Quantification of B-vitamins from different fresh milk samples using ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring methods.

Absolute quantification of B-vitamins in milk is becoming imperative to correlate its impact on child/human health. In today's world of changing food habits and environmental pollution, there is concern if milk is what we think it to be. In the present work, ultra-high performance liquid chromatography mass spectrometry/selected reaction monitoring (UPHPLC-MS/SRM) methods have been developed and validated for quantitative estimation of 21 different B-vitamins [B1-3, B2-3, B3-2, B5-1, B6-5, B8-1, B9-2 vitamins, total choline (betaine, choline and acetylcholine)] from a drop (50 µL) and B12 from 500 µL of milk. These two methods qualify all essential bio-analytical parameters (recovery >80%, accuracy <±15%, variation <±10%) and are highly reproducible. By using these developed methods, B-vitamins in different fresh milk samples from cow, goat, buffalo and pasteurized cow milk (each 10 and total n = 40) were analysed. Total choline is the highest (6.5-10.5 µg/mL) and vitamin B8 is the lowest (16.1-32.9 ng/mL) in all milk samples. Inverse correlation between vitamin B2 and B5 was observed in milk samples. The ratio of vitamin B5/B2 was checked in all milk samples, it is higher in cow (2.64), equal in goat (1.04) and lower in buffalo (0.42) milk. Total B-vitamin content in cow milk is higher (10.5 µg/mL) compared to other three (goat-7.2, buffalo-6.5, pasteurized-8.8 µg/mL). Vitamin B12 is higher in cow milk (3.6 ng/mL) compared to other two fresh milk samples. Different isomers for vitamin B6 were noticed in the fresh milk samples. The complete profile of water soluble vitamins and the ratio of two abundant B vitamins (B5/B2) in milk will be useful to check the nutritional quality and to differentiate the kind of animal milk.