Heated ESI probe
Table 2. LCMS conditions
ESI
Positive
2.0 L / min (nitrogen)
10.0 L / min (nitrogen)
15.0 L / min (zero air)
300 ºC
250 ºC
400 ºC
382.90 > 337.10
MS Interface
Polarity
Nebulizing Gas Flow
Drying Gas Flow
Heating Gas Flow
Interface Temp.
Desolvation Line Temp.
Heater Block Temp.
MRM Transition
B conc. (%)Time (min)
60
100
100
60
0.01
1.50
4.00
4.10
13.00
A conc. (%)
40
0
0
40
Stop
Table 1. LC conditions
Shim-pack XR-ODS (100 mm L x 2.0 mm ID ; 2.2 µm)
A : 0.1% formic acid in water
B : acetonitrile
0.15 mL/min
40 ºC
20 µL
Column
Mobile Phase
Gradient Program
Flow Rate
Oven Temperature
Injection Volume
Low level quantitation of Loratadine from plasma using LC/MS/MS
4
Figure 4A. Mass chromatogram 10 ppb Figure 4B. Mass chromatogram 0.001 ppb
Figure 5. Overlay chromatogram
Results
LC/MS/MS method for Loratadine was developed on ESI +ve ionization mode and 382.90>337.10 MRM transition was optimized for Loratadine. Checked matrix matched plasma standards for highest (10 ppb) as well as lowest (0.001 ppb) concentrations as seen in Figures 4A and 4B respectively. Optimized MS method to ensure no plasma interference at the retention time of Loratadine (Figure 5).
Calibration curve was plotted for Loratadine concentration range. Also as seen in Table 3, % Accuracy was studied to confirm the reliability of method. Linear calibration curves were obtained with regression coefficients R2 > 0.998. % RSD of area was within 15 % and accuracy was within 80-120 % for all calibration levels.
LC/MS/MS Analysis
Speci�city and interference
0.0 2.5 5.0 7.5
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5(x1,000,000)382.90>337.10(+)
LORA
TAD
INE/
3.39
1
0.0 2.5 5.0 7.5-1.0
0.0
1.0
2.0
3.0
4.0
5.0
(x10,000)382.90>337.10(+)
LORA
TAD
INE/
3.37
7
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 min
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2(x10,000)
1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_002.lcd1:LORATIDINE 382.90>337.10(+) CE: -23.0 LORA_PLASMA_003.lcd
------ LOQ Level
------ Blank
5
Low level quantitation of Loratadine from plasma using LC/MS/MS
Figure 6. Loratadine calibration curve
Conclusion• Highly sensitive LC/MS/MS method for Loaratadine was developed on LCMS-8050 system.• Calibration was plotted from 10 ppb to 0.001 ppb, and LOQ was computed as 0.001 ppb.
Table 3. Results of Loratadine calibration curve
Nominal Concentration (ppb)
Measured Concentration (ppb)
% Accuracy(n=3)
% RSD for area counts (n=3)
0.001
0.005
0.05
0.1
0.5
1.0
5.0
10.0
Standard
STD-01
STD-02
STD-03
STD-04
STD-05
STD-06
STD-07
STD-08
Sr. No.
1
2
3
4
5
6
7
8
0.00096
0.0050
0.057
0.095
0.048
0.986
5.077
9.983
0.62
5.24
0.98
1.81
1.40
0.11
1.07
1.96
95.83
100.73
114.83
95.40
95.70
98.53
101.53
99.37
Result Table
0.0 2.5 5.0 7.5 Conc.0.0
1.0
2.0Area (x10,000,000)
1 2 3 4 5
6
7
8
0.05 0.10 Conc.0.0
1.0
2.0
Area (x100,000)
1 2
3
4
Low level quantitation of Loratadine from plasma using LC/MS/MS
For Research Use Only. Not for use in diagnostic procedures.The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice.
© Shimadzu Corporation, 2014
First Edition: June, 2014
www.shimadzu.com/an/
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Volume 48, (2010), 35-44.[2] J. Chen, YZ. Zha, KP. Gao, ZQ. Shi, XG. Jiang, WM. Jiang, XL. Gao, Pharmazie, Volume 59, (2004), 600-603.[3] M. Haria, A. Fitton, and D.H. Peters, Drugs, Volume 48, (1994), 617-637.[4] J. Hibert, E. Radwanski, R. Weglein, V. Luc, G. Perentesis, S. Symchowicz, and N. Zampaglione, J.clin. Pharmacol,
Volume 27, (1987), 694-698.[5] S.P.Clissold, E.M. Sorkin, and K.L. Goa, Drugs, Volume 37,(1989), 42-57.