TN-1205 APPLICATIONS
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TN-1205 For additional technical notes, visit www.phenomenex.com Page 1 of 8 APPLICATIONS A Simple and Effective High pH LC/MS/MS Method for Determination of Underivatized Vitamin B1 and B6 in Human Whole Blood Xianrong (Jenny) Wei, Ramkumar Dhandapani, and Sean Orlowicz Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA Introduction Vitamins B1 and B6 are two water-soluble vitamins with clinical research interest. Thiamine Diphosphate (TDP) is the main biolog- ically active form of Vitamin B1 and is required for various meta- bolic functions. Pyridoxal-5-phosphate (PLP) is the main biologi- cally active form of Vitamin B6 and is a coenzyme for a number of transamination reactions. 1,2 Chromatographically, these are very challenging analytes for a reversed phase separation as they have negative values for the octanol/water partition coefficients. The structure of TDP and PLP are shown in Figure 1. The most com- mon method for TDP and PLP analysis is a HPLC based assay with a pre-column derivatization with alkaline potassium ferricya- nide and semicabazide, followed by fluorescence detection. This procedure is time and labor intensive and uses toxic reagent. An- other approach uses ion pairing reagents, however these are not alternatives that are compatible with mass spectrometry. Present- ed here is a simple method that does not involve derivatization or ion pairing reagents. The method involves whole blood extraction at lower pH, cost effective internal standards, no derivatization and a reversed phase LC/MS/MS compatible method for the anal- ysis of polar TDP and PLP. The assay is evaluated for precision, accuracy, linear range, and the results meet acceptance criteria. Experimental Conditions Optimized Sample Extraction Method Human whole blood samples were frozen immediately at -20 °C after collection. It is important to freeze the sample for at least 24 hours prior to analysis in order to prevent the analyte from decomposition, especially TDP. 1. Pipette 100 µL of thawed hemolyzed blood into a 1.8 mL centrifuge tube 2. Add 300 µL of working internal standard (20 ng/mL of Pyridox ine-D 2 and 50 ng/mL of Thiamine- 13 C 4 in DI water) and mix for 30 seconds 3. Add 30 µL of 70 % HClO 4 and mix for 1 minute to precipitate proteins 4. Centrifuge sample at 14,000 rpms for 10 minutes to pellet the protein 5. Transfer 200 µL of supernatant into an autosampler vial for LC MS/MS analysis Note: Since the analytes are light sensitive, the extraction steps were performed in amber color centrifuge tube and were pro- tected from light. LC/MS/MS Method Parameters Column: Gemini ® 5 µm C18 Dimensions: 50 x 4.6 mm Part No.: 00B-4435-E0 SecurityGuard Cartridge: AJ0-7597 Mobile Phase: A: 10 mM NH 4 HCO 3 in water, pH8.8 B: Methanol Gradient: Time (min) B (%) 0.01 0 1.5 0 5 60 6.5 60 6.51 0 9 0 Flow Rate: 600 µL/min Injection Volume 10 µL Instrument: Agilent ® 1260 LC Detection: MS/MS (ESI+) (SCIEX API 4500 ™ ) Sample: 1. Pyridoxal 5-phosphate (PLP) 2. Thiamine Diphosphate (TDP) 3. Pyridoxine D 2 4. Thiamine- 13 C 4 Ramkumar Dhandapani Technical Manager Ramkumar loves to write poems, and to watch & read Shakespeare’s plays. Figure 1. Structure of TDP and PLP NH 2 N N N P P CH 3 H 2 C C P -O O O O O- H O O O O S + – OH OH OH OH + N H TDP logP: - 5.80 PLP logP: - 2.09 ID Q1 Mass (Da) Q3 Mass (Da) Dwell (msec) DP CE PLP 1 248.1 149.8 125 75 15 PLP 2 248.1 94.1 125 75 25 TDP 1 425.1 122.1 25 60 15 TDP 2 425.1 81.0 25 60 52 TDP 3 425.1 303.9 25 70 20 Thiamine-13C4 1 270.1 122.9 75 40 15 Thiamine-13C4 2 270.1 148.1 75 40 15 Pyridoxine D2 1 172.1 155.0 75 40 15 Pyridoxine D2 2 172.1 136.0 75 40 15 Table 1. MRM Transitions Revision: PHEN-RUO-00078 0
