TN-1208 APPLICATIONS

Xianrong (Jenny) Wei Senior Scientist Separation and Quantitation of Physiologic PhenoLogix applications laboratory. Cobalamins (Vitamin B12) in Dietary Supplements by LC/MS/MS using Synergi™ Fusion-RP Column

Xianrong (Jenny) Wei1, Michael Landesman2, Spencer Carter2, and Sean Orlowicz1 1. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 2. Genysis Lab, 391 S Orange Street Suite F, Salt Lake City, UT 84104

LC/MS/MS Method Parameters Analysis of cobalamins (Vitamin B12 ) on reversed phase columns can be challenging due to the relative chemical instability of the Column: Synergi™ 2.5 µm Fusion-RP analyte, the complexity of matrix effects and the existence of sev- Dimensions: 50 x 2.0 mm Part No.: 00B-4423-B0 eral vitamer forms of B . In this tech note, we describe a selective, 12 SecurityGuard™ Cartridge: AJ0-7844 efficient and reproducible method to separate three B12 vitamers Mobile Phase: A: 10 mM Ammonium Formate pH 3.5 (methylcobalamin, cyanocobalamin and adenosylcobalamin) ap- B: Methanol propriate for the quantitative analysis of dietary supplements via Gradient: Time (min) B (%) LC/MS/MS. 0 2 0.2 2 1.8 98 Introduction 4 98 Cobalamin is a micronutrient that animals and humans obtain 4.01 2 through diet, symbiotic bacteria or vitamin supplements. Vitamin 5.5 2 Flow Rate: 350 µL/min B12 deficiency leads to a degeneration of the sensory in the spinal cord with loss of sensation and paralysis that results in a com- Injection Volume: 10 µL plex spectrum of pathologies, including pernicious anemia, meg- Temperature: 21 C° aloblastic anemia, peripheral neuropathy, depression, impaired Instrument: Agilent 1260 LC cognition, and autoimmune dysfunction. The four vitamer forms Detection: MS/MS (ESI+) (SCIEX Triple Quad™ 4500 and SCIEX 4000 QTRAP) @ 600 ºC of B12 are differentiated based upon the functional group (R) at Sample: 1. Cyanocobalamin (CNB ) the -axial position (Figure 1). The two forms found naturally in 12 β 2. Adenosylcobalamin (AdoB12)

the body are adenosylcobalamin (AdoB12) and methylcobalamin 3. Methylcobalamin (MeB12)

(MeB12), while the two most common vitamin forms are hydroxy-

cobalamin (HOB12) and cyanocobalamin (CNB12). Due to its stabil- ity advantages, cyanocobalamin is the vitamer form that is most Figure 1. Cobalamin Structure commonly used in supplements.

Historically, cobalamin analysis has been performed using mi- crobiological methods that are known to have significant short- comings 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 (AdoB12,

MeB12 and CNB12) that are commonly seen in dietary supplements. We have established a sensitive, selective and reproducible quan- titation method for the nutraceutical industry utilizing HPLC sepa- ration coupled to a triple quadrupole mass spectrometer.

Experimental Conditions Reagent and chemicals All solvents and reagents were HPLC or analytical grade. Refer- ence 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.

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Figure 2. Cobalamin Mobile Phase pH Selection Chart

2.22 Mobile phase at pH 3.5

1.2

Charge 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

Max. 7.4e7 cps. 673.0 [M+2H+] 7.4e7 7.0e7

6.5e7

6.0e7

5.5e7 Methylcobalamin (MeB12) 5.0e7 s p

c 684.0 4.5e7 , y t i

s 4.0e7 n e

t 3.5e7 n

I 695.0 3.0e7

2.5e7 [M-ME+2H+] 2.0e7

1.5e7

1.0e7 665.5 687.5 674.5 762.9 5.0e6 541.0 622.9 700.0 774.0 797.0 831.0 989.51004.5 527.0 882.8 1366.6

0.0 609.2 App ID 23914 23915 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, Da

665.5 1.4e7 1.3e7 1.2e7 1.1e7 Methylcobalamin s 1.0e7 p c

9.0e6 , Product of 673 y t i 8.0e6 s n

e 7.0e6 t n I 6.0e6 5.0e6 4.0e6 673.0 3.0e6 971.5 2.0e6 358.9 1.0e6 147.1 1183.4 0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, Da 665.4 1.27e6 1.20e6 1.10e6 1.00e6 Methylcobalamin

s 9.00e5 p Product of 665.4 c

, 8.00e5 y t i

s 7.00e5 n e

t 6.00e5 n I 5.00e5 358.9 4.00e5 636.2 971.4 3.00e5 2.00e5 147.1 1.00e5 1183.4 340.8 486.3 913.1 1125.4

0.00 App ID 23915 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, Da

Page 2 of 8 TN-1208

Figure 4. Tuning files _ Cyanocobalamin (m.w. 1354.5) by infusion

678.5 [M+2H+] 1.5e7

1.4e7

1.3e7

1.2e7

1.1e7 Cyanocobalamin (CNB12) 1.0e7

9.0e6 s p c

, 8.0e6

y 604.3 t i s

n 7.0e6 e t n

I 689.5 6.0e6 602.2 709.3 5.0e6

4.0e6 609.5 720.3 700.5 + 3.0e6 [M+H ] 697.0 740.5 2.0e6 562.9 616.6657.1 777.8 728.6 845.7 858.5 660.6 903.7 1.0e6 997.4 1023.5 1355.4 638.7 1078.5 1144.5 1206.41281.5 1377.4 1428.2 0.0 App ID 23916 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 1500 m/z, Da

