Validation and robustness testing of a HPLC method for the determination of avermectins and moxidectin in animal liver samples using an alumina column clean-up

A multi-residue method has been developed for the quantitative determination of moxidectin, abamectin, doramectin and ivermectin in liver samples, with capability for qualitative identification of the presence of eprinomectin. Liver samples are extracted with isooctane, followed by clean-up on alumina-N solid phase extraction (SPE) cartridges. Extracts are derivatised and determined by high-performance liquid chromatography (HPLC) with fluorescence detection. The method was validated using bovine liver fortified at levels of 4 and 20 micrograms kg-1 with the drugs. The mean recovery from bovine liver ranged between 90 and 96%. The intra and inter-assay variations showed RSD typically of < 5% and < 10%, respectively. The procedure was applied also to ovine and porcine liver, giving similar results. A robustness study, carried out on the alumina clean-up step, indicated that the step is relatively insensitive to method changes. However, significant differences overall were found for the type of alumina and/or commercial SPE cartridge used. The limit of quantitation of the method is 2 micrograms kg-1 (ppb).     

Determination of fenbendazole and its metabolites in trout by a high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for the simultaneous determination of fenbendazole (FBZ), fenbendazole sulfoxide (FBZ-SO) and fenbendazole sulfone (FBZ-SO2) in trout muscle and skin tissues. The compounds were extracted with acetonitrile and the extract was concentrated and cleaned up by solid phase extraction on C18 and CN cartridges. The extract was analysed by reversed-phase gradient HPLC on a C18 column followed by ultraviolet detection at 290 nm. The method's detection limits were 4.0 micrograms kg-1 for FBZ, 4.5 micrograms kg-1 for FBZ-SO and 3.8 micrograms kg-1 for FBZ-SO2. The mean recovery in muscle was 88% for FBZ, 94% for FBZ-SO and 92% for FBZ-SO2 at a level of 5-150 micrograms kg-1. The corresponding mean recoveries in skin tissue were 88, 81 and 86% at a level of 10-100 micrograms kg-1. The mean relative repeatability standard deviation was 9.2% at a level of 5 micrograms kg-1, 5.9% at a level of 10-100 micrograms kg-1 and 2.3% at a level of 150 micrograms kg-1. The method was applied to trout given feed containing FBZ in an aquaculture pilot plant. The three compounds FBZ, FBZ-SO and FBZ-SO2 were all detected in muscle and skin tissues shortly after administration. The concentrations were generally highest in skin tissue.     

Sensitive GC/MS determination of 15 isomers of chlorobenzoic acids in accelerated solvent extracts of soils historically contaminated with PCBs and validation of the entire method

Chlorobenzoic acids (CBAs) are the major metabolite of aerobic bacterial degradation of polychlorinated biphenyls (PCBs). A rapid and simple simultaneous derivatisation method has been developed for gas chromatography-mass spectrometry determination in historically PCB-contaminated soils for 15 isomers of mono-, di-, tri-, tetra-, and pentachlorobenzoic acids in CBA mixtures. Two derivatisation agents (diazomethane and methyl chloroformate) and various conditions were evaluated (temperature, time, solvents, catalysts) in terms of efficiency. The optimised derivatisation method with diazomethane and 1% methanol running 1 hour at 5°C was used for derivatisation of extracts of soils and river sediment from historically PCB-contaminated sites; the extracts were prepared using accelerated solvent extraction by a previously described method. Methylated CBAs were separated by gas chromatography using a system with two different common columns, DB-5 and DB-200, in series-coupled (tandem) arrangement and detected by EI-MS. A clean-up with a gel permeation chromatography was carried out to remove soil interfering matrix compounds as well as major portion of PCBs. The limits of quantification ranged between 1 and 10 ng g^?1 of individual CBA in the soil. The procedure was applied to various soil samples from Lhenice (Czech Republic) highly contaminated with PCBs. CBAs were found in all tested soils and also in the river sediment. The most contaminated soil contained all CBAs representatives under the study with a total concentration of 3.1 µg g^?1 of dry soil.     

Modified determination of dihydrostreptomycin in kidney, muscle and milk by HPLC.

