A Sensitive and Specific and Comprehensive Assay to Quantify Methylcobalamin, Cyanocobalamin and Adenosylcobalamin in Dietary Supplements using LC/MS/MS



Vitamin B12, also called as cobalamin, has different forms like cyanocobalamin (cB12), methylcobalamin (mB12) and adenosylcobalamin (cobamamide) and plays a key role in the normal functioning of the brain and nervous system. These vitamin B12 present analytical challenges due to it’s unique chemical properties and low concentration in dietary supplements. To our knowledge, there is no published ultra-sensitive LC/MS/MS method to quantitate B12 in finished goods. In 2017, AOAC has an open call for a quantitative vitamin B12 assay in dietary supplement. Here we report a comprehensive, sensitive and specific LC-MS/MS assay for cB12, mB12 and cobamamide (Figure 1) in dietary supplements.


Sample Preparation and Extraction:

About 2 g of sample was extracted with water and methanol. The extracted sample was cleaned by 0.20 μM filtration before analysis.

UPLC‐MS Conditions

UPLC system: Nexera UPLC system including SIL‐30AC auto‐sampler, controller, column heater and binary pump (Shimadzu)

Column: 100×2.1 mm, 1.6 μm CORTECS C18 (Waters)

Mobile Phase A: Formic Acid in water

Mobile Phase B: Formic Acid in acetonitrile

Flow rate: 0.40 mL/min

Pump Gradient Cycle time: 6.0 minutes

MS detector: Triple Quadrupole 4000 MS (AB Sciex)

Results and Discussions

Stability: During method development, standard solution stability was evaluated, and the data indicated that B12 is very sensitive to light and temperature. In order to minimize the effect of the light and temperature, the sample is processed without light exposure at reduced temperature conditions (Figure 2). With light protection and storage at 1−8 oC, the standard solutions and extracted sample are stable for up to 9 days.

Specificity: The specificity results indicated that there is no interference between analyte and IS and the method is specific and the blank sample has no interference at analyte and IS expected retention time.

Linearity: The curve range of 2.00-200 ng/mL was successfully validated. The regression is linear with 1/x as the weighing factor. The Correlation Coefficient R2 is > 0.995. The % variance (deviation from nominal concentration) of LLOQ was 11.1% and all other points on the curve do not exceed 5.00%.

Accuracy and Precision: The accuracy and precision were investigated with post-spiking cobalamin in placebo matrix at lower, medium and high regions of the established range of the calibration curve.


This is the first known published LC‐MS/MS assay for quantification of cB12, mB12 and cobamamide in dietary supplements.