Technical note: Development and validation of a method using ultra performance liquid chromatography coupled with tandem mass spectrometry for determination of vitamin B12 concentrations in milk and dairy products

Abstract

A method using ultra performance liquid chromatography coupled with tandem mass spectrometry was developed to measure cobalamins in naturally enriched raw milk and to evaluate their fate during thermal treatments and along the process of cheese making. After addition of methotrexate as internal standard, samples were submitted to heat treatment in the presence of cyanide, which converts all the less-stable cobalamins into cyanocobalamin; then, purification was performed by a solid-phase extraction step. Reverse-phase ultra performance liquid chromatography separation coupled with tandem mass spectrometry provided a fast and reliable determination. Mass spectrometric analysis was carried out in multiple reaction monitoring mode. The monitored transitions were m/z 678.36 → 147.10 and 678.36 → 359.30 for vitamin B12 and m/z 455.22 → 175.13 and 455.22 → 308.22 for methotrexate (internal standard). The limit of quantification was 2 ng/g. The method showed good linearity from 2 to 20 ng/g (R(2) ≥ 0.98) and intra- and interday precisions were always less than 19%.

Determination of vitamin B12 in dairy products by ultra performance liquid chromatography-tandem mass spectrometry

“…The extraction procedure was conducted on 5 g of the various matrices adding sodium acetate buffer and potassium cyanide; samples were submitted to heat treatment that, in presence of cyanide, converts all the less stable cobalamins in cyanocobalamin (Zironi et al ., 2013 ). For solid matrices it has been necessary to introduce a centrifugation step before solid phase extraction (SPE) clean-up, in order to obtain a better separation between the liquid phase and the solid residue.…”

“…Since milk and dairy products provide a significant dietary cobalamin intake, an ultra performance liquid chromatography-tandem mass spectrometry method (Zironi et al ., 2013 ) was applied to samples collected at several stages along the process of cheese making in order to evaluate the distribution of this molecule in different dairy products.…”

Vitamin B12 determination in milk, whey and different by-products of ricotta cheese production by ultra performance liquid chromatography coupled with tandem mass spectrometry

“…Vitamin B12 determination was performed in accordance with the protocol described by Zironi et al , 2013 , with some modifications due to the different matrices, in particular the solid ones (ricotta).…”

“…A preliminary centrifugation step by 10 minutes at 6000 rpm regarding raw milk and other liquid phase matrices was conducted, in order to remove the fat content. Thus 5 mL or 5 g of matrix were added to 5 mL of sodium acetate buffer (200 mM, adjusted to pH 4 with glacial acetic acid) and 150 μL of 1% potassium cyanide solution in water; finally, 30 μL of methotrexate was also added as internal standard for quantification (Zironi et al , 2013 ).…”

Risk factors for Hyperhomocysteinemia for Specific MTHFR C677T Genotypes and Gender in Chinese Population.

“…Folate concentration was measured using a dual count Solid Phase Boil Radio assay (Diagnostic Products, Los Angeles, CA). The vitamin (Vit) B12 was detected by liquid chromatography-tandem mass spectrometry (18). The blood glucose levels were measured using the hexokinase method; serum levels of bilirubin, Alanine aminotransferase (ALT) and…”