A method is presented for the determination of dihydrostreptomycin in milk, muscle and kidney by reversed-phase ion-pair high-performance liquid chromatography and post-column derivatisation with beta-naphthoquinone-4-sulfonate prior to fluorescence detection. The new sample work-up procedures include acid precipitation of proteins and, in the case of muscle and kidney, removal of fats by solvent extraction followed by solid phase extraction on a cation exchanger. The fluorescence response was linear from 25 to 2000 micrograms l-1 of injected analyte. The detection limits were 10 micrograms kg-1 for milk and 15 micrograms kg-1 for muscle and kidney and the analyte recoveries were on average 93% for milk, 70% for kidney and 75% for muscle.     

Supercritical fluid extraction (SFE) as a multi-residue extraction procedure for beta-agonists in bovine liver tissue

A supercritical fluid extraction procedure has been developed for the extraction of beta-agonists in bovine liver samples. The method is suitable for compounds of different beta-agonist classes: the substituted aniline-type compounds (e.g. clenbuterol) and the phenolic-type compounds (e.g. salbutamol), including conjugated forms of the latter. The developed procedure involves a combination of supercritical fluid extraction with enzyme immunoassay for the determination of clenbuterol and salbutamol residues at the low ppb level. Addition of methanol modifier and removal of sample moisture are necessary for the extraction of more polar analytes such as salbutamol. Method validation incorporating intra- and inter-assays was carried out on fortified liver tissue and showed good recovery and low variation (RSD < 15%). An enzyme hydrolysis procedure was incorporated into the method for the deconjugation of conjugated residues. The developed procedure was shown to be successful for the determination of both clenbuterol and salbutamol in incurred liver tissue.     

A method for the separation of residues of nine compounds in cattle liver related to treatment with oxfendazole

A method for the determination of nine compounds closely related to oxfendazole has been developed for the monitoring of residues in food. The method is based on a multi-residue procedure for basic drug residues and used strong cation exchange solid phase extraction for sample clean-up. These nine compounds include fenbendazole, which is itself a licensed veterinary product. The pro-drug febantel converts quickly to fenbendazole or oxfendazole soon after administration. The method is therefore suitable for monitoring residues following the use of any of these compounds. Some of these analytes have been shown to be present as residues following the treatment of farm animals with oxfendazole. Average recoveries for the nine compounds from tissue fortified with 100 micrograms kg-1 were between 34% and 96% with relative standard deviations between 3% and 22%.     

Analysis of trace levels of pesticides in rainwater by SPME and GC-tandem mass spectrometry after derivatisation with PFFBr

Solid-phase microextraction (SPME) was used for the analysis of some pesticides (bromoxynil, chlorotoluron, diuron, isoproturon, 2,4-MCPA, MCPP and 2,4-D) in rainwater after derivatisation with PFBBr and gas chromatography-ion trap mass spectrometry. The derivatisation procedure was optimized by testing different methods: direct derivatisation in the aqueous phase followed by SPME extraction, on-fibre derivatisation and derivatisation in the injector. The best result was obtained by headspace coating the PDMS/DVB fibre with PFBBr for 10 min followed by direct SPME extraction for 60 min at 68 °C (pH 2 and 75% NaCl). Good detection limits were obtained for all the compounds: these ranged between 10 and 1,000 ng L⁻¹ with a relatively high uncertainty due to the combination of derivatisation and SPME extraction steps. The optimized procedure was applied to the analysis of pesticides in rainwater and results obtained shows that this method is a fast and simple technique to assess the spatial and temporal variations of concentrations of pesticides in rainwater.     

Optimisation of a headspace solid-phase microextraction with on-fiber derivatisation method for the direct determination of haloanisoles and halophenols in wine

A solid-phase microextraction (SPME) procedure for the determination of four haloanisoles (2,4,6-trichloroanisole, 2,3,4,6-tetrachloroanisole, pentachloroanisole and 2,4,6-tribromoanisole), as well as their precursor halophenols (2,4,6-trichlorophenol, 2,3,4,6-tetrachlorophenol, pentachlorophenol and 2,4,6-tribromophenol), involved in the presence of cork taint in wine, was developed. Firstly, analytes were concentrated on a SPME fiber, and then halophenols were derivatised using N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The compounds were desorbed for 5 min in the gas chromatography injector port and then determined with an electron capture detector. The influence of different parameters on the efficiency of extraction (volume of sample, type of fibre coating and time) and derivatisation (time, temperature and volume of MSTFA) steps was evaluated. Polyacrylate (PA) was selected as the extraction fiber, optimised parameters for SPME were 10 ml of wine, temperature 70 degrees C and extraction time 60 min. The optimal conditions identified for the derivatisation step were temperature 25 degrees C, reagent volume 50 microl and extraction time 25 min. Under optimal conditions, the proposed method showed satisfactory linearity, precision and detection limits. The method was applied successfully to the analysis of red wine samples. To our knowledge, this is the first time that headspace (HS) SPME combined with on-fiber derivatisation has been applied to determine cork taint responsible compounds in wine.     