Transcript of TN-1205 APPLICATIONS
TN-1205
For additional technical notes, visit www.phenomenex.com Page 1 of 8
APPLICATIONSA Simple and Effective High pH LC/MS/MS Method for Determination of Underivatized Vitamin B1 and B6 in Human Whole BloodXianrong (Jenny) Wei, Ramkumar Dhandapani, and Sean OrlowiczPhenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA
IntroductionVitamins B1 and B6 are two water-soluble vitamins with clinical research interest. Thiamine Diphosphate (TDP) is the main biolog-ically active form of Vitamin B1 and is required for various meta-bolic functions. Pyridoxal-5-phosphate (PLP) is the main biologi-cally active form of Vitamin B6 and is a coenzyme for a number of transamination reactions.1,2 Chromatographically, these are very challenging analytes for a reversed phase separation as they have negative values for the octanol/water partition coefficients. The structure of TDP and PLP are shown in Figure 1. The most com-mon method for TDP and PLP analysis is a HPLC based assay with a pre-column derivatization with alkaline potassium ferricya-nide and semicabazide, followed by fluorescence detection. This procedure is time and labor intensive and uses toxic reagent. An-other approach uses ion pairing reagents, however these are not alternatives that are compatible with mass spectrometry. Present-ed here is a simple method that does not involve derivatization or ion pairing reagents. The method involves whole blood extraction at lower pH, cost effective internal standards, no derivatization and a reversed phase LC/MS/MS compatible method for the anal-ysis of polar TDP and PLP. The assay is evaluated for precision, accuracy, linear range, and the results meet acceptance criteria.
Experimental ConditionsOptimized Sample Extraction Method
Human whole blood samples were frozen immediately at -20 °C after collection. It is important to freeze the sample for at least 24 hours prior to analysis in order to prevent the analyte from decomposition, especially TDP.
1. Pipette 100 µL of thawed hemolyzed blood into a 1.8 mL centrifuge tube
2. Add 300 µL of working internal standard (20 ng/mL of Pyridox ine-D2 and 50 ng/mL of Thiamine-13C4 in DI water) and mix for 30 seconds
3. Add 30 µL of 70 % HClO4 and mix for 1 minute to precipitate proteins
4. Centrifuge sample at 14,000 rpms for 10 minutes to pellet the protein
5. Transfer 200 µL of supernatant into an autosampler vial for LC MS/MS analysis
Note: Since the analytes are light sensitive, the extraction steps were performed in amber color centrifuge tube and were pro-tected from light.
LC/MS/MS Method ParametersColumn: Gemini® 5 µm C18
Dimensions: 50 x 4.6 mmPart No.: 00B-4435-E0
SecurityGuard Cartridge: AJ0-7597Mobile Phase: A: 10 mM NH4HCO3 in water, pH8.8
B: MethanolGradient: Time (min) B (%)
0.01 01.5 05 606.5 606.51 09 0
Flow Rate: 600 µL/minInjection Volume 10 µL
Instrument: Agilent® 1260 LC
Detection: MS/MS (ESI+) (SCIEX API 4500™)Sample: 1. Pyridoxal 5-phosphate (PLP)
2. Thiamine Diphosphate (TDP)3. Pyridoxine D2
4. Thiamine-13C4
Ramkumar Dhandapani Technical Manager Ramkumar loves to write poems, and to watch & read Shakespeare’s plays.
