蛋白沉淀结合HPLC测定人血浆中尼美舒利及相对生物利用度

Determination of Nimesulide in Plasma by HPLC with Deproteinization and Its Application to Bioavailability

目的建立人血浆中尼美舒利的HPLC-UV测定法,研究两种尼美舒利胶囊的相对生物利用度。方法采用COSMOSIL C_(18)柱(4.6 mm×250 mm,5μm),柱温为30℃,流动相为乙腈-0.015 mol·L^(-1)的磷酸二氢钾缓冲液(氢氧化钠溶液调pH至7.3)(79:21),流速为1.0 mL·min^(-1),检测波长为404 nm。18名健康志愿者采用自身对照随机交叉给药方案,分别单次口服不同厂家尼美舒利胶囊0.1 g,在不同时间点取血,血浆样品以新建立的HPLC-UV测定,比较两药主要药动学参数的差异和相对生物利用度。结果尼美舒利与血浆中内源性杂质分离完全,尼美舒利在0.05～6.0 mg·L^(-1)内与峰面积比线性良好,血浆中尼美舒利最低定量质量浓度为0.05 mg·L^(-1)。方法回收率为96.8%～103.6%,日内精密度(RSD)<13.4%,日间精密度(RSD)<9.9%。单剂量口服尼美舒利0.1g后,两种制剂的ρ_(max)为(5.7±1.5)和(5.4±1.4)mg·L^(-1);t_(max)为(3.4±0.5)和(3.4±0.5)h;AUC_(0→24)为(41.4±12.5)和(40.3±15.7)mg·h·L^(-1);AUC_(0→∞)为(42.9±13.2)和(41.5±10.8)mg·h·L^(-1);t_(1/2)为(4.1±1.6)和(4.1±1.5)h。结论该方法简单快速,灵敏度高,可用于尼美舒利的体内过程研究。方差分析表明,两制剂之间药动学参数无明显差异,试验制剂与参比制剂为生物等效制剂。

OBJECTIVE To develop an HPLC-UV method for the determination of nimesulide in plasma and evaluate the bioequivalence of nimesulide capsules. METHODS After being alkalinized, nimesulide was separated on a COSMOSIL C）8 column （4.6 mm× 250 mm, 5 μm）with acetonitrile-15 mmol· L-1 KH2PO4 as mobile phase at a flow rate of 1.0 mL · min-1. The detection wavelength was at 404 nm. In a randomized crossover design, 18 healthy male volunteers were given nimesulide capsules T （test drug） or capsules R （reference drug）. The plasma samples were obtained and were determined by the developed HPLC method, and the pharmacokinetics and bioavailability were studied. RESULTS The calibration curve of nimesulide were liner over the range of 0.05 -6. 0 mg · L-1 ,and the limit of quantity was 0. 05 mg · L-1. The methodology recoveries were in the range of 96. 8% - 103.6%. Inter-day and intra-day RSDs were less than 13.4% and 9.9%, respectively. Pharmaeokinetic parameters after a single dose of nimesulide capsules T and R were as following ：Pmax were （5.7 ± 1.5 ） and （5.4 ± 1.4） mg · L-1 ; tmax were （3.4 ±0. 5 ） and （3.4 ± 0.5） h; AUC0-24were （41.4±12.5） and （40.3±15.7） mg·h·L-1;AUC0→∞ were （42.9±13.2） and （41.5±10.8） mg·h· L-1 ; tl/2 were （ 4. 1 ± 1.6 ） and （ 4. 1 ± 1.5 ） h. CONCLUSION The method is simple, sensitive and rapid for nimesulide determination. The results of ANOVA and two one-side test analysis showed that there were no significant differences between the pharmaeokinetie parameters of the two formulations. The two formulations were bioequivalent.