Determination of cymoxanil in drinking water and soil using high-performance liquid chromatography

We describe a method for determination of cymoxanil, 1-2-cyano-2-methoxy(iminoacetyl)-3-ethylurea, in drinking water and in soil, using reversed-phase HPLC with UV detection at 240 nm and a mobile phase of acetonitrile-water (30:70, v/v). Fortified water samples (1.0 L) were extracted with solid-phase extraction on Strata X. Soil samples (20 g) were extracted with acetone and the extracts were transferred onto Strata C18E. The recoveries of cymoxanil from water and soil samples were over 85% for each fortification level. The RDS were within the range 1.7-4.1% for water and 0.9-1.2% for soil samples. After optimization of the extraction and separation conditions, the method was validated.     

Determination of methylbenzoquate in premixes and animal feeds using liquid chromatography with fluorescence detection

A rapid analytical procedure was developed and tested for routine identification and quantification of methylbenzoquate in feeds by liquid chromatography (LC). The ground feed samples were extracted using methanol-water (80 + 20, v/v) at 65 degrees-70 degrees C in a water bath for premixes and in dichloromethane at 45 degrees C in a water bath for final feeds, respectively. The extract of final feeds was cleaned using solid-phase extraction on silica columns. Both the final feed and premix extracts were analyzed by reversed-phase LC on a NovaPak C18 column (3.9 x 150 mm; 4 microm) with methanol-acetonitrile-water-phosphoric acid (340 + 350 + 308 + 2, v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 265 and 390 nm, respectively. Alternatively, post-column addition of sulfuric acid solution was used to decrease the determination limit. The recovery of methylbenzoquate, in a concentration range of 0.5-10 mg/kg, was 105.0 +/- 7.3%. The limit of quantitation, based on a signal-to-noise ratio of 10:1, was 48 microg/kg. The developed LC method was tested in an interlaboratory study. The interlaboratory repeatibility for both samples ranged from 7.1 to 10.6%; the interlaboratory reproducibility ranged from 11.7 to 15.2%. With the post-column addition of sulfuric acid, the limit of quantification was decreased by a factor of 50. Overall, the developed method is highly selective and can be used in routine analysis.     

Determination of lafutidine in human plasma by high-performance liquid chromatography-electrospray ionization mass spectrometry: application to a bioequivalence study

A rapid, sensitive and specific high-performance liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) method was developed and validated for the first time to determine the concentration of lafutidine in human plasma. After the addition of diazepam (the internal standard, IS) and 1 M sodium hydroxide solution to 0.5-ml plasma sample, lafutidine was extracted from plasma with n-hexane : isopropanol (95 : 5, v/v). The organic layer was evaporated and the residue was redissolved in 200-microl mobile phase. The analyte was chromatographically separated on a prepacked Shimadzu Shim-pack VP-ODS C(18) column (250 x 2.0 mm i.d.) using a mixture of methanol-water (20 mM CH(3)COONH(4)) = 80 : 20 (v/v) as mobile phase. Detection was performed on a single quadrupole mass spectrometer using an electrospray ionization interface and the selected-ion monitoring (SIM) mode. The method showed excellent linearity (r = 0.9993) over the concentration range of 5-400 ng/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation. The limit of detection was 1 ng/ml. The validated LC/ESI-MS method has been successfully applied to the bioequivalence study of lafutidine in 24 healthy male Chinese volunteers.     

Determination of microcystins in fish by solvent extraction and liquid chromatography

A liquid chromatography electrospray mass spectrometry (LC/ESI/MS) method has been developed to identify and quantify microcystins in fish liver and intestine. Microcystins (MCs) were extracted from 500 mg sample with methanol-water (85:25, v/v) and the extracts concentrated to 250 microl. The parameters were optimized by a full factorial 2(3) design. Neither laborious pre-treatment nor clean up were necessary. MCs were separated using conventional C18 column and an acetonitrile-acidified water (pH 3) gradient. Negative samples (without MCs) were discriminated by liquid chromatography diode array detection (LC/DAD). The limits of detection (LOD) and the limits of quantification (LOQ) resulted equal for MC-RR, MC-YR, and MC-LR and were 0.1 and 0.5 microg g(-1), respectively. MCs recoveries at three levels in spiked samples (0.5-3.0 microg g(-1)) were > 92%, with relative standards deviations (RSDs) < 16% for liver samples and > 68% with RSDs < 18% for intestine samples. The proposed method was applied to determine MC-LR in exposed fish to evaluate the bioaccumulation risk. The results showed the transference of MC-LR from cyanobacterial cells to fish tissues.     

