APPLICATIONS Separation and Quantitation of Physiologic Cobalamins (Vitamin B 12 in Dietary Supplements by LC/MS/MS using Synergi Fusion-RP Column Xianrong (Jenny Wei Senior Scientist PhenoLogix applications laboratory. Xianrong (Jenny Wei 1, Michael Landesman 2, Spencer Carter 2, and Sean Orlowicz 1 1. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 2. Genysis Lab, 391 S Orange Street Suite F, Salt Lake City, UT 84104 Analysis of cobalamins (Vitamin B 12 on reversed phase columns can be challenging due to the relative chemical instability of the analyte, the complexity of matrix effects and the existence of several vitamer forms of B 12. In this tech note, we describe a selective, efficient and reproducible method to separate three B 12 vitamers (methylcobalamin, cyanocobalamin and adenosylcobalamin appropriate for the quantitative analysis of dietary supplements via LC/MS/MS. Introduction Cobalamin is a micronutrient that animals and humans obtain through diet, symbiotic bacteria or vitamin supplements. Vitamin B 12 deficiency leads to a degeneration of the sensory in the spinal cord with loss of sensation and paralysis that results in a complex spectrum of pathologies, including pernicious anemia, megaloblastic anemia, peripheral neuropathy, depression, impaired cognition, and autoimmune dysfunction. The four vitamer forms of B 12 are differentiated based upon the functional group (R at the β-axial position (Figure 1. The two forms found naturally in the body are adenosylcobalamin (AdoB 12 and methylcobalamin (MeB 12, while the two most common vitamin forms are hydroxycobalamin (HOB 12 and cyanocobalamin (CNB 12. Due to its stability advantages, cyanocobalamin is the vitamer form that is most commonly used in supplements. LC/MS/MS Method Parameters Column: Synergi 2.5 µm Fusion-RP Dimensions: 50 x 2.0 mm Part No.: 00B-4423-B0 SecurityGuard Cartridge: AJ0-7844 Mobile Phase: A: 10 mm Ammonium Formate ph 3.5 B: Methanol Gradient: Time (min B (% 0 2 0.2 2 1.8 98 4 98 4.01 2 5.5 2 Flow Rate: 350 µl/min Injection Volume: 10 µl Temperature: 21 C Instrument: Agilent 1260 LC Detection: MS/MS (ESI+ (SCIEX Triple Quad 4500 and SCIEX 4000 QTRAP @ 600 ºC Sample: 1. Cyanocobalamin (CNB 12 2. Adenosylcobalamin (AdoB 12 3. Methylcobalamin (MeB 12 Figure 1. Cobalamin Structure Historically, cobalamin analysis has been performed using microbiological methods that are known to have significant shortcomings for accuracy and specificity. More recently, analysis has been done with HPLC separation and various modes of detection including UV, fluorescence and MS. LC/MS/MS analysis can be challenging due to the chemical instability and insource ionization issues for many of the vitamer forms. In this study, we assessed the selectivity, specificity and separation of three forms (AdoB 12, MeB 12 and CNB 12 that are commonly seen in dietary supplements. We have established a sensitive, selective and reproducible quantitation method for the nutraceutical industry utilizing HPLC separation coupled to a triple quadrupole mass spectrometer. Experimental Conditions Reagent and chemicals All solvents and reagents were HPLC or analytical grade. Reference standards were supplied by Genysis Lab. Equipment and materials Agilent 1260 pumps and autosampler were used along with a SCIEX QTRAP 4000 and SCIEX 4500, positive polarity, ESI for detection. For additional technical notes, visit www.phenomenex.com Page 1 of 8
