Determination of metronidazole and hydroxymetronidazole in trout by a high-performance liquid chromatographic method

Abstract

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for determination of metronidazole (MNZ) and its metabolite hydroxymetronidazole (MNZ-OH) in muscle and skin tissues of rainbow trout. Tinidazole (TNZ) was used as internal standard. The compounds were extracted with acetonitrile and the extract was evaporated and redissolved in a mixture of ethyl acetate and hexane (1:2). The extract was cleaned up by solid phase extraction on a silica cartridge. The extract was analysed by reverse phase gradient HPLC on a C18 column followed by ultraviolet detection at 325 nm. The limit of detection was 2.8 micrograms/kg for both compounds in muscle. The estimated limits of detection in skin tissue were 3 micrograms/kg for MNZ and 5 micrograms/kg for MNZ-OH. The mean recoveries of MNZ in muscle calculated without use of internal standard were 93% and 81% at levels of 10 micrograms/kg and 25-100 micrograms/kg respectively. The mean recovery of MNZ-OH in muscle was 79% at a level of 10-100 micrograms/kg. The mean relative repeatability standard deviations on spiked muscle tissue were 3.3% for MNZ and 3.2% for MNZ-OH at a level of 10-100 micrograms/kg. The method was applied to trout given feed containing MNZ in an aquaculture pilot plant. Residues of MNZ and MNZ-OH were detected in muscle and skin tissues shortly after the administration period but not 3 weeks later.

Simultaneous multiresidue determination of metronidazole and spiramycin in fish muscle using high performance liquid chromatography with UV detection

“…Relatively high levels of MET are present in the muscles during dosing; these levels decrease during withdrawal, as might be expected. It is interesting to note that the levels observed in this study are similar to those in which MET-dosed catfish muscle samples were analyzed by another method [15]. The fraction of HMET to MET was 2% or less, as was also observed in previous work [15].…”

“…It is important, whenever possible, to test a method for detection of veterinary drug residues using actual dosed animal (incurred) samples as these samples are closer to what would be found in an actual monitoring situation than samples which have only been fortified [15,24]. This was accomplished, and the results obtained are summarized in Table 4.…”

“…In previous work [9,17] describing the simultaneous determination of macrolide antibiotics in animal tissues, the samples were extracted with metaphosphoric acid-methanol and the cleanup procedure was performed using different types of solid phase extraction (SPE) cartridges, e.g., a pre-packed cation exchange resin cartridge [9] or C18 (ODS) cartridge [17]. Also analytical methods based on SPE technique have been reported for the determination of MET and HMET in fish tissues where C 18 cartridges were used for the cleanup procedure [15]. Since the latter is the most widely used solid-stationary phase for extraction and cleanup, a reversed phase C18 Sep-Pak ® light column (Waters Sep-Pak ® Vac, USA) which is an octadecyl silane bonded to silica was used in this work together with methanol as eluting solvent.…”

“…So it is very important to extract as much as possible of the determined analytes and also to exclude interfering substances present in biological matrices. Different extraction and cleanup methods have been developed [9,15,17].…”

“…Therefore, efficient analytical methods are required to monitor these drug residues in the edible tissues of livestock animals including fish tissues. High performance liquid chromatography (HPLC) is by far the most widely applied quantitative technique for the analysis of MET [15] or SPY residues [16,17] in fish tissues.…”