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (μLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 μl injection volumes with on-column focusing. A 350×0.32 mm capillary column packed with Kromasil 100-C₁₈ of 5 μm particle size was used with a mobile phase of acetonitrile–10 mM ammonium acetate (85:15, v/v) at a flow of 5 μl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90°C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile–water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 μm poly(vinylidene difluoride) syringe filter before μLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 μg/kg. The within-day and between-day repeatabilities were in the range 4–18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.     

Stability indicating methods for the determination of norfloxacin in mixture with tinidazole

Three stability indicating assay methods are developed for the determination of norfloxacin (Nor) in the presence of its decarboxylated degradation product and in mixture with tinidazole (Tnd). The proposed methods are reversed phase ion pair liquid chromatography (LC), thin layer densitometry (TLC) and second derivative ratio spectra zero crossing spectrophotometry ((2)DD). Chromatographic separation was achieved on mu-Bondapack C18 column 5 microm (300 mm x 3.9 mm, I.D.) and precoated silica gel TLC stationary phases for LC and TLC methods, respectively. Mobile phases consisting of phosphate buffer pH 3.2 : methanol (3 : 1, v/v) containing 0.005 M pentane sulfonic acid sodium salt and isopropanol : butanol : concentrated ammonia : water (25 : 50 : 5 : 25, v/v/v/v) were used for resolution of Nor and Tnd by both techniques, respectively. Detection was carried at 280 nm. In the ratio spectra method, detection of Nor was carried at 282 nm. Linearity, accuracy and precision were found to be acceptable over concentration ranges of 20-225 microg/ml, 0.8-4 microg/spot and 1-7 microg/ml for Nor by LC, TLC and (2)DD methods and over concentration ranges of 37.5-375 microg/ml and 4.8-20 microg/spot for Tnd by LC and TLC methods respectively. The suggested methods were successfully applied for the determination of both drugs in bulk powder, laboratory prepared mixtures and in commercial samples. Statistical comparison between the results obtained by the proposed and the reference methods was carried out using Student t-test, F ratio and one way ANOVA.     

Monitoring of the pesticide diazinon in soil, stem and surface water of rice fields.

Diazinon is an organophosphorus insecticide (OPP) that is used as a pesticide for Chilo suppressalis (WLK) (Lep., Pyralidae) in rice fields. The extraction of diazinon from soil and the stems of rice plants has been carried out by microwave-assisted extraction (MAE) and the results compared with ultrasonic extraction (USE). The best parameters for MAE are hexane-acetone (8:2 v/v) as a solvent, a 2.5 min extraction time, and 20 ml of the solvent volume. Also, surface-water samples of the rice fields were extracted by solid phase extraction (SPE) using a C18 disc. The optimum conditions of SPE were a sample volume of 750 ml, a pH of 7 and high ionic strength of water. The extracted samples were analyzed by gas chromatography-mass spectrometry (GC-MS). The relative standard deviation (RSD) and regression coefficients related to the linearity were <3.5% (n = 5) and 0.99, respectively. The limit of detection (LOD) is 0.1 ng ml(-1) with selected ion monitoring (SIM) at 137 m/z. The average recoveries of diazinon in soil and stem samples by MAE and surface-water by SPE were 98% (+/-3), 94% (+/-5) and 87% (+/-3), respectively. In June, the concentration of diazinon in soil and stem samples of the rice plants in Guilan province is high (55 ng ml(-1)) and in September is low (2 ng ml(-1)). In surface-water samples, the results are converse. In November, diazinon can not be detected in soil, stem or surface-water samples. Diazinon is degraded to diethylthiophosphoric acid. Also, three microorganism genera (Pseudomonas sp, Flavobacterium sp and Agrobacterium sp) have been found to degrade diazinon in soil and surface water.     