Figure 2. Cobalamin Mobile Phase ph Selection Chart 2.22 Mobile phase at ph 3.5 Charge 1.2 0.18-0.84-1.86 0 2 4 6 8 10 12 14 ph Figure 3. Tuning files _ Methylcobalamin (m.w. 1343.59 by infusion 7.4e7 7.0e7 6.5e7 6.0e7 673.0 [M+2H + ] Max. 7.4e7cps. 5.5e7 5.0e7 4.5e7 4.0e7 3.5e7 3.0e7 684.0 695.0 Methylcobalamin (MeB 12 2.5e7 2.0e7 [M-ME+2H + ] 1.5e7 1.0e7 665.5 687.5 674.5 762.9 5.0e6 70 774.0 797.0 831.0 527.0 541.0 622.9 882.8 989.5 1004.5 609.2 1366.6 23915 500 600 700 800 900 1000 1100 1200 1300 1400 1500 App ID 23914 1.4e7 1.3e7 1.2e7 1.1e7 1.0e7 9.0e6 8.0e6 7.0e6 665.5 6.0e6 5.0e6 4.0e6 673.0 3.0e6 2.0e6 358.9 971.5 1.0e6 147.1 1183.4 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1.27e6 1.20e6 1.10e6 1.00e6 9.00e5 8.00e5 7.00e5 6.00e5 5.00e5 4.00e5 3.00e5 2.00e5 1.00e5 0 147.1 358.9 636.2 665.4 Methylcobalamin Product of 673 Methylcobalamin Product of 665.4 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 971.4 913.1 340.8 486.3 1125.4 1183.4 App ID 23915 Page 2 of 8
Figure 4. Tuning files _ Cyanocobalamin (m.w. 1354.5 by infusion 1.5e7 1.4e7 1.3e7 1.2e7 678.5 [M+2H + ] 1.1e7 Cyanocobalamin (CNB 12 1.0e7 9.0e6 8.0e6 7.0e6 6.0e6 5.0e6 604.3 689.5 602.2 709.3 4.0e6 3.0e6 2.0e6 1.0e6 609.5 700.5 720.3 697.0 562.9 616.6 657.1 740.5 728.6 777.8 845.7 858.5 638.7 903.7 660.6 1355.4 997.4 1023.5 1078.5 1144.5 1206.41281.5 1377.4 1428.2 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 [M+H + ] App ID 23916 1.0e6 9.5e5 9.0e5 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 147.0 359.0 636.0 678.4 Cyanocobalamin Product of 678.5 1.5e5 912.6 340.6 1.0e5 1209.6 664.7 5.0e4 457.0 184.9 278.8 499.2 725.2 833.7 930.6 1015.3 1124.5 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 997.5 App ID 23917 For additional technical notes, visit www.phenomenex.com Page 3 of 8
Figure 5. Tuning files _ Adenosylcobalamin (m.w. 1579.58 by infusion 2.9e7 2.8e7 790.6 [M+2H + ] 2.6e7 2.4e7 Adenosylcobalamin (AdoB 12 2.2e7 2.0e7 1.8e7 1.6e7 1.4e7 1.2e7 1.0e7 [M-Ado+2H + ] 665.5 801.5 812.5 [M+H + Na + ] [M+2Na + ] 8.0e6 6.0e6 527.5 4.0e6 676.5 687.5 880.5 2.0e6 550.7 566.8 673.0 823.5 948.6 580.7 755.0 838.7 989.4 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 App ID 23918 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 Adenosylcobalamin 1.4e6 1.3e6 Product of 790.6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 801.6 3.0e5 603.5 2.0e5 1.0e5 234.9 362.8 728.7 971.4 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 791.0 I n t e n s i t y, c p s I n t e n s i t y, c p s I n t e n s i t y, c p s 4.2e6 4.0e6 3.8e6 3.6e6 3.4e6 3.2e6 3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 676.5 665.5 358.9 971.4 147.0 1183.3 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 358.9 636.0 147.3 971.4 665.3 912.3 Adenosylcobalamin Product of 801.4 Adenosylcobalamin Product of 665.5 2.0e5 1.5e5 1.0e5 5.0e4 341.0 486.2 184.1 278.8 457.0 528.8 930.8 1125.3 1183.2 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 App ID 23919 Page 4 of 8
Table 1. Mass Transitions ID Q1 Mass (Da Q3 Mass (Da Dwell (msec DP CE MeB 12 1 673.0 665.5 50 50 25 MeB 12 2 673.0 971.5 50 50 38 CNB 12 1 678.5 359.0 50 90 32 CNB 12 2 678.5 997.5 50 90 28 AdoB 12 1* 801.5 676.3 50 70 31 AdoB 12 2 790.6 665.6 50 70 31 *An adduct ion [M+H + +Na + ]/2 Figure 6. Representative Chromatogram of Blank Sample 880 850 800 750 700 650 600 550 500 450 400 350 300 250 200 150 100 50 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min App ID 23920 For additional technical notes, visit www.phenomenex.com Page 5 of 8
