Determination of fluoroquinolones in aquaculture products by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of fluoroquinolones-ciprofloxacin (CIPRO), danofloxacin (DANO), enrofloxacin (ENRO) and sarafloxacin (SARA)-in aquaculture products, specifically salmon, shrimp and tilapia. After initial sample extraction with an acidic acetonitrile solution, the extract was diluted with dichloromethane and centrifuged, then an aliquot was concentrated and applied to a C18 solid-phase extraction cartridge and concentrated for a second time. The resultant residue was dissolved in acetonitrile, diluted with water, and then further defatted with hexane. The fluoroquinolone residues were determined by UPLC with an HSS T3 C18 reverse-phase column using an ammonium hydroxide-formic acid buffer in an acetonitrile gradient with MS/MS detection using multiple reaction monitoring. Average recoveries for salmon tissue ranged from 73% for DANO to 95% for SARA, for shrimp from 71% for DANO to 109% for SARA, and from 62% for DANO to 111% for SARA in tilapia, fortified at the 1.0 ng g(-1) level. Standard curves were linear between 0.002 and 0.5 ng injected for all compounds. Detection limits of 0.2 ng g(-1) for CIPRO, DANO, ENRO, and SARA were easily obtainable. The operational errors, interferences, and recoveries for fortified samples demonstrate that this described method is suitable for routine use in a regulatory programme. The recommended method is simple, rapid, specific and reliable for the routine monitoring of fluoroquinolone residues in aquatic species such as salmon, tilapia and shrimp.

Simultaneous determination of emamectin and ivermectin residues in Atlantic salmon muscle by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method for determining residues of the antiparasitic drugs emamectin (EMA) and ivermectin (IVR) in fish tissues has been developed. EMA and IVR residues are extracted with acetonitrile and cleaned up on a C18 solid-phase extraction column. Extracts are derivatized with 1-methylimidazole and trifluoroacetic anhydride and the components are determined by LC on a C18 reversed-phase column with fluorescence detection (excitation: 365 nm, emission: 470 nm). The mobile phase is 94% acetonitrile-water run isocratically. Calibration curves were linear between 1 and 32 ng injected for both EMA and IVR. The limit of detection for both analytes was 0.5 ng/g, with a limit of quantitation of 1.5 ng/g. Recoveries of EMA and IVR added to salmon muscle averaged 96 +/- 9% and 86 +/- 6%, respectively, at levels between 5 and 80 ng/g. The percent relative standard deviation for the described method was less than 7% over the range of concentrations studied. The operational errors, interferences, and recoveries for fortified samples compare favorably with an established IVR method. The recommended method is simple, rapid, and specific for monitoring residues of EMA and IVR in Atlantic salmon muscle.

Liquid chromatographic determination of hypoxanthine content in fish tissue.

A liquid chromatography (LC) method for determining the hypoxanthine content in fish tissues has been developed. Hypoxanthine is extracted with 0.6M perchloric acid, and determined by LC on a reverse phase microparticulate column with UV absorbance detection. The mobile phase is 0.01M potassium phosphate buffer (pH 4.5). The percent relative standard deviation for measurements by the recommended method was less than 7% with a detection limit of 10 ng. Recoveries of hypoxanthine added to various fish tissues were better than 90%. The operational errors, interferences, and recoveries for spiked samples have been investigated and compare favorably with an established xanthine oxidase enzyme method. The described LC method is simple, rapid, and specific for measuring hypoxanthine content in various fish tissues. Some post-mortem studies have indicated the method may also be used for the determination of adenosine monophosphate, inosine monophosphate, and inosine.

Isolation and identification of testosterone from the serum and testes of the American lobster (Homarus americanus).

Testosterone was isolated and quantified from male lobster serum and testes by solvent extraction, sequential thin-layer chromatography, and high-performance liquid chromatography. The identity of the isolated steroid was established by its isopolarity and isomorphicity with authentic radiolabeled testosterone and its acetate derivative. The concentrations of testosterone were determined by high-performance liquid chromatography, by a double-isotope derivative assay, and by radioimmunoassay. The concentrations of testosterone as determined by the three methods were the same and were 0.3 ng/ml and 14.3 ng/g in lobster serum and testes, respectively.