Determination of six kavalactones in dietary supplements and selected functional foods containing Piper methysticum by isocratic liquid chromatography with internal standard

Kava (Piper methysticum) dietary products have been sold worldwide for treatment of nervous anxiety, tension, and restlessness. Recent reports showed potential association of kava usage and liver injuries. This study was conducted to develop simple and reliable methodologies for the extraction and determination of 6 major kavalactones: (+)-methysticin, (+)-dihydromethysticin, (+)-kavain, (+)-dihydrokavain, yangonin, and desmethoxyyangonin. Ultrasonic extraction techniques and isocratic reversed-phase liquid chromatography (LC) were optimized for different types of samples, including capsules containing kava root extract or root powder, raw root material, tea bags, and snack bar. A suitable internal standard, 5,7-dihydroxyflavone, was used for LC calibration. Kavalactones were completely separated in 30 min using a Luna C18-2 column at 60 degrees C with an isocratic mobile phase consisting of 2-propanol-acetonitrile-water-acetic acid (16 + 16 + 68 + 0.1, v/v/v/v). Within-laboratory, intraday, and interday method variation (% relative standard deviation) for most samples extracted by methanol or methanol-water mixture were <5%. Lower levels of kavalactone contents and higher variations were observed for tea bags from water extraction or infusion as compared to methanol extraction. Labeling information of tea bags based on methanol extraction could be misleading to consumers. Analytical recoveries of snack bar fortified at 10 and 20 microg/g were >84% with RSD values <8%. Methods developed in this study offer a simple and reproducible means for analysis of kavalactones in various matrixes of dietary products.     

Analysis of Metribuzin and transformation products in soil by pressurized liquid extraction and liquid chromatographic-tandem mass spectrometry

A method developed for study of metribuzin degradation in soil is presented. LC-MS-MS and electrospray ionisation was used for analysis of metribuzin and the metabolites deaminometribuzin (DA), diketometribuzin (DK) and deaminodiketometribuzin (DADK). Soil samples were extracted by pressurized liquid extraction using methanol-water (75:25) at 60 degrees C. In general, recoveries were about 75% for metribuzin, DA and DADK and their detection limit in soil was 1.25 microg/kg. Lower sensitivity was observed for DK, with detection limit at 12.5 microg/kg and recovery about 50%.     

Amnesic shellfish poisoning toxins in shellfish: estimation of uncertainty of measurement for a liquid chromatography/tandem mass spectrometry method

A liquid chromatography/mass spectrometry (LC/MS) method for amnesic shellfish poisoning toxins in shellfish was developed and validated. Tissue homogenate (4 g) was extracted with 16 mL methanol-water (1 + 1, v/v). Dilution into acetonitrile-water (1 + 9, v/v) was followed by C18 solid-phase extraction cleanup. Domoic acid (DA) and epi-domoic acid were determined by LC/MS/MS with electrospray ionization and multiple reaction monitoring. External calibration was performed with dilutions of a certified reference standard. Advantages of this method include speed, lower detection limits, and a very high degree of specificity. The LC/MS response was highly linear, and there were no significant interferences to the determination of DA. Formal method validation was performed on 4 shellfish species. Fortification studies gave recoveries (mean +/- SD; n = 24) of 93 +/- 14% at 1 mg/kg, and 93.3 +/- 7.6% at 20 mg/kg over all the species. Analysis of a mussel certified reference material showed the bias as < 5%. The limits of detection and quantitation were 0.15 and 0.5 mg/kg, respectively. Routine application of the method over 4 months gave a recovery for the QC sample (1 mg/kg fortified blank mussel homogenate) run with each batch of 88.9 +/- 5.5% (mean +/- SD; n = 37). The total uncertainty of measurement results were estimated as 0.12 (12%) at 0.25-5 mg/kg and 0.079 (7.9%) at 5-50 mg/kg. The major contribution to the uncertainty was the repeatability of the LC/MS determination, probably arising from subtle matrix effects.     

