| Low protein diet with keto acid supplementation is one of the important diet treatments of chronic renal failure(CRF).It can modify the body's amino acids and protein metabolic disturbance,so as to delay chronic renal failure.a-keto acid as precursor of the amino acid is transformed into the corresponding L-amino acids by transamination in the body,which provides raw materials for protein synthesis and reduces urea generated at the same time,relieving symptoms of uremia.Moreover,keto calcium salt supplementation can increase the intake of calcium.Compound a-ketoacid tablet is a medicine which contains five keto-or hydroxy-amino acids in the form of calcium salts including D,L-?-Hydroxymethionine calcium(HMACa),D,L-?-ketoisoleucine calcium(KILCa),a-ketovaline calcium(KVCa),?-ketoleucine calcium(KLCa)and a-ketophenylalanine calcium(KPACa).Due to its complicated composition,like high polarity and so on,it is difficult to carry out quantitative analysis for a-ketoacid tablet by general performance liquid chromatography.To date,there have been a few methods being reported for the simultaneous determination of these five analytes.As a new sample pretreatment technology,hollow fiber liquid-phase microextraction(HF-LPME)has been environmentally-friendly and efficient technology in many fields,such as food,biological samples and environmental samples in recent years.This method possesses the advantages of traditional three phases LPME,such as high enrichment factor,less organic solvent consumption.HF-LPME has been widely used in sample pretreatment for gas chromatography(GC),liquid chromatography(LC)and capillary electrophoresis(CE),but rarely for ion pair chromatography(IPC).In this work,application of HF-LPME combined with IPC in determination of a-keto acid was investigated.The main work and achievement of this thesis are as follows:1.A method by ion-pair HPLC-DAD was developed for the content determination of HMACa,KVCa,KILCa,KLCa and KPACa in compound a-ketoacid tablet.These analytes were separated on C18 column(250 mm×4.6 mm,5?m),the mobile phase was acetonitrile-20 mM ammonium acetate buffer(containing 30 mM n-amylamine,and pH 7.0)at a flow rate of 0.6 mL/min with gradient elution,and the column temperature was 35?.Good results were obtained under the optimum conditions:the linear range was 20-150 mg/L,and the linear correlation coefficient was 0.9994,1,1,0.9999 and 0.9999,respectively.The limits of detection(LODs)were 4.8,4.06,2.4,4 and 1.5 mg/L(based on S/N=3),respectively.The spike recoveries were ranged from 92.85.45%to 103.2%and the RSD%was less than 3.42%(n=5).This method was proved to be rapid,accurate and sensitive,proving an effective way to detect five analytes in compound a-ketoacid tablet and related medicines.2.A method by ion-pair HPLC-ELSD was developed for the content determination of HMACa,KVCa,KILCa,KLCa and KPACa in compound a-ketoacid tablet.These analytes were separated on C18 column(250 mm x 4.6 mm,5 the mobile phase was acetonitrile-20 mM ammonium acetate buffer(containing 30 mM n-amylamine,and pH 7.0)at a flow rate of 0.6 mL/min with gradient elution,and the column temperature was 35?.An evaporative light-scattering detector(PL-ELS2100)was used,its parameters were optimized as follows:drift tube temperature was 50?,and the flow rate of carrier gas was 1.0 mL/min.Good results were obtained under the optimum conditions:the linear range was 20-150 mg/L with r>0.999 and the limits of detection(LODs)were 7.05,16.66,8.00,8.00 and 14.28 mg/L(based on S/N=3),respectively.The spike recoveries were ranged from 87.45%to 109.82%and the RSD%was less than 3.16%(n=5).This method was proved to be rapid,accurate and sensitive,proving an effective way to detect five analytes in compound a-ketoacid tablet.3.A three-phase hollow fiber microextraction combined with ion-pair HPLC-DAD method was established for determination of five components(HMACa,KILCa,KILCa,KLCa and KPACa)in compound a-ketoacid tablet.The extraction conditions were optimized:hexyl acetate was selected as organic extracting solvent,and 6 ?KL of aqueous solution with 0.5%tetrabutylammonium hydroxide(TBAH)was selected to be acceptor solution,and then five analytes in 10 mL aqueous solution(pH 1)were extracted in 30 min of shaking(150 rpm).The analytes were separated on C18 column with the mobile phase of acetonitrile-50 mM sodium dihydrogen phosphate buffer(containing 14 mM TBAH,and pH 7.0)at a flow rate of 0.6 mL/min.The DAD detection wavelength was set at 205 nm.Under the optimal conditions,the enrichment factors were 21,175,285,400,420 respectively,and the calibration curve for these analytes was linear in the range of 0.1?10 mg/L and 0.5?7 mg/L(HMACa)with r>0.99,and the limits of detection(LODs)were 42.1,8.13,12.3,5,4?g/L(based on S/N=3),respectively.Good spike recoveries(84-108.83%)were obtained and the RSD%was less than 5.65%.The proposed method was proved to be rapid and sensitive for five target analytes in compound?-ketoacid tablet. |
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