Pavan Kumar Venkata V, Vinu Menon C A, Ramani Addepalli V, Mullangi Ramesh, Srinivas Nuggehally R
Drug Metabolism and Pharmacokinetics, Discovery Research, Dr Reddy's Laboratories Ltd, Miyapur, Hyderabad- 500 049, India.
Biomed Chromatogr. 2006 Jan;20(1):125-32. doi: 10.1002/bmc.539.
A specific, accurate, precise and reproducible high performance liquid chromatography (HPLC) method was developed and validated for the simultaneous quantitation of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib in human plasma. The method employed a simple liquid-liquid extraction of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide and celecoxib and internal standard (IS, DRF-4367) from human plasma (500 microL) into acetonitirile. The organic layer was separated and evaporated under a gentle stream of nitrogen at 40 degrees C. The residue was reconstituted in the mobile phase and injected onto a Kromasil KR 100-5C18 column (4.6 x 250 mm, 5 microm). The chromatographic separation was achieved by gradient elution consisting of 0.05 M formic acid (pH 3)-acetonitrile-methanol-water at a flow rate of 1.0 mL/min. The eluate was monitored using an ultraviolet (UV) detector set at 235 nm. The ratio of peak area of each analyte to IS was used for quantification of plasma samples. Nominal retention times of etoricoxib, salicylic acid, valdecoxib, ketoprofen, nimesulide, IS and celecoxib were 15.63, 17.20, 21.66, 24.95, 26.27, 30.24 and 32.22 min, respectively. The standard curve for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib was linear (r2 > 0.999) in the concentration range 0.1-50 microg/mL and for nimesulide (r2 > 0.999) in the concentration range 0.5-50 microg/mL. Absolute recovery was >83% from human plasma for all the analytes and IS. The lower limit of quantification (LLOQ) of nimesulide was 0.5 microg/mL and for etoricoxib, salicylic acid, valdecoxib, ketoprofen and celecoxib the LLOQ was 0.1 microg/mL. The inter- and intra-day precisions in the measurement of QC samples, 0.1, 0.3, 15.0 and 40.0 microg/mL (for all analytes except nimesulide), were in the range 2.29-9.37% relative standard deviation (RSD) and 0.69-10.28% RSD, respectively. For nimesulide the inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.5, 1.5, 15.0 and 40.0 microg/mL, were in the range 3.21-7.37% RSD and 0.97-7.06% RSD, respectively. Accuracy in the measurement of QC samples for all analytes was in the range 91.03-106.38% of the nominal values. All analytes including IS were stable in the battery of stability studies, viz. bench top, autosampler and freeze-thaw cycles. Stability of all analytes was established for 21 days at -20 degrees C. The application of the assay in an oral pharmacokinetic study in rats co-administered with celecoxib and valdecoxib is described.
建立并验证了一种特异性强、准确、精密且可重现的高效液相色谱（HPLC）方法，用于同时定量测定人血浆中的依托考昔、水杨酸、伐地考昔、酮洛芬、尼美舒利和塞来昔布。该方法采用简单的液-液萃取法，从500微升人血浆中提取依托考昔、水杨酸、伐地考昔、酮洛芬、尼美舒利和塞来昔布以及内标（IS，DRF - 4367）至乙腈中。分离有机层，并在40℃的氮气缓流条件下蒸发。残渣用流动相复溶后，注入Kromasil KR 100 - 5C18色谱柱（4.6×250 mm，5微米）。通过梯度洗脱实现色谱分离，流动相为0.05 M甲酸（pH 3）-乙腈-甲醇-水，流速为1.0 mL/min。使用设置在235 nm的紫外（UV）检测器监测洗脱液。各分析物与内标的峰面积比用于血浆样品的定量分析。依托考昔、水杨酸、伐地考昔、酮洛芬、尼美舒利、内标和塞来昔布的标称保留时间分别为15.63、17.20、21.66、24.95、26.27、30.24和32.22分钟。依托考昔、水杨酸、伐地考昔、酮洛芬和塞来昔布的标准曲线在0.1 - 50微克/毫升浓度范围内呈线性（r2>0.999），尼美舒利在0.5 - 50微克/毫升浓度范围内呈线性（r2>0.999）。所有分析物和内标从人血浆中的绝对回收率均>83%。尼美舒利的定量下限（LLOQ）为0.5微克/毫升，依托考昔、水杨酸、伐地考昔、酮洛芬和塞来昔布的LLOQ为0.1微克/毫升。质量控制（QC）样品0.1、0.3、15.0和40.0微克/毫升（尼美舒利除外的所有分析物）的日间和日内精密度，相对标准偏差（RSD）范围分别为2.29 - 9.37%和0.69 - 10.28%。对于尼美舒利，质量控制（QC）样品0.5、1.5、15.0和40.0微克/毫升的日间和日内精密度，RSD范围分别为3.21 - 7.37%和0.97 - 7.06%。所有分析物的QC样品测量准确度在标称值的91.03 - 106.38%范围内。包括内标在内的所有分析物在一系列稳定性研究中均稳定，即台式、自动进样器和冻融循环条件下。所有分析物在-20℃下的稳定性均确定为21天。描述了该测定法在与塞来昔布和伐地考昔共同给药的大鼠口服药代动力学研究中的应用。