The derivatisation of avermectins and milbemycins in milk: new insights and improvement of the procedure

Derivatisation of the avermectines ivermectin (IVM), doramectin (DOR), abamectin (ABA) and eprinomectin (EPR), and the milbemycin moxidectin (MOX) to fluorescent derivatives is commonly used for quantitative analysis at relevant levels using high performance liquid chromatography (HPLC) with fluorescence detection. Problems associated with the differences in reactivity towards derivatisation (EPM) and limited stability of the derived products (IVM, DOR, ABA) may seriously hamper the applicability of the method and the reliability of the obtained results. A study was performed to obtain more insight in this derivatisation process from an organic chemistry point of view. This study demonstrated the occurrence of two main fluorescent derivatives: the trifluoroacetyl esters (flu-TFA) and the derivatives with a free hydroxy group at the glycosidic ring (flu-OH). Optimisation of the derivatisation conditions resulted in a fast and reproducible formation of the fluorescent derivatives for all analytes including EPM. The improved procedure involves the addition of 1-methylimidazole (MI), trifluoroacetic anhydride (TFAA), triethylamine (TEA) and trifluoroacetic acid (TFA) with a subsequent incubation for 30 min at 70 °C. With this procedure for IVM, DOR and ABA flu-TFA derivatives are obtained instead of flu-OH derivatives as generally described in literature. The derivatisation is reproducible in different milk samples and the derivatives proved to be stable for at least 80 h at room temperature. Using the optimised procedure a limit of detection (LoD) of 0.1 μg kg⁻¹ in milk was readily obtained.     

Development of a liquid chromatography-tandem mass spectrometry method for the identification of natural androgen steroids and their conjugates in urine samples

An analytical procedure for the determination of natural steroids and their conjugates was developed and applied to bovine and human urine. Fifteen compounds (free and conjugated) after extraction by solid-phase were identified by liquid chromatography-tandem mass spectrometry (LC-MS-MS) in multiple reaction monitoring (MRM). Free and conjugated steroids were detected all together in the same chromatographic analysis respectively in positive and negative ionization. This procedure reveals to be time-saving, because it overcomes the need of further sample pre-treatment steps, such as enzymatic hydrolysis and derivatisation.The recovery for each compound was around 80%. Limit of detection of each steroid free or conjugated has been evaluated.     

Simultaneous Determination of Avermectin and Milbemycin Residues in Bovine Tissue by Pressurized Solvent Extraction and LC with Fluorescence Detection

A rapid and sensitive method has been developed for the simultaneous determination of four avermectins and one milbemycin residues in bovine tissue. The isolation of the analytes from muscle and liver samples was accomplished utilizing a pressurized solvent extractor. The optimized extraction procedure using acetonitrile/water (40:60, v/v) as extraction solvent, was automatically carried out at 100 °C and 10 MPa, applying two static cycles for 3 min. The extracts were cleaned up on a C₁₈ solid-phase extraction cartridge and analyzed by liquid chromatography with fluorescence detection after derivatization. Mean recoveries of the five analytes from fortified samples were between 84.8 and 101.8%, with relative standard deviations lower than 10.8%. The limit of detection and quantification were in the ranges of 0.1–0.2 and 0.5–0.6 μg kg^–1, respectively. The application of the newly developed method was demonstrated by analyzing bovine meat samples from market.     

Determination of sulfonylurea degradation products in soil by liquid chromatography-ultraviolet detection followed by confirmatory liquid chromatography-tandem mass spectrometry.

A method based on liquid extraction followed by sample enrichment on reversed-phase solid-phase extraction was developed for the extraction of five degradation products of four sulfonylurea herbicides (chlorsulfuron, metsulfuron-methyl, thifensulfuron-methyl and tribenuron-methyl) from soil. The compounds have been quantified by LC-UV and identified by tandem LC-MS with electrospray ionization or atmospheric pressure chemical ionization. The limits of detection for the five compounds were between 10 and 50 micrograms kg-1. The method has been applied to the extraction of soil samples after microbial degradation of sulfonylurea herbicides.     

