Establishment Of LC-MS/MS And LC-MS Method For Quantitative Detection Of Vitamin B₆ In Foods

Vitamin B₆（B₆）includes pyridoxamine（PM）,pyridoxal（PL）,pyridoxine（PN）,and their respective phosphate forms 5’-pyridine phosphate Pyridoxamine（PMP）,5’-pyridoxal phosphate（PLP）and 5’-pyridoxine phosphate（PNP）.B₆ is widely present in various types of food,but in the latest version of"China Food Composition",there is no B₆ data.The reason is that our country has not established a reliable official method to determine the content of B₆in natural foods.In addition,various special complex diets continue to appear today,and the possibility of matrix interference will further affect the accuracy of high-performance liquid chromatography（HPLC）method in the current GB 5009.154-2016"China Food Safety Standard for Determination of Vitamin B₆".Therefore,in the existing GB 5009.154-2016,it is urgent to add a method with higher sensitivity,stronger specificity,more accurate and reliable.Based on the above background and commissioned by the National Standards Committee,GB 5009.154-2016 was revised to establish isotope dilution liquid chromatography-tandem mass spectrometry（ID-LC-MS/MS）and isotope dilution liquid chromatography-mass spectrometry（ID-LC-MS）method applied to the determination of B₆ in various foods（natural food+fortified food）and fortified food,respectively.Its significance lies mainly in the improvement of the existing national standards and the support of the detection method for the establishment of the B₆ database in Chinese food.The main research results of this topic are as follows:The ID-LC-MS/MS method and the ID-LC-MS method were initially established,which were used to determine the content of B₆ in various foods（natural food+fortified food）and fortified food,respectively.In terms of optimizing the chromatographic conditions,the columns selected by the two methods were HSS PFP,3.0×150 mm,1.8μm,and the mobile phase was 2%formic acid+10 mmol/L ammonium formate and 0.1%formic acid+methanol solution.In terms of mass spectrometry,the former method uses triple quadrupole mass spectrometry multiple reaction monitoring（MRM）mode scanning to obtain quantitative ions of PM,PL,PN and their corresponding isotope internal standards PM-IIS,PL-IIS,PN-IIS.They are 169.0→134.0,168.0→150.0,170.0→134.0,172.0→136.0,171.0→153.0,174.0→138.0.The latter method uses single quadrupole mass spectrometry selective ion reaction（SIR）monitoring mode scanning,and the monitoring ions of the above six target objects are 169.1,168.1,170.1,172.2,171.1,and 175.2,respectively.In determining the extraction of B₆（converting the combined state to the free state）,the natural food is treated by acid hydrolysis+acid phosphatase enzymatic hydrolysis overnight.For the fortified food,the pure water is used for direct treatment.The linearity of the former（0.5～50 ng/m L）and the latter（10～250 ng/m L）method both performed ideally.The correlation coefficient of each test was>0.999.The actual sample test results show that the recovery rate of the total amount of B₆ under the two methods is within 88.54～106.24%,indicating a high accuracy.In terms of method precision,the RSD of PL and PN is always<10%,but the PM is sometimes>10%.The sample solution was stored at 4°C for 7 days and tested again,the content did not change significantly.The limit of quantification of the former method can meet the detection requirements of lower content of B₆ in natural food.For fortified food,the two methods of quantitation limit can meet the detection requirements of the lowest content of B₆.From the matrix effect（ME）level,the ME of PM,PL,PN is mostly between 80%and 105%,indicating that the ME is negligible.Finally,the quantitative results of ID-LC-MS/MS,ID-LC-MS and HPLC methods show that the measurement results between different methods are comparable.