Loewen G R, Macdonald J I, Verbeeck R K
College of Pharmacy, University of Saskatchewan, Saskatoon, Canada.
J Pharm Sci. 1989 Mar;78(3):250-5. doi: 10.1002/jps.2600780317.
A direct high-performance liquid chromatographic (HPLC) assay was developed to simultaneously quantitate diflunisal and its three known metabolites (i.e., the phenolic and acyl glucuronides and the sulfate conjugate) in human urine. Chromatographically pure standards of the diflunisal conjugates were isolated from urine of volunteers following ingestion of multiple doses of diflunisal (500 mg twice daily). Diflunisal, its three conjugates, and an internal standard (naproxen) were separated on a reversed-phase column using gradient elution. The column eluate was monitored fluorometrically (excitation: 258 nm; emission: 428 nm). Urine samples were diluted with phosphate buffer (pH 5.75) and injected onto the column. The limit of detection was approximately 1 microgram/mL for each conjugate and 0.1 microgram/mL for diflunisal. Due to the presence in most urine samples of significant concentrations of rearrangement products of the biosynthetic 1-O-acyl glucuronide of diflunisal, the acyl glucuronide could not be reliably quantitated by direct injection of diluted urine samples. Instead, diflunisal acyl glucuronide was quantitated indirectly following alkaline hydrolysis of the urine samples. The method has been successfully used to investigate the dose-dependent glucuronidation and sulfation of diflunisal in humans.
建立了一种直接高效液相色谱（HPLC）分析法，用于同时定量人尿中双氯芬酸及其三种已知代谢物（即酚醛葡萄糖醛酸苷、酰基葡萄糖醛酸苷和硫酸酯共轭物）。在多次服用双氯芬酸（每日两次，每次500毫克）后，从志愿者尿液中分离出色谱纯的双氯芬酸共轭物标准品。双氯芬酸及其三种共轭物和内标（萘普生）在反相柱上采用梯度洗脱进行分离。柱洗脱液用荧光法监测（激发波长：258纳米；发射波长：428纳米）。尿液样品用磷酸盐缓冲液（pH 5.75）稀释后注入柱中。每种共轭物的检测限约为1微克/毫升，双氯芬酸的检测限为0.1微克/毫升。由于大多数尿液样品中存在大量双氯芬酸生物合成的1-O-酰基葡萄糖醛酸苷重排产物，因此不能通过直接注入稀释后的尿液样品来可靠地定量酰基葡萄糖醛酸苷。相反，双氯芬酸酰基葡萄糖醛酸苷是在尿液样品碱性水解后间接定量的。该方法已成功用于研究双氯芬酸在人体内的剂量依赖性葡萄糖醛酸化和硫酸化。