359.0 1.0e6 9.5e5 9.0e5 Cyanocobalamin 8.5e5 Product of 678.5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 s p

c 5.5e5 147.0

, y t i s

n 5.0e5 e t n I 4.5e5 678.4 997.5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 636.0 1.5e5 912.6 340.6 1.0e5 1209.6 664.7 457.0 5.0e4 184.9 278.8 499.2 725.2 833.7 930.6 1015.3 1124.5

0.0 App ID 23917 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z, Da

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Figure 5. Tuning files _ Adenosylcobalamin (m.w. 1579.58) by infusion

790.6 [M+2H+] 2.9e7 2.8e7

2.6e7

Adenosylcobalamin (AdoB12) 2.4e7

2.2e7

2.0e7

1.8e7 s p c

1.6e7 + , [M-Ado+2H ] + + y 801.5 [M+H Na ] t i s 1.4e7 n e t n I 1.2e7 665.5 + 1.0e7 812.5 [M+2Na ]

8.0e6

6.0e6 527.5 4.0e6 676.5 687.5 880.5 2.0e6 566.8 673.0 755.0 823.5 948.6 550.7 580.7 838.7 989.4 0.0 App ID 23918 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z, Da

676.5 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 Adenosylcobalamin

s 1.4e6

p 1.3e6 Product of 790.6 c

1.2e6

, 1.1e6 y

t 1.0e6 i 9.0e5 s

n 8.0e5

e 7.0e5 t 6.0e5 n

I 5.0e5 4.0e5 801.6 3.0e5 603.5 2.0e5 728.7 1.0e5 234.9 362.8 971.4 0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z, Da 791.0 4.2e6 4.0e6 3.8e6 3.6e6 3.4e6 3.2e6 3.0e6 s 2.8e6 p 2.6e6 c 665.5 Adenosylcobalamin 2.4e6 ,

y 2.2e6 Product of 801.4 t

i 2.0e6

s 1.8e6 n 1.6e6 e

t 1.4e6

n 1.2e6 I 1.0e6 8.0e5 6.0e5 4.0e5 358.9 971.4 2.0e5 147.0 1183.3 0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 358.9 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 s 636.0 p 5.5e5 Adenosylcobalamin c

5.0e5 , Product of 665.5 y 4.5e5 t

i 4.0e5 s 147.3 n 3.5e5 665.3 971.4 e 3.0e5 t

n 2.5e5 912.3 I 2.0e5 1.5e5 1.0e5 341.0 486.2 1125.3 1183.2 5.0e4 184.1 278.8 457.0 528.8 930.8

0.0 App ID 23919 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 m/z, Da

Page 4 of 8 TN-1208

Table 1. Mass Transitions ID Q1 Mass (Da) Q3 Mass (Da) Dwell (msec) DP CE

MeB12 1 673.0 665.5 50 50 25

MeB12 2 673.0 971.5 50 50 38

CNB12 1 678.5 359.0 50 90 32

CNB12 2 678.5 997.5 50 90 28

AdoB12 1* 801.5 676.3 50 70 31

AdoB12 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 s p

c 500

, y t i 450 s n e t 400 n I 350 300 250 200 150 100 50

0 App ID 23920 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

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23121 Figure 7. Representative Chromatogram of 15 ng/mL standard

3 9370 9000

8500

8000

7500 1. Cyanocobalamin 2. Adenosylcobalamin 7000 3. Methylcobalamin

6500

6000 2 s

p 5500 c

,

y 1

t 5000 i s n e

t 4500 n I 4000

3500

3000

2500

2000

1500

1000

500

0 App ID 23921 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 min

Figure 8. Representative Methylcobalamin Calibration Curve

1.3e5

1.2e5

1.1e5 Curve range: 1.5 - 50 ng/mL 1.0e5 r = 0.9992

9.0e4

8.0e4 s t n u

o 7.0e4 c

, a

e 6.0e4 r A 5.0e4

4.0e4

3.0e4

2.0e4

1.0e4

0.0 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 TN-1208

Table 2. Assay Reproducibility at LLOQ (1.5 ng/mL)

Sample Adenosylcobalamin Cyanocobalamin Methycobalamin 3 B12s at 1.50 ng/mL 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 0.00 189 48 0.00 112 48 0.00 %RSD 8.00% 13.09% 0.18% 8.44% 10.72% 0.16% 3.33% 6.98% 0.17%

Results and Discussion Conclusion All monitored cobalamins showed instability at the mass spec- In this technical note, a quantitative method for the analysis of cy- trometer source. TFA added to the mobile phase is reported to anocobalamin, methylcobalamin and adenosylcobalamin useful increase ionization and peak shape, but TFA is not a suitable for for the analysis of dietary supplements was developed. This as- mass spectrometry. A 10 mM ammonium formate at pH 3.5 mo- say was successfully evaluated and transferred to a customer lab. bile phase was used to effectively ionize the analytes while pro- viding sufficient separation and sensitivity, Figure( 2). The assay References was run on a Phenomenex Synergi Fusion-RP 2.5 µm 50 x 2.0 mm column which provided reasonable selectivity. 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 During method development, all cobalamins showed poor re- (2011). sponse 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 MeB12 and AdoB12 at m/z of 665.2. To bet- ter understand the mass selectivity, please refer to all compound tuning files Figures( 3-5).

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 cobal- amins was 1.5 – 50 ng/mL (Figure 8). Representative chromato- grams 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.

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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) SecurityGuardSecurityGuard 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

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