Determination of imidacloprid in paddy water and soil by liquid chromatography electrospray ionization-tandem mass spectrometry

A method for the determination of imidacloprid in paddy water and soil was developed using liquid chromatography electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS). Separation of imidacloprid was carried out on a Shimadzu C18 column (150 mm × 4.6 mm, 4.6 μm) with an acetonitrile-water (50: 50, v/v) mobile phase containing 0.1% of acetic acid. The flow rate was 0.3 mL/min in isocratic mode. The product ion at 209 m/z was selected for quantification in multiple-reaction monitoring scan mode. Imidacloprid residues in soil were extracted by a solid-liquid extraction method with acetonitrile. Water samples were filtered and directly injected for analysis without extraction. Detection limits of 0.5 μg/kg and 0.3 μg/L were achieved for soil and water samples, respectively. The method had recoveries of 90 ± 2% (n = 4) for soil samples and 100 ± 2% (n = 4) for water samples. A linear relationship was observed throughout the investigated range of concentrations (1–200 μg/L), with the correlation coefficients ranging from 0.999 to 1.000.     

Determination of benzimidazole fungicides in soil samples using microwave-assisted micellar extraction and liquid chromatography with fluorescence detection

A simple and fast analytical method was developed for the determination of benzimidazole fungicides (benomyl, carbendazim, thiabendazole, and fuberidazole) in soil samples. The analytes were extracted from the soil samples by means of conventional microwave-assisted extraction, using the non-ionic surfactants polyoxyethylene 10 lauryl ether (POLE) and oligoethylene glycol monooalkyl ether (Genapol X-080) as extractants. Determinations were made by using liquid chromatography with direct fluorescence detection. The use of an analytical column Symmetry C-18 offered short retention times of analytes without the need of any pH regulators with mobile phase methanol-water (50 + 50, v/v). The best results were obtained using 5% (v/v) POLE as extractant with recoveries of the fungicides in spiked soil samples between 71 and 105%. The results were compared with those obtained when Soxhlet extraction was applied to the same soil samples.     

Rapid method for the simultaneous determination of six ionophores in feed by liquid chromatography/mass spectrometry

A simple and highly sensitive LC/MS method was developed for the simultaneous determination of six ionophores--lasalocid, monensin, laidlomycin, maduramycin, salinomycin, and narasin--in feed. The procedure involved extraction of 1 g of feed with 4 mL of methanol-water (9 + 1, v/v) by shaking on a platform shaker for 45 min. After centrifugation, the extracts were diluted with methanol-water (75 + 25, v/v) and analyzed without any cleanup. The analysis was performed on a Betasil C18 column (150 x 4.6 mm id, 5 pm particle size) connected to an LC/MS system operated in the atmospheric pressure chemical ionization (APCI) mode. We believe this to be the first method that uses the APCI mode for the analysis of ionophores. The mobile phase consisted of 50 mM ammonium acetate as solvent A and acetonitrile-methanol (7 + 3, v/v) as solvent B in a gradient run. Excellent recoveries of 81-120% were found for all compounds at fortification levels of 1-200 microg/g, with RSD < or =15% (except 17% for maduramycin at 2 and 5 microg/g, and 16% for salinomycin at 1 microg/g). At 0.5 microg/g, recoveries of 87-119% were obtained, with RSD < or =20%. However, recovery of lasalocid was 133% and salinomycin 79% in sow and horse feed, respectively. Average RSD values of lasalocid and salinomycin were 22 and 21%, respectively. Finally, proficiency test samples analyzed with the method demonstrated favorable agreement with the certified values.     

Determination of trace amounts of off-flavor compounds in drinking water by stir bar sorptive extraction and thermal desorption GC-MS.

A method for the determination of trace amounts of off-flavor compounds including 2-methylisoborneol, geosmin and 2,4,6-trichloroanisole in drinking water was developed using the stir bar sorptive extraction technique followed by thermal desorption-GC-MS analysis. The extraction conditions such as extraction mode, salt addition, extraction temperature, sample volume and extraction time were examined. Water samples (20, 40 and 60 ml) were extracted for 60-240 min at room temperature (25 degrees C) using stir bars with a length of 10 mm and coated with a 500 microm layer of polydimethylsiloxane. The extract was analyzed by thermal desorption-GC-MS in the selected ion monitoring mode. The method showed good linearity over the concentration range from 0.1 or 0.2 or 0.5 to 100 ng l(-1) for all the target analytes, and the correlation coefficients were greater than 0.9987. The detection limits ranged from 0.022 to 0.16 ng l(-1). The recoveries (89-109%) and precision (RSD: 0.80-3.7%) of the method were examined by analyzing raw water and tap water samples fortified at the 1 ng l(-1) level. The method was successfully applied to low-level samples (raw water and tap water).     