Figure 1. Structure of TDP and PLP
NH2
N N
N
PP
CH3
H2C
C
P-O
OO
OO-
H
O
OO
OS
+
– OHOH
OH
OH
+NHTDP
logP: - 5.80PLP
logP: - 2.09
ID Q1 Mass (Da)
Q3 Mass (Da)
Dwell (msec)
DP CE
PLP 1 248.1 149.8 125 75 15
PLP 2 248.1 94.1 125 75 25
TDP 1 425.1 122.1 25 60 15
TDP 2 425.1 81.0 25 60 52
TDP 3 425.1 303.9 25 70 20
Thiamine-13C4 1 270.1 122.9 75 40 15
Thiamine-13C4 2 270.1 148.1 75 40 15
Pyridoxine D2 1 172.1 155.0 75 40 15
Pyridoxine D2 2 172.1 136.0 75 40 15
Table 1. MRM Transitions
Rev
isio
n:
PH
EN
-RU
O-0
0078
0
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Table 2. Accuracy and Precision
Run 1 Run 2TDP PLP TDP PLP
Nominal Conc. 100 ng/mLSample ID Conc. Found (ng/mL) Conc. Found (ng/mL)QC1_1 103 88.5 118 95.8QC1_2 80.5 103 89.1 74.5QC1_3 94.8 98.5 105 88.3QC1_4 87.2 110 102 106QC1_5 85.5 102 86.7 88QC1_6 93.6) 73.6 110 85.8
Nominal Conc. 200 ng/mLQC2_1 222 170 215 176QC2_2 226 201 217 190QC2_3 218 196 259 260QC2_4 211 215 192 209QC2_5 216 202 228 205QC2_6 255 228 227 160
Mean Conc. Found (ng/mL) 96.3 92.8 224 201STDV 11.3 11.7 18.2 26.8CV% 11.8 12.6 8.13 13.3
Accuracy (%) 96.3 92.8 112 101
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.50.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
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3.0e5
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Inte
nsity
, cp
s
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nsity
, cp
s
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min
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nsity
, cp
s
Figure 2. Representative Chromatogram in Whole Blood at LLOQ (20 ng/mL)
Ap
p ID
235
49
Thiamine 13C4 (IS)
Pyridoxine D2 (IS)
TDP
PLP
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, cp
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nsity
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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.50.0
2.0e4
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nsity
, cp
s
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nsity
, cp
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min
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tens
ity, c
ps
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nsity
, cp
s
Figure 3. Representative Chromatogram in Whole Blood at ULOQ (250 ng/mL)
Figure 5. Plot Demonstrating Suitability of Internal Standard
5 10 15 20 25 30 35 40 45 50 55 60Index
5 10 15 20 25 30 35 40 45 50 55 60Index
Pyridoxine D2 HCL
Thiamine13C4 HCL
25 % of mean response
1.0e5
2.0e5
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1.4e61.5e6
IS P
eak
Are
a, c
ount
s
0.0
5.0e41.0e51.5e52.0e52.5e53.0e53.5e54.0e54.5e55.0e55.5e56.0e56.5e57.0e57.5e58.0e58.5e59.0e59.5e5
IS P
eak
Are
a, c
ount
s
0.0
Figure 4. Interference Peak Analysis during TDP Detection
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Inte
nsity
, cp
s
Interference 425.1 > 303.9 mz
Ap
p ID
235
56
Ap
p ID
235
50
Thiamine13C4 (IS)
Pyridoxine D2 (IS)PLP
TDP
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Figure 6. Representative Calibration Curve of TDP in Whole Blood
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250Analyte Conc. / IS Conc.
0.000
5.000e-3
0.010
0.015
0.020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.064
Ana
lyte
Are
a /
IS A
rea
Figure 8. Matrix Effects on PLP Analysis: Water vs. Whole Blood
Whole Blood
Water
0.02000.04000.06000.08000.0
1.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e43.2e43.4e43.6e4
Inte
nsity
, cp
s
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
Step Total Time (min)
Flow Rate (µL/min) A (%) B (%)
0 0.01 500 100 0
1 1.5 500 100 0
2 1.8 500 40 60
3 5 500 40 60
4 5.5 500 100 0
5 8 500 100 0
Figure 9. Flow Rate Alteration to Separate Interference Peaks During PLP Detection
Ap
p ID
235
57
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250Analyte Conc. / IS Conc.
