Simultaneous Determination Of Five Components Of Compound ?-ketoacid Tablets In Urine By Ion-pair HPLC

In recent years,chronic kidney disease(CKD)has become the global health challenge.Low protein diet supplemented with compound?-ketoacid tablet is one of the important diet treatments of CKD Due to its complicated composition and high polarity,it is difficult to carry out quantitative analysis for a-ketoacid tablet by general high performance liquid chromatography(HPLC),so ion-pair HPLC is employed in following work.The pretreatment of body fluid samples,which have complex matrix,is usually cumbersome and the sensitivity is low.As a new sample pretreatment technology,hollow fiber liquid-phase microextraction(HF-LPME)has the advantages of simple operation,high enrichment factors and low cost,and it is easy to combine with liquid chromatography(LC),gas chromatography(GC)and so on.Based on the advantages above,HF-LPME has been widely used in the pretreatment of body fluid samples,such as urine,plasma and saliva.In this research,an ion-pair HPLC method combined with HF-LPME has been established for the simultaneous determination of five components of compound a-ketoacid tablets in human urine,which can provide some basis for the therapy of CKD.The main work and achievement of this thesis are as follows:1.A method of ion-pair HPLC-DAD was developed for the simultaneous determination of HMACa,KVCa,KILCa,KLCa and KPACa in compound a-ketoacid tablet.A mixed solution of these five standard substances was employed to optimize the concentration of ion-pair reagents,the pH value of the mob.ile phase and so on.The optimized mobile phase was acetonitrile-20 mmol/L phosphate buffer(containing 15 mmol/L TBAH,pH 7.0)(25/75,V/V)at a flow rate of 1.0 mL/min.The results showed that the linear range was 10-150 mg/L,and the linear correlation coefficient of the five components was above 0.9993.The relative standard deviation(RSD)was less than 3.63%(n=5),and the limits of detection(LODs)of these components were between 3.3 and 5.2 mg/L(based on S/N=3).The spike recoveries were ranged from 85.28%to 105.77%.This method was proved to be simple,rapid,accurate and sensitive for the quality control of compound ?-ketoacid tablet.2.A method of ion-pair HPLC-DAD was developed for the simultaneous determination of HMACa,KVCa,KILCa,KLCa and KPACa in human urine.The mobile phase was acetonitrile-20 mmol/L phosphate buffer(containing 15 mmol/L TBAH,pH 7.0)at 1.0 mL/min flow rate with gradient elution.The calibration curve for the five components was linear in the range from 20 to 200 mg/L(r>0.9990).The limits of detection(LODs)were 3.0,5.0,3.6,5.7 and 2.5 mg/L(S/N=3)for HMACa,KVCa,KILCa,KLCa and KPACa,respectively.Intra-day and inter-day precisions were less than 6.61%,and the average recoveries were between 86.79%and 111.12%in the human urine with RSDs lower than 8.62%(n=5).The method was proved to be precise and specific,and the pretreatment of biosamples was simple while the influence of endogenous substrate was small.So it was appropriate to be used for the determination of the five components in human urine.3.An optimized three-phase hollow fiber liquid-phase microextraction(HF-LPME)combined with ion-pair HPLC method was applied for the determination of the five components(HMACa,KVCa,KILCa,KLCa,KPACa)in human urine.Under optimal conditions,the obtained enrichment factors(EFs)were up to 11,110,198,202 and 50-fold,respectively.The calibration curves for these analytes were linear over the range of 0.1-10 mg/L for KVCa,KILCa and KLCa,0.5-10 mg/L for HMACa and KPACa with r>0.9903.The RSDs(n=5)were less than 6.27%and the limits of detection(LODs)were 100.7,10.0,5.8,7.8 and 8.6 ?g/L(based on S/N=3),respectively.Good recoveries from spiked urine samples(91.71-118.00%)were obtained.The proposed method demonstrated excellent sample clean-up and selectivity.It was convenient and easy to determine the low concentration of five analytes of compound ?-ketoacid tablets in human urine.