Simultaneous determination of chlorzoxazone and ketoprofen in binary mixtures and in ternary mixtures containing the chlorzoxazone degradation product by reversed-phase liquid chromatography

New, simple, rapid, and precise reversed-phase high-performance liquid chromatographic (LC) methods were developed for the simultaneous determination of chlorzoxazone (CH) and ketoprofen (KT) in binary mixtures and in ternary mixtures containing the CH degradation product, 2-amino-4-chlorophenol (CD). The analytes were separated by LC on a Lichrosphere 60 C18 column (250 x 4 mm, 5 microm). The mobile phases, methanol-water (40:60, v/v) at 1 mL/min and methanol-0.05% phosphoric acid (60:40, v/v, pH 2.81) at 1.5 mL/min, satisfactorily resolved the binary and ternary mixtures, respectively. The UV detector was operated at 280 nm for the determination of CH and at 254 nm for the determination of KT and CD. Linearity, accuracy, and precision were found to be acceptable over the concentration ranges of 20-240 and 5-60 microg/mL for CH and KT, respectively, in the binary mixtures and 50-300, 10-60, and 20-160 microg/mL for CH, KT, and CD, respectively, in the ternary mixtures. The optimized methods proved to be specific, robust, and accurate for the quality control of CH and KT in pharmaceutical preparations.     

Liquid chromatographic determination of fosthiazate residues in environmental samples and application of the method to a fosthiazate field dissipation study

A method was developed and validated for the determination of residues of the organophosphorus nematicide fosthiazate in soil and water by using reversed-phase liquid chromatography with UV detection. Good recoveries (>85%) of fosthiazate residues were obtained from water samples (drinking water, groundwater, and liquid chromatography water) after passage of 0.5-2 L water through solid-phase extraction (SPE) C-18 cartridges and subsequent elution with ethyl acetate. Residues in soil were extracted with methanol-water (75 + 25, v/v) on a wrist-action shaker, and the extract was cleaned up on C-18 SPE cartridges before analysis. The method was validated by analysis of a range of soils with different physicochemical characteristics; recoveries exceeded 87% at fortification levels ranging from 0.02 to 5.0 mg/kg. The precision values obtained for the method, expressed as repeatability and reproducibility, were satisfactory (<11%). Fosthiazate detection limits were 0.025 microg/L and 0.005 mg/kg for water and soil samples, respectively. The decline in the levels of fosthiazate residues in soil was measured after application of fosthiazate to protected tomato cultivation. The dissipation of fosthiazate residues followed first-order kinetics with a calculated half-life of 21 days.     

Simultaneous determination of spironolactone and its active metabolite canrenone in human plasma by HPLC-APCI-MS

A sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the simultaneous determination of spironolactone and its active metabolite canrenone in human plasma has been developed and validated. After the addition of estazolam as the internal standard (IS), plasma samples were extracted with methylene chloride : ethyl acetate mixture (20 : 80, v/v) and separated by high-performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol-water (57 : 43, v/v). Analytes were determined in a single quadrupole mass spectrometer using an atmospheric pressure chemical ionization (APCI) source. LC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.25 for spironolactone and canrenone, m/z 295.05 for estazolam. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of spironolactone and canrenone were linear over the range 2-300 ng/ml. The within- and between-batch precisions (relative standard deviation (RSD)%) were lower than 10% and the accuracy ranged from 85 to 115%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2 ng/ml. The proposed method was successfully applied to study the pharmacokinetics of spironolactone and its major metabolite in healthy male Chinese volunteers.     

Determination of benzoylurea insecticide residues in tomatoes by high-performance liquid chromatography with ultraviolet-diode array and atmospheric pressure chemical ionization-mass spectrometry detection

