Badar Shaikh

Liquid chromatographic analysis of antibacterial drug residues in food products of animal origin

This paper reviews recent developments in the liquid chromatographic (LC) methods of analysis for the residues of antibiotics (aminoglycosides, chloramphenicol, sulfonamides, tetracyclines, macrolides, beta-lactams, etc.) in food products of animal origin. The review also covers clean-up procedures, such as, ultrafiltration, liquid-liquid partition, solid-phase extraction, immunoaffinity, and matrix solid-phase dispersion, for use as extraction, deproteination, and concentration steps. The LC methods offer considerable potential for rapid automated analysis, and some may be used as direct screening for residues in meat and milk.

Determination of gentamicin in bovine milk using liquid chromatography with post-column derivatization and fluorescence detection.

A method capable of separating and quantifying the three major and one minor components of gentamicin in milk has been developed. The method is capable of detecting 15 ng/ml gentamicin, based on a total of the four components. Milk samples are centrifuged at 4 degrees C, the fat layer removed, and the samples deproteinated with 30% trichloracetic acid. After a second centrifugation, the supernatant is passed through a C18 solid-phase extraction column. The column is washed with water, water-methanol (50:50) and methanol. Ammonium hydroxide (16%) in methanol is used to elute the gentamicin. The eluent is evaporated to near dryness and taken up with water. An aliquot of the sample is then mixed with an ion-pairing reagent for chromatography. Separation is achieved using pentanesulfonic acid in a water-methanol mobile phase on a C18 reversed-phase column. The o-phthalaldehyde fluorescence derivatives of gentamicin are formed post-column and are detected with excitation at 340 nm and emission at 430 nm. The percent recovery of gentamicin averaged 72, 78 and 88% from milk samples fortified at 15, 30 and 60 ng/ml, respectively.

Kinetics of hepatic phase I and II biotransformation reactions in eight finfish species.

Hepatic microsomes and cytosols of channel catfish (Ictalurus punctatus), rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), red tilapia (Oreochromis sp.), largemouth bass (Micropterussalmoides), striped bass (Morone saxatilis), hybrid striped bass (M. saxatilis x M. crysops), and bluegill (Lepomis macrochuris) (n=8) were used to study the kinetics of phase I (ECOD, EROD, PROD, BROD) and phase II (UDP-glucuronosyltransferase (UDPGT)-, sulfotransferase (ST)- and glutathione-s-transferase (GST)-mediated) reactions. The best catalytic efficiency for ECOD and GST activities was performed by channel catfish, Atlantic salmon, rainbow trout and tilapia. The highest EROD catalytic efficiency was for Atlantic salmon. None of the species had either PROD or BROD activities. Rainbow trout had very similar UDPGT catalytic efficiency to tilapia, channel catfish, Atlantic salmon, largemouth bass and bluegill. Sulfotransferase conjugation had no significant differences among the species. In summary, tilapia, channel catfish, Atlantic salmon and rainbow trout had the best biotransforming capabilities; striped bass, hybrid striped bass and bluegill were low metabolizers and largemouth bass shared some capabilities with both groups.

DETERMINATION OF ALBENDAZOLE, FENBENDAZOLE, AND THEIR METABOLITES IN MOUSE PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY USING FLUORESCENCE AND ULTRAVIOLET DETECTION

A rapid and reliable high performance liquid chromatographic (HPLC) method has been developed and validated for the determination of albendazole (ABZ), albendazole sulfoxide (ABZSO), albendazole sulfone (ABZSO2); fenbendazole (FBZ), fenbendazole sulfoxide (FBZSO), and fenbendazole sulfone (FBZSO2) in mouse plasma. The mouse plasma was made alkaline with potassium carbonate and extracted with ethylacetate. The extracts were evaporated, reconstituted in mobile phase, and analyzed by HPLC. The chromatography was carried out on a reversed-phase column using acetonitrile-methanol-buffer as the mobile phase. The fenbendazole and its metabolites were detected by using UV detector set at the wavelength of 290 nm. The albendazole and its metabolites were detected by a fluorescence detector with excitation and emission wavelengths of 290 and 330 nm, respectively. The average recoveries of ABZ, ABZSO, and ABZSO2 from fortified control plasma samples were 95, 82, and 92%, respectively. Similarly, the average recoveries of FBZ, FBZSO, and FBZSO2 were 64, 90, and 94%, respectively. The average CVs were ≤15% for all of the compounds. The method was applied to incurred mouse plasma samples to determine ABZ, FBZ, and their metabolites.

