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

•For the first time 21 B-vitamins were quantified from miniscule volume of milk.•Three different fresh milk samples from cow, goat, buffalo were compared.•Total choline is the highest and vitamin-B8 is the lowest in all milk samples.•Inverse correlation between vitamin B2 and B5 was observed in milk samples.•Total B-vitamin and B12 contents are higher in cow milk compared to other two. 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