Smith P C, Benet L Z
J Chromatogr. 1984 Mar 9;306:315-21. doi: 10.1016/s0378-4347(00)80893-2.
Quantitation of total amounts (i.e., free compound plus glucuronide conjugate) of indomethacin (INDO) and its deschlorobenzoyl (DBI) and desmethyl (DMI) metabolites in human urine is described. An aliquot (0.4 ml) of urine is incubated with glucuronidase (1000 U, 2 h, 37 degrees C) and extracted with 5 ml of dichloromethane containing the internal standards: the fluoro analogue of INDO, F-INDO, and indole-3-propionic acid (IPA). The organic phase is concentrated, dissolved in mobile phase and aliquots are injected onto the high-performance liquid chromatograph. INDO and DMI are measured with UV detection at 254 nm over a linear range of 0.25-125 micrograms/ml. Retention times for DMI, F-INDO and INDO are 4.0, 6.8 and 12.1 min, respectively, using a C8 reversed-phase column with an acetonitrile-0.1 M acetate, pH 5.0 (30:70) mobile phase at a 2.5 ml/min flow-rate. DBI is measured using fluorescence detection (excitation = 305 nm, emission = 370 nm) over a linear range of 0.25-12.5 micrograms/ml. Retention times for DBI and IPA are 4.5 and 7.8 min, respectively, on the same C8 column with an acetonitrile-0.025 M acetate, pH 4.0 (22:78) mobile phase at a 2.0 ml/min flow-rate. Inter- and intra-day precision were smaller than 10% for INDO, DMI and DBI over the concentration ranges indicated.
本文描述了人尿中吲哚美辛（INDO）及其去氯苯甲酰（DBI）和去甲基（DMI）代谢物的总量（即游离化合物加上葡萄糖醛酸缀合物）的定量方法。取0.4 ml尿液等分试样，与葡萄糖醛酸酶（1000 U，2小时，37℃）一起孵育，并用5 ml含有内标物的二氯甲烷萃取：INDO的氟类似物F-INDO和吲哚-3-丙酸（IPA）。有机相浓缩后，溶解于流动相中，取等分试样注入高效液相色谱仪。在0.25 - 125μg/ml的线性范围内，通过254 nm的紫外检测测定INDO和DMI。使用C8反相柱，以乙腈 - 0.1 M醋酸盐（pH 5.0，30:70）为流动相，流速为2.5 ml/min时，DMI、F-INDO和INDO的保留时间分别为4.0、6.8和12.1分钟。在0.25 - 12.5μg/ml的线性范围内，通过荧光检测（激发 = 305 nm，发射 = 370 nm）测定DBI。在同一C8柱上，以乙腈 - 0.025 M醋酸盐（pH 4.0，22:78）为流动相，流速为2.0 ml/min时，DBI和IPA的保留时间分别为4.5和7.8分钟。在所示浓度范围内，INDO、DMI和DBI的日间和日内精密度均小于10%。
