A Specific, Sensitive and High-throughput Assay to Simultaneously Quantify Methylcobalamin, Cyanocobalamin and Cobamamide in Finished Goods using LC/MS/MS



Vitamin B12, including cyanocobalamin (cB12), methylcobalamin (mB12), adenosylcobalamin (cobamamide) and hydroxocobalamin, has a key role in the normal functioning of the brain and nervous system, and in the formation of red blood cells. Recently, mB12 is rapidly gaining popularity as the supplemental B12 form due to its higher bioavailability, longer retention time in tissue and it does not contain toxic cyanide. The Daily Value (%DV) of vitamin B12 has been revised from 6.0 μg downward to 2.4 μg as of May 2016, and its low content in supplements requires ultra-sensitive quantitation method. In this paper, we developed a specific, sensitive and highthroughput quantitative method for cB12, mB12 and cobamamide in finished goods using LC/MS/MS.


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 and water

Mobile Phase B: Formic Acid and acetonitrile

Flow rate: 0.40 mL/min

Pump Gradient Cycle time: 6.0 minutes

MS detector: Triple Quadrupole 4000 MS (AB Sciex)

results and discussion

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 (Fig. 2). Both protein and non-protein matrix samples were investigated, and it was found that the different diluents were necessary for different types of matrix. The method was successfully validated over the range of 2.00-200 ng/mL in both protein and non-protein matrix with the target concentration of sample preparation at 100 ng/mL. The specificity experiment showed that there was no significant contribution between analytes/IS and no visible interference peaks showed in blank diluent at the expected retention time (Fig. 2). The LLOQ has sufficient sensitivity (S/N> 10) (Fig. 3). System suitability consisted of six replicate injections of the middle standard solution and was injected before sample analysis, and RSD was ≤6% (Table 2). The response linearity study revealed that quadratic regression with 1/X weighing factor provides the best fit, and the correlation coefficient r2 is ≥0.995 (Fig. 4 and Table 3). The accuracy experiment showed that the spiking recovery is within ±20% (Table 4). The %RSD of precision and repeatability was <3.6% (Table 4). The standard solution stability and extracted sample stability was established for up to 19 and 3 days at 1-8°C in light protection, respectively.


This is the first known published specific, fast and high-throughput LC/MS/MS assay for quantification of cB12, mB12 and cobamamide in finished goods.