0.0002.000e-34.000e-36.000e-38.000e-3
0.0100.0120.0140.0160.0180.0200.0220.0240.0260.0280.0300.0320.0340.0360.0380.0400.0420.044
Ana
lyte
Are
a /
IS A
rea
Dynamic range: 20-250 ng/mLr = 0.9953
Dynamic range: 20-250 ng/mLr = 0.9939
Figure 7. Representative Calibration Curve of PLP in Whole Blood
Flow Rate: 500 µL/min
Ap
p ID
235
55
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
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1000.02000.03000.04000.05000.06000.07000.08000.09000.0
1.0e41.1e41.2e41.3e41.4e4
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nsity
, cps
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2000.04000.06000.08000.0
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Inte
nsity
, cp
s
Interference 248.1 > 149.8 mz
Interference 248.1 > 149.8 mz
min
Flow Rate: 600 µL/min
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Figure 10. Matrix Effect on PLP Detection
PLP (200 ng/mL)Solvent (blank)
Blood (blank)
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min0.0
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23551
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Inte
ns
ity
, c
ps
Ap
p ID
235
51
Figure 11. Matrix Effect on TDP Detection
Solvent (blank)
Blood (blank)
TDP(200 ng/mL)
min0.0
1.5e4
1.0e4
5000.00
2.0e4
2.5e4
3.0e4
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7.0e47.3e4
Inte
ns
ity
, c
ps
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23552
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p ID
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Figure 12. Endogenous Levels of PLP in Six Individual Lots of Blank Whole Blood
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.0 5.0 5.5 min0
50
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Inte
nsity
, cps
Average Endogenous Level: ~400 cps (Height)
Average Endogenous Level: ~ 6000 cps (Height)
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1.10e4
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Ap
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54
Ap
p ID
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Figure 13. Endogenous Levels of TDP in Six Individual Lots of Blank Whole Blood
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Results and DiscussionPresented here is a simplified extraction procedure for whole blood samples followed by a LC/MS/MS compatible reversed phase method for both TDP and PLP. Both analytes are highly polar (refer to structures in Figure 1.) and retaining both com-pounds under reversed phase conditions is challenging. Gemini® 5 µm C18 is a reversed phase HPLC column with an extreme pH resistance that offers flexibility to explore pH beyond conventional silica columns capabilities. For the present work, Gemini 5 µm C18, 50 x 4.6 mm was employed under high pH mobile phase conditions. The gradient started with 100 % aqueous mobile phase to promote retention of TDP and PLP and representative chromatogram at Lower Limit Of Quantitation (LLOQ ) and Upper Limit Of Quantitation (ULOQ) are presented in Figures 2 and 3. These chromatograms provide evidence of successful retention of both the analytes on Gemini C18 column despite the use of any ion pairing reagent. In addition to improved reversed phase retention, interference peaks were also resolved from the analyte peaks as shown in Figure 4.
The extraction was performed under acidic conditions prior to LC/MS/MS which not only helped with the stability of TDP and PLP, but also allowed the extraction to take place at room temperature. Two sets of evaluation batches were run individually on 2 different days, on each day 2 concentration of QCs 100 ng/mL and 200 ng/ml were run 6 times to ensure precision and accuracy. Accuracy and precision data are presented in Table 2. The assay is accu-rate with a recovery of 92.8-112.0 % and precise with % CV of 8.13-12.6 % for human whole blood matrix. The method utilized cost effective alternative for internal standard which are commer-cially available. The response of the isotope internal standards is proven consistent within ±25 % of the mean response of the standards and QCs for all samples as shown in Figure 5.
The dynamic ranges of both TDP and PLP in whole blood are presented in Figures 6 and 7 and the developed method proved to be linear from 20 to 250 ng/mL. Water as a matrix was also analyzed and was compared with chromatograms of whole blood extract as shown in Figure 8. It is clearly evident that, DI water matrix will have clean chromatography but when calibration curve is performed on DI water to quantitate TDP and PLP from whole blood, there will be matrix interference that will not be accounted as shown in Figure 8. For the same reason, we highly recommend using the whole blood calibration curve as it is the best practice for TDP and PLP analysis.
Challenging matrices, such as whole blood, usually require a rigorous sample cleanup due to the presence of matrix interfer-ences. Especially due to the fact that whole blood as a matrix offers variability. In order to account for this variability and to demonstrate method flexibility, experiments were performed with change in mobile phase flow rate. The respective chromatograms are shown in Figure 9, which shows the separation of interfer-ence peaks with changes in the mobile phase flow rate. Further to demonstrate the success of the sample cleanup, matrix ef-fects were monitored during the method development process by performing post column infusion tests as shown in Figures 10 and 11. Certainly, endogenous levels of TDP and PLP in the blank whole blood can affect the LLOQ of the assay and for the same reason, it is important to evaluate the level of endogenous interference. Six individual lots of commercially purchased human whole blood blanks were evaluated to determine the endogenous levels of TDP and PLP. The average endogenous level of six lots was ~6000 cps (peak height) for TDP and ~400 cps (peak height) for PLP when quantified by MS/MS (SCIEX API 4500™) as shown in Figures 12 and 13.
Overall, the method development involved changing the extraction pH, selecting the correct reversed phase column, and optimiza-tion of gradient profile. In addition, selection of cost effective internal standards produced a time and cost efficient alternative for the separation of TDP and PLP. Aside from being efficient, the method proved to be reproducible and gave consistent results for more than 500 injections on the LC/MS/MS.