In situ ethylation of organolead,organotin and organomercury species by bromomagnesium tetraethylborate prior to GC‐ICP‐MS analysis

An analytical method for simultaneous in situ ethylation, of organolead, organotin and organomercury compounds in aqueous samples was developed using a new derivatisation agent, bromomagnesium tetraethylborate (BrMgEt₄B). The determination of lead, tin and mercury compounds was done by species‐specific isotope dilution, derivatisation and GC–inductively coupled plasma MS (GC‐ICP‐MS) or by GC‐MS. The recovery and accuracy of the derivatisation were evaluated. The effect of pH and the relative quantity of derivatisation agent were studied.     

Simultaneous Analysis of Avermectins in Bovine Tissues by LC-MS-MS with Immunoaffinity Chromatography Cleanup

A rapid, simple and sensitive LC-MS-MS method was developed to simultaneously measure residues of avermectin, ivermectin, doramectin, and eprinomectin in bovine liver and muscle. The extracted samples were cleaned by an immunoaffinity column which was prepared by coupling anti-avermectin polyclonal antibodies with CNBr-activated Sepharose 4B. The residues eluted from the column were analyzed by high-performance liquid chromatography with tandem mass spectrometry. Recoveries fortified at levels 5?50 ng g^?1 ranged from 62.93 to 84.03%, with relative standard deviations from 6.02 to 17.39% for all four compounds in liver tissue. The limits of quantification were 5 ng g^?1, and limits of detection were 2.5 ng g^?1 in liver for the four drugs. The procedure was also applied to bovine muscle, giving similar results.     

Determination of dimetridazole, ronidazole and their common metabolite in poultry muscle and eggs by high performance liquid chromatography with UV detection and confirmatory analysis by atmospheric pressure chemical ionisation mass spectrometry.

A method was developed for the determination of the nitroimidazole compounds dimetridazole (DMZ) and ronidazole (RNZ) and their common metabolite, 2-hydroxymethyl-1-methyl-5-nitroimidazole (2-OH-M). Extracts obtained from a clean-up process using strong cation exchange (SCX) solid phase extraction (SPE) can be analysed either by high performance liquid chromatography with UV detection (HPLC-UV) or by high performance liquid chromatography with atmospheric pressure chemical ionisation mass spectrometry (HPLC-APCI-MS) as a confirmatory method. Up to 20 samples can be extracted in approximately 4 h. The HPLC-UV analysis had a limit of detection of 0.5 microgram kg-1. Validation in chicken muscle fortified at a concentration of 5 micrograms kg-1 gave recoveries of 75% DMZ, 77% RNZ and 81% 2-OH-M with RSDs of 16.4, 11.3 and 14.0%, respectively (n = 17). Validation in egg fortified at the same concentration gave recoveries of 77% DMZ, 80% RNZ and 80% 2-OH-M, with RSDs of 14.9, 22.0 and 18.2%, respectively (n = 18). The limit of detection of the HPLC-APCI-MS method was 0.1 microgram kg-1 for DMZ and RNZ and 0.5 microgram kg-1 for 2-OH-M. This method gave mean recoveries in fortified egg samples of 65% DMZ, 87% RNZ and 75% 2-OH-M with RSDs of 22, 11 and 14%, respectively (n = 10). The ratios of the peak areas of the molecular ion and a fragment ion were monitored as added confirmation of the presence of the analyte. Both the HPLC-UV screening procedure and the HPLC-APCI-MS confirmatory method have subsequently been used for the analysis of several hundred samples as part of UK surveillance programmes.     

Thin-layer chromatographic screening method for the tranquillizers azaperone, propiopromazine and carazolol in pig tissues

A procedure is described for the detection of azaperone, propiopromazine and carazolol in pig muscle, liver and kidney tissue. The method comprises extraction from an alkaline tissue homogenate with diethyl ether, followed by cleaning up and concentration of the extract on a silica gel solid-phase extraction column. Two-dimensional thin-layer chromatography on a silica plate was used for the detection of the tranquillizers. Detection levels were 25 micrograms kg-1 for propiopromazine, 50 micrograms kg-1 for azaperone (or its metabolite azaperol) and 125 micrograms kg-1 for carazolol. In pigs treated with the usual doses the presence of propiopromazine and azaperol could be established in kidney tissue 8 h after administration, whilst in injection sites all three tranquillizers could be detected.     

Reversed-phase high-performance liquid chromatographic determination of dexamethasone in bovine tissues.

