Moncrieff J
Department of Pharmacology, Faculty of Medicine, University of Pretoria, South Africa.
J Chromatogr. 1992 May 20;577(1):185-9. doi: 10.1016/0378-4347(92)80618-z.
The author describes a method of using reversed-phase high-performance liquid chromatography with fluorimetric detection for the assay of diclofenac sodium in serum. The method is sensitive down to 20 ng/ml (250-microliters loop). Elution is at pH 6.2 with methanol in 0.05 M phosphate buffer (43:57, v/v) on a 25-cm Spherisorb S5 ODS2 column. Detection is at an excitation wavelength of 282 nm and an emission wavelength of 365 nm. Serum sample size is 100 microliters. Sample protein, to which diclofenac is highly bound, is first denatured by heat and then with methanol to release the diclofenac prior to centrifugation and injection of 100 microliters (or 250 microliters) of the clear supernatant. Harmol, with similar fluorescence and polarity characteristics to diclofenac, is a satisfactory internal standard. At the 1 micrograms/ml level intra-sample reproducibility is better than 2%, whilst inter-sample reproducibility is 4.6%. Detector response is linear from 40 ng/ml to 20 micrograms/ml (100-microliters loop).
作者描述了一种采用反相高效液相色谱法并结合荧光检测来测定血清中双氯芬酸钠的方法。该方法灵敏度高达20 ng/ml（250微升定量环）。在一根25厘米长的Spherisorb S5 ODS2柱上，以pH 6.2的0.05 M磷酸盐缓冲液与甲醇（43:57，v/v）进行洗脱。检测时激发波长为282 nm，发射波长为365 nm。血清样本量为100微升。双氯芬酸与之高度结合的样本蛋白质，首先通过加热使其变性，然后再用甲醇处理以释放双氯芬酸，之后进行离心并注入100微升（或250微升）清亮的上清液。哈尔满与双氯芬酸具有相似的荧光和极性特征，是一种令人满意的内标物。在1微克/毫升水平，样本内重现性优于2%，而样本间重现性为4.6%。检测器响应在40 ng/ml至20微克/毫升（100微升定量环）范围内呈线性。
