Study On Determination Of Potassium Sodium Hydrogen Citrate Granules In Human Urinary And Pharmacokinetic In Healthy Chinese Volunteers

Objective:1. To establish a high specificity and sensitivity HPLC-MS methods for determination of citrate concentration in human urine.2. To study the pharmacokinetics of potassium sodium hydrogen citrate granules in healthy volunteers and evaluate excretion kinetics characteristics, which may1) provide evidence for its scientific and reasonable clinical dosing regimen, The bioavailability of two potassium sodium hydrogen citrate granules, provide test data for the examination and approval of new drugs;2) evaluate bioequivalence between test and reference formulations;3) provide test data for new drug approval.Methods:1. Urine samples were disposed and determined by HPLC-MS and with acetaminophen as internal standard. Chromatographic conditions:with1%formic acid-methanol (90:10, V/V) as the mobile phase; using Diamonsil C18column (150mm×4.6mm,5μm) separation; flow rate was0.6mL·min-1; column temperature was maintained at25℃; injection volume was10μL. MS conditions:AP-ESI ion source, the positive ion mode detection, dry gas (N2) pressure and temperature were50psi,350℃, drying gas flow rate11.0L·min-1, capillary voltage was4000V, quantitative analysis for positive ions [M+H]+:citrate m/zl90.9, internal standard (acetaminophen) m/s150.1, debris voltages were70V and90V.2. Thirty-six healthy male volunteers were randomly administered with oral single dose of0.5g test and reference preparations of potassium sodium hydrogen citrate granules in a crossover study with a washout period of7days. Urine was collected before or after oral doses at the scheduled time (0-2,2-4,4-6,6-8,8-10,10-12,12-24h,24-36h,36-48h) and the urine volumes were recorded. Repeat test1week later. Urinary citrate concentration was determined by HPLC-MS method. And48-h accumulative urinary excretion of citrate was calculated before and after administration.Results:1. A good linearity of citrate concentration in human urine was observed in the range of10～600μgmL～(-1)(r=0.99876), with the low limit of quantization was 10μg·mL～(-1). The absolute recovery with different urinary citrate concentration samples was≥92%. Intra-day and inter-day RSD were both<15%. Citrate in urine was stable stored at-20℃for24h,7-day,27-day and two freeze-thawing cycles later.2.36healthy volunteers were administered the test and reference preparations of5.0g potassium sodium hydrogen citrate granules respectively for a single dose.8h cumulative urinary citrate excretion were:were(264,84±333,77)and(416.50±409.49) mg before and after taking test preparation;(265.84±392.24) and (400.98±434.15) mg before and after taking reference preparation, respectively.Conclusions:1. The HPLC-MS method developed in this paper was sensitive, accurate and with high selectivity and good specificity, in addition, it is easy to use and suitable for determination of citrate concentration in human urine.2.36healthy volunteers were administered the test and reference preparations of5.0g potassium sodium hydrogen citrate granules respectively.48h cumulative urinary citrate excretion increased (151.66±144.78) and (135.14±142.66) mg respectively, compared with before administration. The incremental is no significant difference (p>0.05) by paired t-test and there is bioequivalent between test and reference preparations.