In vitro kinetics of hepatic albendazole sulfoxidation in channel catfish (Ictalurus punctatus), tilapia (Oreochromis sp.), rainbow trout (Oncorhynchus mykiss) and induction of EROD activity in ABZ-dosed channel catfish

Liver microsomes from market-size (n = 6) rainbow trout, channel catfish and tilapia were used to investigate in vitro biotransformation kinetics of albendazole (ABZ). ABZ was transformed to a single metabolite, ABZ sulfoxide (ABZ-SO). Catfish displayed the highest maximal velocity (V(max) = 264.0 +/- 58.6 pmols ABZ-SO/min/mg protein) followed by tilapia (112.3 +/- 8.2) and rainbow trout (73.3 +/- 10.3). V(max) in catfish was significantly different (P < 0.05) from the other two species. Michaelis-Menten constant (K(m)) values (microm) varied significantly among the species: rainbow trout (3.9 +/- 0.5), tilapia (9.2 +/- 1.7) and catfish (22.0 +/- 3.2). However, V(max)/K(m) ratios showed no difference among the three species, making them equally efficient performing this phase I biotransformation reaction. In a second series of experiments, channel catfish (n = 6 per treatment) were dosed in vivo with gel-food containing ABZ (10 mg/kg, p.o.). Fish were killed at 24, 48, 72 and 120 h after dosage. Control fish were fed ABZ-free feed. Induction of ethoxyresorufin-o-deethylase activity was significant (P < 0.05) in all ABZ-dosed treatments as compared with controls.

Residue depletion of albendazole and its metabolites in the muscle tissue of large mouth and hybrid striped bass after oral administration

The residue depletion profiles of albendazole (ABZ) and its major metabolites: albendazole sulfoxide (ABZ-SO), albendazole sulfone (ABZ-SO(2)) and albendazole aminosulfone (ABZ-2-NH(2)SO(2)) were studied in the muscle tissues of large mouth (LMB) and hybrid striped bass (HSB). A single oral dose of 10mg/kg albendazole was given to the two fish species via intra-gastric tube. The muscle tissues with adhering skin were collected at 8, 16, 24, 48, 72, 96 and 120h post dose from both species. The samples were homogenized in dry ice and subjected to extraction and cleanup procedures. The final sample extracts were analyzed by high performance liquid chromatography. The results indicate that both ABZ and its pharmacologically active metabolite ABZ-SO were retained longer in LMB than in HSB after oral treatment. Albendazole was detectable until 8h or 6.7 degree days ( degrees D) and 48h (40 degrees D) in HSB and LMB, respectively. However, ABZ-SO was detectable up to 48h (40 degrees D) and 96h (80 degrees D) in HSB and LMB, respectively. Among the inactive metabolites, ABZ-SO(2) was present in both fish species; however, ABZ-2-NH(2)SO(2) was detected only in LMB.

Marker Residue Determination of Tritium-Labeled Ivermectin in the Muscle of Aquacultured Largemouth Bass, Hybrid Striped Bass, and Yellow Perch following Oral Treatment

The residue depletion profiles of tritium-labeled ivermectin and its metabolites in the muscle of aquacultured largemouth bass (LMB), hybrid striped bass (HSB), and yellow perch (YP) following oral treatment are reported. Fish were administered ³H-ivermectin at the dose level of 0.1 mg/kg body weight (7-9 μCi) in a gel capsule via stomach tube. At each postdose withdrawal time, six fish of each species were sedated with buffered MS-222 and blood samples taken. Fish were then euthanized, and fillets with adhering skin (scales removed) and bile samples were collected. The muscle fillets were homogenized in dry ice to a fine powder. Aliquots of tissue, plasma, and bile were assayed for total radioactive residue (TRR). The homogenized muscle was extracted in acetonitrile or methanol followed by high-performance liquid chromatographic (HPLC) analysis to determine the presence of parent ivermectin and its potential metabolites. The highest TRR concentrations (ivermectin equivalents) of 53, 45, and 44 ng/g (ppb) were obtained on postdose day 1 for HSB, LMB, and YP, respectively. The TRR depleted most slowly in HSB to 25 ppb at day 91, followed by YP to 19 ppb at day 42 and then by LMB to 22 ppb at day 35. The total residue of ivermectin and its metabolites by HPLC analysis followed the same depletion pattern in the three species. Additionally, the depletion rate of TRR of ³H-ivermectin in the three species followed the pattern bile > plasma > muscle. The results further indicate that one of the polar metabolites of ivermectin could serve as a potential marker residue as an indication of use, rather than the parent ivermectin.