The Study On The Determination Of 18 Kinds Of AAs By PITC-HPLC Based On Oxidization With Performic Acid

Proteins,the molecular basis for the organism structures and biological activities,are composed of amino acids.Therefore,amino acids can affect the functions of proteins and thus are indispensable to life.With the increasingly rapid pace of our society,many kinds of fast food which will lead to malnutrition become more popular.Eventually,various functional foods containing amino acids are consumed to meet the nutrition desire for human health and diseases treatment.For these products,the kinds of amino acid,their contents and the ratio of each component in products are of important quality indicators.Therefore,it is necessary to establish a rapid and accurate determination method for amino acid determination.High-performance liquid chromatography(HPLC)is a commonly used detection method for amino acids.A derivation method is usually operated before HPLC analysis because most amino acids are lack of chromophores.In this paper,phenyl-isothiocyanate(PITC)was taken as derivatization reagent,a method for determination of aminio acids by HPLC was establish.The oxidation of performic acid was carried out to improve the traditional PITC derivative method,which widened the detection range by PITC.Meanwhile,This method was expected to offer some ideas for other new amino acid derivative method.Some factors affection the derivative reaction,including the PITC concentration,derivative time and temperature,were optimized to test the determination of aminio acids.Meanwhile,the effects of buffer concentration and pH of mobile phase,and column temperature on separation of 18 kinds of amino acids were investigated.The results showed that 18 kinds of amino acid were separated by using gradient elution with acetonitrile-water(80/20,v/v)as mobile phase A and 50 mmol/Lol/L pH=6.5sodium acetate-acetonitrile(93/7,v/v)buffer solution as mobile B coupling with UV detector at 246 nm and column temperature at 40?.Performic acid was prepared using formic acid oxidized by hydrogen peroxide,of which,the factors influencing this reaction,including time,temperature,and concentration of performic acid on the preparation was investigated.The bestpreparation conditions had been achieved through orthogonal experiment,which was made by 88% formic acid and 30% hydrogen peroxide at a ratio of 9:1 at room temperature,and then stand for 90 min.The results showed that cystine and methionine were completely oxidized to cysteic acid and methionine sulfone,respectively.To test the stability of oxidative products,a same stand solution was injected for six times.The result showed that their RSD(n=6)were 1.81% and 1.65%,respectively,which means the derivation products were stable and can be applied to the determination of cystine and methionine with PITC.The method was applied to determine the real samples,and the results showed that 18 kinds of amino acids had good linear correlation in 125 ?g/mL-1500 ?g/mL with R2>0.99,and the LOD was between 0.62 and 6.60 ?g/mL with the recovery of67.79% to 109.69%,which was lower when compared to traditional PITC derivative method.The results showed that the content in real sample were 0.15 g/g in compound methionine and choline bitartrate tablets,0.20 g/g in cystine tablets and40.14 mg/mL total amino acid in amino acid oral liquid,which was close to instruction book.