Bioconversion of steroids by the testes of the American lobster, Homarus americanus, in vitro.

Lobster testes have been demonstrated to contain steroid 20-ketone reductase by their capacity to convert [14C]progesterone to 20 alpha-dihydroprogesterone (20 alpha-DHP). The major product was isopolar and identical with 20 alpha-DHP during thin-layer chromatography, high-pressure liquid chromatography, mass spectrometry, and acetate derivative formation. The vas deferens from the lobster was also capable of the same conversions but to a lesser extent. Lobster testes converted [14C]pregnenolone to a major product identified as 20-dihydropregnenolone (20-DHPe) by mass spectrometry after purification by thin-layer chromatography and high-pressure liquid chromatography. The presence of delta 5,3 beta-ol dehydrogenase and delta 5,delta 4-isomerase in the lobster were also indicated. [14C]Cholesterol was not transformed to steroid hormones by lobster testes under the same experimental conditions.

Novel cleanup method for quantitative gas chromatographic determination of trace amounts of Di-2-ethylhexyl phthalate in fish lipid.

A new, simple, and rapid cleanup procedure is described for di-2-ethylhexyl phthalate (DEHP) residues in fish lipids. Extracted are chromatographed on small alumina:sulfuric acid-impregnated alumina (layered) columns after gel permeation chromatography of the fish lipid extracts on BioBeads SX-3. Quantitative analyses were carried out by electron capture gas chromatography using 2 columns. Spiked DEHP recoveries varied from 79.3% in mackerel to 86.3% in herring. DEHP concentrations were determined in plaice, eel, redfish, herring, cod, and mackerel tissues and ranged from trace (less than 0.001 microgram/g) to approximately 10 microgram/g (wet wt basis). Ethanolic KOH saponification of the purified DEHP fraction, resulting in the disappearance of the DEHP peak, was used as a confirmatory test. Limited studies with dibutyl, di-n-heptyl, and diethyl phthalates suggest that the method can be made more versatile.

Molt induction in lobsters (Homarus americanus) by intramuscular injection of ecdysterone triacetate.

The principal component of the successful, molt-inducing ecdysterone acetate mixture was established as ecdysterone triacetate and shown to have the same activity as the acetate mixture. The triacetate induced ecdysis in male lobsters collected in winter and summer and in female lobsters collected in winter. Intramuscular injections of triacetate in ethanol were as effective as those with oil emulsions.

The successful induction of molting in the adult male lobster (Homarus americanus) with a slow-release form of ecdysterone.

In an initial, and then a confirmatory experiment, adult, male lobsters were injected with solvent, ecdysterone (E, 2.0 mug/g live weight) or ecdysterone acetate (EAc, 2.5 or 5.0 mug/g live weight) emulsions in Freund's incomplete adjuvant (FIA). Control lobsters underwent no molts and only one death in the two cases. The E treated animals all died (average: exp. 1, 19.2 +/-2.1 days; exp. 2, 25.3 +/- 8 days). After the lobsters were treated twice with 2.5 mug EAc/g live weight, in the first experiment, four out of five molted and one died; in the second experiment six out of eight molted, one died and one remained refractory. The high EAc dose resulted in five deaths, one molt and two pseudomolts after one treatment. It is concluded that the use of the oil emulsion and EAc sufficiently slowed the release of free ecdysterone to allow complete premolt development in the lobster.

Organochlorine pesticide residues in a farming area, Nova Scotia--1972-73.

Soil, silt, and water samples from the Habitant Creek water-shed, Nova Scotia, a tobacco-growing area, have been monitored for organochlorine insecticides. Most samples contain measurable quantities of many persistent pesticides used in farming during the past decade. Sediment levels indicate that residues settle in sluggish parts of the stream. Drainage ditches show highest residual content caused in part by mass transport of soil in runoff. Residue content of water samples is normally one-tenth to one-hundredth that of silt, but is much higher during periods of heavy runoff. Levels vary with the seasons and are highest in the fall, decrease through the spring and summer, and are lowest in the winter. Although samples of well water taken fairly close to the stream showed virtually no residual content, a natural drainage reservior had a pesticide content similar to that in the stream.