Figure 7. Representative Chromatogram of 15 ng/ml standard 9370 9000 3 8500 8000 7500 7000 1. Cyanocobalamin 2. Adenosylcobalamin 3. Methylcobalamin 6500 6000 5500 5000 4500 4000 1 2 3500 3000 2500 2000 1500 1000 500 0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min App ID 23921 Figure 8. Representative Methylcobalamin Calibration Curve 1.3e5 1.2e5 1.1e5 1.0e5 Curve range: 1.5-50 ng/ml r = 0.9992 Area, counts 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 Concentration, ng/ml Page 6 of 8
Table 2. Assay Reproducibility at LLOQ (1.5 ng/ml Sample 3 B12s at 1.50 ng/ml Adenosylcobalamin Cyanocobalamin Methycobalamin Peak Area Peak Height RT (min Peak Area Peak Height RT (min Peak Area Peak Height RT (min Injection 1 2264 443.8 2.512 2421 491.0 2.469 3104 628.0 2.618 Injection 2 2428 479.8 2.509 2094 401.3 2.471 3450 662.9 2.622 Injection 3 2401 485.6 2.513 2034 419.0 2.468 3314 691.0 2.621 Injection 4 2361 439.0 2.512 2372 450.5 2.474 3341 659.2 2.617 Injection 5 2447 466.1 2.508 2298 428.2 2.466 3391 765.8 2.610 Injection 6 2824 601.2 2.505 2061 392.7 2.466 3382 650.6 2.614 Injection 7 2072 369.4 2.507 2087 403.3 2.462 3486 740.8 2.612 Injection 8 2322 425.0 2.512 2167 463.4 2.473 3268 618.7 2.619 Injection 9 2481 441.2 2.52 2618 548.0 2.473 3462 713.5 2.622 Injection 10 2517 507.8 2.516 2236 448.0 2.473 3359 684.4 2.622 AVG 2412 466 2.51 2239 445 2.47 3356 681 2.62 SD 193 61 0 189 48 0 112 48 0 %RSD 8.00% 13.09% 0.18% 8.44% 10.72% 0.16% 3.33% 6.98% 0.17% Results and Discussion All monitored cobalamins showed instability at the mass spectrometer source. TFA added to the mobile phase is reported to increase ionization and peak shape, but TFA is not a suitable for mass spectrometry. A 10 mm ammonium formate at ph 3.5 mobile phase was used to effectively ionize the analytes while providing sufficient separation and sensitivity, (Figure 2. The assay was run on a Phenomenex Synergi Fusion-RP 2.5 µm 50 x 2.0 mm column which provided reasonable selectivity. During method development, all cobalamins showed poor response at [M+H + ]. It was found that cobalamins are often multiply charged and/or form strong adduct ions. Furthermore, insource fragmentation can result in the loss of the β-axial ligand leading to adjusted parent masses (Table 1. This fragmentation produces identical parent ions for MeB 12 and AdoB 12 at m/z of 665.2. To better understand the mass selectivity, please refer to all compound tuning files (Figures 3-5. Conclusion In this technical note, a quantitative method for the analysis of cyanocobalamin, methylcobalamin and adenosylcobalamin useful for the analysis of dietary supplements was developed. This assay was successfully evaluated and transferred to a customer lab. References 1. Shawn C. Owen, Manfai Lee and Charles B. Grissom. Ultra-Performance Liquid Chromatographic Separation and Mass Spectrometric Quantitation of Physiologic Cobalamins. J. Chromatographic Science, Vol 49: 228-233 (2011. The assay was further evaluated by Genysis Lab to assess the dynamic range and the assay reproducibility at the LLOQ (lower limit of quantitation level. The dynamic range of all three cobalamins was 1.5 50 ng/ml (Figure 8. Representative chromatograms are presented in Figures 6 and 7. The reproducibility was assessed by injecting LLOQ standards 10 times and tracking the peak area, peak height and retention times for each analyte. For additional technical notes, visit www.phenomenex.com Page 7 of 8
