Determination of Pantothenic Acid in Food by LC-MS/MS

Direct extraction method was proposed to separate pantothenic acid from those food samples, such as milk powder, rice flour and etc.which contain pantothenic acid in the free state. In this case, 5 g of the sample were taken and 0.2 g of amylase to gether with 50 mL of water were added, and the mixture was enzymolyzed by shaking in a water bath heated at (55±5)℃ for 10 min. An aliquot equivalent to 0.5-5.0 g of the sample was taken, and 100 μL of the isotopic calcium pantothenate internal standard solution were added, and the solution was diluted to 20 mL. 0.4 mL each of the 2 precipitants, 300 g·L^-1 zinc acetate solution and 150 g·L^-1 potassium ferrocyanide solution, was added and extra-pure water was added to make its volume to 25.0 mL. After mixing, standing for 30 min and centrifuging for 2 min, the supernatant was taken and filtered through 0.22 μm filtering membrane. The filtrate was used for LC-MS/MS analysis under the instrumental working conditions. For food samples containing pantothenic acid in its combined states of acetyl coenzyme and acyl carriex protein, such as cereals, potato, meat, eggs and beans (taking 1-5 g of sample), fresh fruits and vegetables (taking 5-10 g of sample), the sample should be first hydrolyzed for 15 min in 10 mL of Tris-HCl buffer solution (pH 8.1±0.1) and 40 mL of water at 121℃ under pressure. After cooling, the solution was diluted to 100.0 mL with extrapure water and filtered on a dry-filter paper. An aliquot of 10.0 mL was taken, and after adding 100 μL of the isotopic internal standard solution and 5 mL of the Tris-HCl buffer solution, the mixture was placed into an ice-bath, and after adding 0.1 mL of 0.08 mol·L^-1 sodium carbonate solution, 0.4 mL of 0.02 g·mL^-1 alkaline phosphatese solution, 0.2 mL of 0.5 g·L^-1 pigeon liver extract and 0.4 mL of extrapure water were well mixed, where one drop of toluene was added, the reaction mixture was enzymolyzed for at least 8 h at 37℃ to liberate pantothenic acid from its combined state to free state. After the reaction was completed, the solution was diluted to 20 mL, and the produre was carried on as described in the direct extraction method starting from the addition of the 2 precipitants. Waters BEH C₁₈ column was selected as stationary phase and mixtures of (A) 1% (volum fraction) formic acid solution and (B) acetonitrile in various ratlos were used as mobile phases in gradient elution, and peak areas of pamtothenic in the respective eluants in the 2 testing solution obtained as described above were measured by MS under the prescribed condition. Linearity range of the standard curve of pantothenic acid was found between 10-1 500 μg·L^-1. Detection limit (3S/N) found was 3.0 μg·kg^-1. Recovery was tested by standard addition method, giving results of recovery in the range of 91.0%-105%. Values of RSDs (n=6) found were ranged from 0.46%-3.0%. 53 food samples (including milk powder and other foodstuffs) were analyzed by the present method and by the microbial method given in GB/T 5009.210-2016 for checking. It was found that the relative deviations between the results obtained by this method and the results given by the microbial method were less than 10%; and that on the base of the results of t-test, no significant discrepancy was found between the results of the 2 methods.