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35

TN-1208

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

Adenosylcobalamin

Cyanocobalamin

Methycobalamin

Sample 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 Injection 2 Injection 3 Injection 4 Injection 5 Injection 6 Injection 7 Injection 8 Injection 9 Injection 10

2264 2428 2401 2361 2447 2824 2072 2322 2481 2517

443.8 479.8 485.6 439.0 466.1 601.2 369.4 425.0 441.2 507.8

2.512 2.509 2.513 2.512 2.508 2.505 2.507 2.512

2421 2094 2034 2372 2298 2061 2087 2167 2618 2236

491.0 401.3 419.0 450.5 428.2 392.7 403.3 463.4 548.0 448.0

2.469 2.471 2.468 2.474 2.466 2.466 2.462 2.473 2.473 2.473

3104 3450 3314 3341 3391 3382 3486 3268 3462 3359

628.0 662.9 691.0 659.2 765.8 650.6 740.8 618.7 713.5 684.4

2.618 2.622 2.621 2.617 2.610 2.614 2.612 2.619 2.622 2.622

2.52

2.516

AVG

2412

466

2.51 0.00

2239

445

2.47 0.00

3356

681

2.62 0.00

SD

193

61

189

48

112

48

%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 spec- trometer 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 mo- bile phase was used to effectively ionize the analytes while pro- viding sufficient separation and sensitivity, ( Figure 2 ). The assay was run on a Phenomenex Synergi Fusion-RP 2.5 µm 50 x 2.0mm column which provided reasonable selectivity. During method development, all cobalamins showed poor re- 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  -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 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.

Conclusion In this technical note, a quantitative method for the analysis of cy- anocobalamin, methylcobalamin and adenosylcobalamin useful for the analysis of dietary supplements was developed. This as- say 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).

04/05/2019