ConclusionA simple and rapid assay method is presented for the quantitation of Vitamin B1 and B6 in human whole blood by LC/MS/MS. The method is accurate with recoveries from 92.8-112.0 % and with %CV of 8.13-12.6 % it proves to be precise and the method is linear from 20-250 ng/mL of the analytes. Matrix effects and res-olution of interferences from analyte peaks were also monitored for TDP and PLP, resulting in an acceptable detection of TDP and PLP. This assay is simple, time saving, cost effective and automa-tion friendly.
References
1 Puts J, de Groot M, Haex M, Jakobs B (2015) Simultaneous Determination of Underivatized Vitamin B1 and B6 in Whole Blood by Reversed Phase Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry. PLoS ONE 10(7): e0132018. doi:10.1371/journal. pone.0132018
2 Trang, Hung Khiem, “Development of HPLC methods for the determination of water-soluble vitamins in pharmaceuticals and fortified food products” (2013). All Theses. Paper 1745.
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APPLICATIONS
TN45
5402
17_W
www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected]
Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions.
Trademarks Gemini is a registered trademark, MidBore and SecurityGuard are trademarks of Phenomen-ex. Agilent is a registered trademark of Agilent Technologies, Inc. API 4500 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX™ is being used under license.
Gemini is patented by Phenomenex. U.S. Patent No. 7, 563, 367 and 8,658,038 and foriegn counterparts.SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362.
Disclaimer FOR RESEARCH ONLY. Not for use in diagnostic procedures.
CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep, PREP or ULTRA holders, or to any cartridges.
© 2017 Phenomenex, Inc. All rights reserved.
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Luxembourgt: +31 (0)30-2418700 f: +31 (0)30-2383749
Mexicot: 01-800-844-5226f: 001-310-328-7768
The Netherlandst: +31 (0)30-2418700 f: +31 (0)30-2383749
New Zealandt: +64 (0)9-4780951f: +64 (0)9-4780952
Norwayt: +47 810 02 005f: +45 4810 6265
Puerto Ricot: +1 (800) 541-HPLCf: +1 (310) 328-7768
Spaint: +34 91-413-8613f: +34 91-413-2290
Swedent: +46 (0)8 611 6950f: +45 4810 6265
United Kingdomt: +44 (0)1625-501367f: +44 (0)1625-501796
USAt: +1 (310) 212-0555f: +1 (310) 328-7768
All other countries Corporate Office USA
t: +1 (310) 212-0555f: +1 (310) 328-7768
Ordering Information Gemini® HPLC Columns
For Gemini Capillary or Preparative HPLC Column and Guards contact your Phenomenex techical consultant or local distributor.
If Phenomenex products in this technical note do not provide at least an equivalent separation as compared to other products of the same phase and dimensions, return the product with compar-ative data within 45 days for a FULL REFUND.
3 μm Analytical Columns (mm) SecurityGuard™ Cartridges (mm)Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*
10/pkC18 00A-4439-E0 00B-4439-E0 00D-4439-E0 00F-4439-E0 00G-4439-E0 AJ0-7597
for ID: 3.2-8.0 mm
5 μm Analytical Columns (mm) SecurityGuard™ Cartridges (mm)Phases 30 x 4.6 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0*
10/pkC18 00A-4435-E0 00B-4435-E0 00D-4435-E0 00F-4435-E0 00G-4435-E0 AJ0-7597
for ID: 3.2-8.0 mm
3 μm Microbore‚ Minibore and MidBore™ Columns (mm) SecurityGuard™ Cartridges (mm)Phases 50 x 1.0 20 x 2.0 30 x 2.0 50 x 2.0 100 x 2.0 150 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 4 x 2.0*
10/pkC18 00B-4439-A0 00M-4439-B0 00A-4439-B0 00B-4439-B0 00D-4439-B0 00F-4439-B0 00B-4439-Y0 00D-4439-Y0 00F-4439-Y0 AJ0-7596
for ID: 2.0-3.0 mm
5 μm Minibore and MidBore Columns (mm) SecurityGuard™ Cartridges (mm)Phases 30 x 2.0 50 x 2.0 150 x 2.0 250 x 2.0 50 x 3.0 100 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0*
10/pkC18 00A-4435-B0 00B-4435-B0 00F-4435-B0 00G-4435-B0 00B-4435-Y0 00D-4435-Y0 00F-4435-Y0 00G-4435-Y0 AJ0-7596
for ID: 2.0-3.0 mm
*SecurityGuard™ Analytical Cartridges require holder‚ Part No.: KJ0-4282
