A. Gulaid, G. Houghton, O. Lewellen
Nov 1, 1978
Citations
0
Influential Citations
48
Citations
Journal
British journal of clinical pharmacology
Abstract
Metronidazole [ 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole; Flagyl®] is an antibacterial agent which has been widely used in the treatment of a variety of anaerobic infections (Templeton, 1977). The major urinary metabolites which have been reported for man are 1-(2-hydroxyethyl)-2-hydroxymethyl-5-nitroimidazole and 2-methyl-5-nitroimidazole-1-acetic acid (Stambaugh, Feo & Manthei, 1968). The analytical methods which are most commonly used to determine this drug in biological fluids are polarography (Kane, 1961), microbiological assay (Levison, 1974) or gas chromatography (Midha, McGilveray & Cooper, 1973; Wood, 1975). The first two methods are nonspecific, and the gas chromatographic methods, although specific, are designed for the assay of metronidazole alone, and have a sensitivity which is more appropriate for the measurement of therapeutic levels than for pharmacokinetic studies of metronidazole. The specific and sensitive high pressure liquid chromatographic (HPLC) method described herein was devised for the simultaneous determination of metronidazole and its two major human oxidative metabolites in pharmacokinetic studies. During the final stages in the preparation of this letter, it was noted that an HPLC method for the quantification of metronidazole and its metabolites in plasma was published by Wheeler, DeMeo, Halula, George & Heseltine (1978). This method is similar in some regards to that described here. However, our method has a considerably lower limit of sensitivity for compounds in plasma, may be used to quantify metronidazole and its metabolites in urine, employs an internal standard for greater accuracy, and has a different sample work-up procedure which is likely to lead to a longer HPLC column life. The extraction and chromatographic procedures used in the assay method are described below. An aliquot (100 gl) of the HPLC mobile phase (a mixture of 0.1 M diammonium hydrogen phosphate and methanol, 5 :1 v/v) containing a known concentration (0.1 to 40 gg/ml) of internal standard [ 1-(2hydroxyethyl)-2-ethyl-5-nitroimidazole] was added to heparinized human plasma (5 ml). Ammonium sulphate (3.3 g; prewashed with methyl ethyl ketone) was added and the mixture shaken for 5 min. The pH of the mixture was adjusted to 3.5 with 2M hydrochloric acid. Methyl ethyl ketone (25 ml) was added and the mixture shaken for a further 5 min, centrifuged and the organic layer removed and retained. This was dried over anhydrous sodium sulphate (5 g; prewashed with methyl ethyl ketone), filtered and evaporated to dryness under vacuum. The residue was dissolved in anhydrous methanol (3 ml), transferred to a vial and evaporated to dryness under a stream of oxygen-free-nitrogen. The residue was dissolved in mobile phase (100 pl) and an aliquot (10 to 25 ,ul) injected onto the HPLC column. The extraction method described above was also used for the quantification of the three compounds of interest in urine, except that the quantity of internal standard was increased to a known concentration in