Determination of flumequine and a hydroxy metabolite in biological fluids by high-pressure liquid chromatographic, fluorometric, and microbiological methods

Abstract

A sensitive and specific high-pressure liquid chromatographic method is described for the determination of the antibacterial drug flumequine and a major metabolite, 7-hydroxyflumequine, in human plasma and urine. The assay was linear over a concentration range of 1 to 120 pLg/ml for both compounds. This method is compared with fluorometric and microbiological assays for flumequine. These latter methods did not differentiate between flumequine and any fluorescent or antimicrobiologically active metabolites. However, because essentially all drug in the plasma was found to be flumequine in radiolabeled studies, levels of unchanged drug in the plasma could be quantitated by either high-pressure liquid chromatography or fluorometry. Although only high-pressure liquid chromatography was able to specifically measure flumequine in the urine, the antimicrobial activity of the urine, which is more therapeutically relevant due to antimicrobially active metabolites, could be quantitated by either the fluorometric or the microbiological assay.Flumequine (I) is a quinolone derivative active against a broad spectrum of gram-negative bacteria, some of which are associated with or responsible for urinary tract infections. It is commercially available in Europe for human and veterinary use. Chemically, I is 9-fluoro-6,7-dihydro-5-methyl-1-oxo-lH,5H-benzo[ii]quinolizine-2-carboxylic acid.Some chemical, microbiological, and metabolic aspects of I have been reported previously (1, 2).In the earlier stages of I development, two microbiological assays and a fluorometric method were employed for analysis of urine and plasma samples. For pharmacokinetic and biopharmaceutical studies, a specific and sensitive highpressure liquid chromatographic (HPLC) method was developed which can simultaneously quantitate I and a 7-hydroxy metabolite, 7-hydroxyflumequine (II) (Fig. 1 hydroxide. The mobile phase was pumped at 1.5 ml/min through a stainless steel column (61 cm by 2.3-mm inside diameter) containing a strong anion-exchange resin (Zipax SAX, Du Pont Co., Wilmington, Del.). The best results were obtained with prepacked columns purchased from the supplier.To 1 ml of plasma were added 100 ,ug of the internal standard, quinaldic acid (100 ,ul of a stock solution of 1 mg of quinaldic acid in 1 ml of methanol), 0.5 ml of 0.6 N HCl, and 5 ml of chloroform. The tubes were shaken on a reciprocal shaker for 5 min (125 cycles/min) and then centrifuged for 10 min at 750 x g. The aqueous phase was discarded. To the chloroform phase was added 0.5 ml of 0.6 N NaOH. The tubes were shaken and centrifuged, and a 10-to 25-,ul sample of the aqueous phase was injected. Urine samples were extracted with 5 ml of chloroform after 300 ,ug of quinaldic acid (300 pAl of the stock solution described above) and 1.0 ml of 0.5 M phosphate buffer, pH 6.0, were added to the original 1 ml of urine sample. A larger amount of internal standard was added to the urine because the larger aqueous volume and higher pH of the urine extraction reduced the recovery of this compound. ...

Fish antibiotherapy: bioencapsulation of flumequine using adult brine shrimp (Artemia salina)

“…The determination of flumequine concentration in Artemia was firstly performed in a biological assay based on the antibiotic MIC determination following NCCLS guidelines (NCCLS 2002). Escherichia coli (ATCC 25922) with MIC values of 0.25 μg mL −1 was chosen as control strain due to its sensitivity to flumequine (Harrison, Schuppan, Rohlfing, Hansen, Hansen, Funk, Collins & Ober 1984, Guillemin, Jarlier & Cambau 1998) and because it may be considered as a representative strain of Enterobacteriaceae , among which is the majority of fish pathogens.…”

“…For the quantification of the flumequine content of fortified Artemia , a microbiological method was chosen (Harrison et al 1984; Majack et al 2000; San Martín, Cañón, Quiróz & Hernández 2000). This method is based on the macrodilution assay for the determination of MIC for E. coli (NCCLS 2002), allowing the indirect quantification of flumequine, through the evaluation of the ability of the bioencapsulated drug to inhibit microbial growth.…”

Pharmacokinetics, metabolism and renal clearance of flumequine in veal calves

“…The methods for treating the samples in plasma and urine, and for the HPLC analysis of flumequine and 7-hydroxy-flumequine in the samples, have been described previously (Harrison et al, 1984;Decolin et al, 1987;Mevius et al, 1989).…”

“…Flumequine is extensively metabolized to glucuronide conjugates in man, dogs, calves and rats, and in man it is metabolized to a smaller extent to 7-hydroxy-flumequine (Lenwland et al, 1981;Dorrestein et al, 1983;Harrison et al, 1984Harrison et al, , 1986Decolin et al, 1987). After flumequine was administered orally to homing pigeons a 'first-pass' effect was observed which may be due either to its biotransformation in the intestinal mucosa and/or the liver, or to its inactivation in the digestive tract before absorption (Dorrestein et al, 1983).…”

Clinical pharmacokinetics of flumequine in calves

“…administration and expressed reservations about the utility of the two-compartment model because of earlier reports indicating extensive metabolism of flumequine in men, dogs, and rats (Rohlfing & Gerster, 1974;Schuppan et al, 1974;Benothmane, 1979). More recently it was established (Harrison et al, 1984) that although most of flumequine is excreted in human urine as the 7-hydroxyflumequine metabolite, which has lower antimicrobial activity than the parent compound, the quantitative microbiological assays of flumequine in plasma should provide a reasonable estimate of unchanged drug even though these assays are nonspecific. Furthermore, chloroform extracts of plasma from human subjects receiving radiolabelled flumequine were subjected to thin-layer chromatography and shown to contain predominantly unchanged drug (Schuppan el al., 1974).…”