A sensitive and selective high-performance liquid chromatographic procedure is described for the determination of the synthetic corticosteroid dexamethasone (DXM), in bovine muscle, kidney, liver and fat tissues, using methylprednisolone as the internal standard. Following extraction with ethyl acetate (muscle, kidney and liver) or diethyl ether (fat) and clean-up of the tissue extract, the drug residue was isolated using a C18 solid-phase extraction column. Separation of DXM was achieved by reversed-phase high-performance liquid chromatography with ultraviolet detection at 254 nm. By using this procedure, DXM levels as low as 0.01 mg kg-1 can be detected in muscle, kidney, liver and fat.     

Screening and confirmation of triphenylmethane dyes and their leuco metabolites in trout muscle using HPLC-vis and ESP-LC-MS.

An extraction and clean-up protocol for the determination of Malachite Green and Crystal Violet and the corresponding leuco compounds in trout muscle has been developed. Final determination is by HPLC with visible (screening) or ESP-MS (confirmation) detection. In both cases lead(IV) oxide was used on-line to oxidise the leuco compounds back to the parent after chromatographic separation and prior to detection. The procedure was validated down to 2 micrograms kg-1. Intra- and inter-batch precision was measured at 3 levels for all compounds. Recoveries were in the range 66-116% with RSD of 1-17% for determination by HPLC with visible detection. For LC-MS determination, recoveries were in the range 61-94% with RSD of 4-15%. Limited surveillance data indicated that Malachite Green usage was more effectively monitored by including the leuco compound as well as the parent (9 positives for the leuco compound as opposed to 1 for Malachite Green out of 31 samples analysed).     

Residue study of doxycycline and 4-epidoxycycline in pigs medicated via-drinking water.

A study was performed to determine the residues in edible tissues of healthy pigs after continuous administration of doxycycline with drinking water for five consecutive days at a dose rate of 10.5 mg doxycycline kg-1 body weight (BW) per day. Quantitation was performed using a validated HPLC method with fluorescence detection. The method was able to separate doxycycline and its 4-epimer, 4-epidoxycycline. This epimer was found in kidney, liver, skin, fat and muscle tissue. The method was validated at the maximum residue limit (MRL), at half the MRL and at double the MRL for both doxycycline and 4-epidoxycycline. Linear calibration curves were obtained in spiked tissues (r > 0.99). The accuracy of the calibrators of the calibration curves was within -20% to +10%. The accuracy and precision (expressed as the within-run repeatability) were found to be within the required ranges for the specific concentration. The limits of detection and limits of quantification were below one-half of the MRL. The quantification limits were 50 micrograms kg-1 for doxycycline and 100 micrograms kg-1 for 4-epidoxycycline in kidney and liver, 20 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in skin and fat and 10 micrograms kg-1 for doxycycline and 50 micrograms kg-1 for 4-epidoxycycline in muscle tissue. The withdrawal time was calculated according to the recommendations of the European Agency for the Evaluation of Medicinal Products (EMEA/CVMP/036/95) and was set at 3 days. The plasma concentration of doxycycline and the stability of doxycycline in drinking water were also determined during the treatment period. The mean plasma concentration of doxycycline during the treatment period ranged from 0.83 to 0.96 microgram ml-1. Thirty-six hours after the withdrawal from medicated drinking water, no plasma levels could be detected. Samples of medicated water were taken at time zero and at 24 h after addition of doxycycline to the drinking water. No statistically significant difference in the mean drinking water concentration was seen at time zero and at time 24 h (Student's t-test, alpha = 0.05).     

High-performance liquid chromatographic method for the determination of a potential memory-enhancing compound (CL 275,838) and its desbenzyl metabolite in rat plasma or serum.

A rapid, selective, precise reversed-phase liquid chromatographic method has been developed for the determination of a potential memory-enhancing agent (CL 275,838) and its main metabolite (CL 286,527) in plasma and serum. The procedure includes isolation of compounds from proteins precipitated with acetonitrile, subsequent resolution by reversed-phase (Whatman Partisphere C8) high-performance liquid chromatography and ultraviolet detection. The assay was linear over the range 0.12-1.25 micrograms/ml of plasma or serum. The detection limit was 0.12 micrograms/ml, using 0.2 ml of plasma or serum. Intra- and inter-day validation studies indicated an acceptable precision and reproducibility of the method within the concentration range investigated, the overall coefficient of variation being less than 10%. The method is currently applied in support of pharmacological and toxicity studies of the compound in rodents.