A simple and sensitive method using high-performance liquid chromatography/ mass spectrometry (LC/MS) was developed and validated for simultaneous determination of 5 benzoylurea insecticides-diflubenzuron, triflumuron, teflubenzuron, lufenuron, and flufenoxuron-in tomatoes. Residues were successfully separated on a C18 column by methanol-water isocratic elution. Detection was carried out by an ultraviolet diode array detector (UV-DAD) coupled with a quadrupole mass spectrometer, using atmospheric pressure chemical ionization (APCI) in negative-ion mode. The main ions were the deprotonated molecules [M-H]- for triflumuron, and the anions formed by elimination of hydrofluoric acid [M-H-HF]- for diflubenzuron and flufenoxuron, and [M-2H-HF] for lufenuron and teflubenzuron. The calibration plots were linear for both detectors over the range 0.05 to 10 microg/mL, and the method presented good quality parameters. The limits of detection for standard solutions were 0.008-0.01 mg/L (equivalent to 0.08-0.1 ng injected) for both detectors, and the limits of quantification (LOQs) were approximately 10 times lower than national maximum residue levels (MRLs). Depending on the compound and the detector, the LOQ values ranged from 0.2 to 0.4 ng injected. The optimum LC-UV-DAD/APCI-MS conditions were applied to the analysis of benzoylureas in tomatoes. The obtained recoveries from fortified tomato samples (50 g), extracted with ethyl acetate and purified by solid-phase extraction on silica sorbent, were 88-100 and 92.9-105% for the UV-DAD and MS detectors, respectively, with precision values (relative standard deviations) of 2.9-11 and 3.7-14%, respectively. The method was applied to 12 tomato samples from local markets, and diflubenzuron and lufenuron were detected in only one sample at concentrations lower than the MRLs. The results indicate that the developed LC/MS method is accurate, precise, and sensitive for quantitative and qualitative analysis at low levels of benzoylureas required by legislation.     

Determination of mepiquat chloride residues in cotton and soil by liquid chromatography/mass spectrometry

A liquid chromatographic/mass spectrometric (LC/MS) method is reported for the determination of the onium-type plant growth regulator mepiquat chloride in cotton and soil. The pesticide was extracted from the sample with ethanol and water containing 2% NH4Cl. The extract was cleaned up on a solid-phase extraction C18 column, and the pesticide was determined by LC/MS. The average recoveries were 85.9-93.8, 81.3-91.7, and 78.1-94.7%, with corresponding relative standard deviations of 3.4-9.6, 3.7-12.3, and 4.0-9.8%, from soil, cotton leaves, and cotton seeds, respectively. A coefficient of determination of R2 = 0.9964 was obtained for the analyte calibration graph, from 0.05 to 100 microg/mL. Decision limits, CCalpha, and detection capability, CCbeta, were calculated. Electrospray ionization LC/MS in the positive-ion mode (ESI+) was used to detect mepiquat chloride in extracts of soil, cotton leaves, and cotton seeds. The ion at m/z 114 in the mass spectrum was monitored.     

Single-laboratory validation of a modified liquid chromatographic method with UV detection for determination of trenbolone residues in bovine liver and muscle

Trenbolone acetate is a synthetic testosterone analog registered for use in a number of countries as a growth-promoting hormone, applied as an implant in the ears of feedlot cattle. The method is intended for the detection and quantitation of trace amounts of alpha- and beta-trenbolone in bovine tissues (muscle, liver) by liquid chromatography (LC) with UV detection and eliminates the use of the structural analog, 19-nortestosterone, as an internal standard. Trenbolone residues are extracted from tissues that have been homogenized in sodium acetate with a 3-phase liquid-liquid extraction by adding a mixture of water-acetonitrile-dichloromethanehexane, with trenbolone residues preferentially partitioned into the middle acetonitrile layer. The extract is passed through solid-phase extraction cartridges (both C18 and silica gel) using, respectively, methanol-water and acetone-toluene as eluents. Reversed-phase high-performance LC separation is performed, an octadecyl-bonded column with methanol-acetonitrile-water used as mobile phase for sample analysis. The limit of detection is 0.2 ng/g in muscle tissue and 0.6 ng/g in liver tissue, with coefficients of variation of 3.5-12.1% for alpha- and beta-trenbolone at concentrations from 0.2 to 4.0 ng/g fortified in muscle and 3.3-26.0% from liver fortified at 0.6-10.0 ng/g. Absolute recoveries of 40-130% were observed, but the use of fortified matrix curves eliminated recovery correction. Critical control points were identified in a pH adjustment step and an evaporation step during method validation, which included ruggedness testing. Analysis of incurred tissues (bovine liver and muscle) stored at -20 degrees C for over 25 weeks did not identify any significant loss of residues.