APPLICATIONS Ordering Information Synergi Fusion-RP HPLC Columns 2.5 µm High Speed Technology (HST Columns (mm Phase 30 x 2.0 50 x 2.0 100 x 2.0 50 x 3.0 100 x 3.0 50 x 4.6 Fusion-RP 00A-4423-B0 00B-4423-B0 00D-4423-B0 00B-4423-Y0 00D-4423-Y0 00B-4423-E0 4 μm Capillary Columns (mm Guard Column (mm Phase 50 x 0.30 150 x 0.30 150 x 0.50 20 x 0.30 Fusion-RP 00B-4424-AC 00F-4424-AC 00F-4424-AF 03M-4424-AC 4 µm Microbore and Minibore Columns (mm SecurityGuard Cartridges (mm Phase 50 x 1.0 150 x 1.0 30 x 2.0 50 x 2.0 75 x 2.0 150 x 2.0 250 x 2.0 4 x 2.0* Fusion-RP 00B-4424-A0 00F-4424-A0 00A-4424-B0 00B-4424-B0 00C-4424-B0 00F-4424-B0 00G-4424-B0 AJ0-7556 for ID: 2.0-3.0 mm 4 µm MidBore Columns (mm SecurityGuard Cartridges Cartriges (mm Phase 50 x 3.0 150 x 3.0 250 x 3.0 4 x 2.0* Fusion-RP 00B-4424-Y0 00F-4424-Y0 00G-4424-Y0 AJ0-7556 for ID: 2.0-3.0 mm 4 µm Analytical Columns (mm SecurityGuard Cartridges (mm Phase 50 x 4.6 75 x 4.6 150 x 4.6 250 x 4.6 4 x 3.0* Fusion-RP 00B-4424-E0 00C-4424-E0 00F-4424-E0 00G-4424-E0 AJ0-7557 for ID: 3.2-8.0 mm * SecurityGuard Analytical cartridges require holder, Part No.: KJ0-4282 Australia t: +61 (02-9428-6444 f: +61 (02-9428-6445 auinfo@phenomenex.com Austria t: +43 (01-319-1301 f: +43 (01-319-1300 anfrage@phenomenex.com Germany t: +49 (06021-58830-0 f: +49 (06021-58830-11 anfrage@phenomenex.com India t: +91 (040-3012 2400 f: +91 (040-3012 2411 indiainfo@phenomenex.com Norway t: +47 810 02 005 f: +45 4810 6265 nordicinfo@phenomenex.com Puerto Rico t: +1 (800 541-HPLC f: +1 (310 328-7768 info@phenomenex.com Belgium t: +32 (02 503 4015 (French t: +32 (02 511 8666 (Dutch f: +31 (030-2383749 beinfo@phenomenex.com Canada t: +1 (800 543-3681 f: +1 (310 328-7768 info@phenomenex.com China t: +86 (022 2532-1032 f: +86 (022 2532-1033 chinainfo@phenomenex.com Denmark t: +45 4824 8048 f: +45 4810 6265 nordicinfo@phenomenex.com Finland t: +358 (09 4789 0063 f: +45 4810 6265 nordicinfo@phenomenex.com France t: +33 (01 30 09 21 10 f: +33 (01 30 09 21 11 franceinfo@phenomenex.com www.phenomenex.com Phenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at international@phenomenex.com Page 8 of 8 Ireland t: +353 (01 247 5405 f: +44 1625-501796 eireinfo@phenomenex.com Italy t: +39 051 6327511 f: +39 051 6327555 italiainfo@phenomenex.com Luxembourg t: +31 (030-2418700 f: +31 (030-2383749 nlinfo@phenomenex.com Mexico t: 01-800-844-5226 f: 001-310-328-7768 tecnicomx@phenomenex.com The Netherlands t: +31 (030-2418700 f: +31 (030-2383749 nlinfo@phenomenex.com New Zealand t: +64 (09-4780951 f: +64 (09-4780952 nzinfo@phenomenex.com Spain t: +34 91-413-8613 f: +34 91-413-2290 espinfo@phenomenex.com Sweden t: +46 (08 611 6950 f: +45 4810 6265 nordicinfo@phenomenex.com United Kingdom t: +44 (01625-501367 f: +44 (01625-501796 ukinfo@phenomenex.com USA t: +1 (310 212-0555 f: +1 (310 328-7768 info@phenomenex.com All other countries Corporate Office USA t: +1 (310 212-0555 f: +1 (310 328-7768 info@phenomenex.com 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 comparable dimensions, return the product with comparative data within 45 days for a FULL REFUND. Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/termsandconditions. Trademarks Synergi, SecurityGuard, and MidBore are trademarks of Phenomenex. Triple Quad is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license. SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362 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. 2016 Phenomenex, Inc. All rights reserved. TN47721116_W