Determination of residues of azamethiphos in salmon tissue by liquid chromatography with fluorescence detection.

A liquid chromatographic (LC) method with fluorescence detection (FLD) is described for determining residues of the pesticide azamethiphos (AZA) in salmon tissue. The sample is extracted with ethyl acetate, centrifuged, dehydrated with anhydrous sodium sulfate, evaporated, reconstituted in water, and defatted with hexane. The aqueous phase is passed through a C18 solid-phase extraction (SPE) column. The SPE column is eluted with methanol, and the eluate is evaporated to dryness and then taken up in 10% acetonitrile (ACN) in water. The analyte is determined by LC using a C18 column, ACN-H2O (32 + 68) mobile phase, and FLD with excitation at 230 nm and emission at 345 nm. Composited salmon tissues were fortified with AZA at 5, 10, 21, 42, and 83 ng/g or ppb (target level, X = 10 ng/g). Overall recoveries were 86%, with between-day variability of 5.3%. The method detection limit was calculated as 1.2 ppb AZA based on a 5 g sample. The limit of quantitation as determined empirically by this method is the lower limit of the standard curve, approximately 5 ppb.     

Multiresidue chromatographic method for the determination of macrolide residues in muscle by high-performance liquid chromatography with UV detection.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of tilmicosin, tylosin, spiramycin, and its major metabolite neospiramycin was developed that is suitable for porcine, bovine, and poultry muscles. Macrolide residues were extracted from muscle with acetonitrile, fat was removed by liquid-liquid extraction with isooctane, and the extract was then cleaned on Bond Elut C18 cartridges. The HPLC separation was performed on an Inertsil ODS3 C18 column (150 x 4 mm) with 0.05% trifluoroacetic acid-acetonitrile in a gradient mode. Two different chromatographic gradients were used for tilmicosin-tylosin and spiramycin-neospiramycin, and the detection wavelengths were 287 and 232 nm, respectively. The method was validated from 1/2 the maximum residue limit (MRL) to 4 times the MRL with pork muscle samples. Mean recoveries were 60, 63.5, 51, and 42% for tilmicosin, tylosin, spiramycin, and neospiramycin, respectively. The detection limits are 15 micrograms/kg for tilmicosin and tylosin, 30 micrograms/kg for spiramycin, and 25 micrograms/kg for neospiramycin. Linearity, precision, and accuracy of the method were also tested.     

Determination of benomyl residues in shiitake mushrooms (Lentinula edodes) by liquid chromatography with UV detection

A method is optimized to determine benomyl (as carbendazim derivative) in shiitake mushrooms. It is based on the extraction of the fungicide with an ethyl acetate-hexane mixture and a further analysis of the extract by high-performance liquid chromatography-UV. Mean recoveries are evaluated and range from 76% to 86% with relative standard deviation between 1.1% and 5.8%. The limit of quantitation (0.5 mg/Kg) is lower than the maximum residue level established by European legislation. The method is successfully applied to the analysis of shiitake mushrooms cultivated on eucalyptus logs treated with Benlate 500 (benomyl as active ingredient) under natural environmental conditions.     

Determination of polyphenols in wines by liquid chromatography with UV spectrophotometric detection

This paper describes a new chromatographic method for the determination of polyphenolic compounds in wines. The method is based on the separation of analytes by reversed-phase mode in a C18 column (2.6 μm particle size) and UV absorption spectroscopy. The elution gradient is generated from 0.1% formic acid aqueous solution and acetonitrile as an organic modifier. Experimental conditions including pH, percentage of organic modifier and elution gradient profile have been thoroughly optimized using experimental design. A multi-objective function has been defined as a criterion for obtaining a satisfactory compromise among number of compounds separated, resolution and analysis time. Multi-detection at 280, 310 and 370 nm has been utilized in order to work under the most appropriate wavelengths for each compound. Figures of merit including linearity ranges, precisions, detection limits and recoveries have been established under selected experimental conditions using synthetic standards and commercial red wines. The method has been applied to analyze red wines from various Spanish regions.     

Pyraflufen-ethyl residues in soil by solid phase extraction and high-performance liquid chromatography with UV detection.

Solid-phase extraction (SPE) procedure for cleanup followed by HPLC-UV method has been investigated for the determination of pyraflufen-ethyl residues in soil. The pesticide is extracted from the sample with acetone-water (80:20, v/v) and the extract is loaded onto an octadecyl (C(18)) column. The pesticide is eluted with acetonitrile and determined by HPLC with a UV detector. Using an acetone-water extraction followed by a C(18) cleanup, this method is characterized by recovery >90.1%, precision <5.8% RSD and sensitivity of 0.01 mg/kg. The proposed method has been successfully employed for the determination of the degradation dynamics of pyraflufen-ethyl in four agricultural soil samples under laboratory conditions.     

Determination of tranquilisers and carazolol residues in animal tissue using high-performance liquid chromatography with electrochemical detection.

A multi-residue method for the determination of tranquiliser residues in animal tissue is described. The procedure may be used to determine residues of the tranquilisers acepromazine, azaperone, chlorpromazine, haloperidol, propionylpromazine, xylazine, the metabolite of azaperone, azaperol, and the beta-adrenoreceptor blocking agent carazolol. Existing methods of analysis for tranquilisers are based on ultraviolet and fluorescence detection and have been used for pig kidney analysis. Determination in this method was by high-performance liquid chromatography with electrochemical detection in the screen mode. The enhanced selectivity offered by the electrochemical detector allowed determination in liver extracts, which often give rise to more interferences on chromatographic traces when using conventional methods of detection. The method offers up to a ten-fold improvement in limits of determination over methods using ultraviolet and fluorescence detection. Recoveries and coefficients of variation have been determined in the range 2-25 micrograms/kg in pig kidney and liver. This electrochemical detection method has been used to measure residues in routine surveillance programmes.     

Determination of ethambutol by ion-pair reversed phase liquid chromatography with UV detection

An ion-pair reversed phase liquid chromatography method for the antituberculosis drug ethambutol hydrochloride was developed using sodium 1-heptanesulfonate (4.0 mg/ml) as an ion-pairing (IP) reagent. To enable detection of the ethambutol with a UV detector without sample pretreatment, the pH 4.5 aqueous tetrahydrofuran (THF) (25%, v/v) mobile phase contained 1.0 mM Cu(II), which forms a UV-absorbing complex with the analyte. At a column temperature of 35 °C, ethambutol gives a symmetrical peak with a retention time of 5 min. Chromatographic conditions were optimized through study of the effects of mobile phase composition and pH, Cu(II) and IP reagent concentration, and column temperature. The method is shown to be simple, precise, efficient, robust, linear up to at least 0.25 mg/ml, and to have a limit of quantitation of 6 μg/ml.     

Determination of atazanavir in human plasma by high-performance liquid chromatography with UV detection

A high-performance liquid chromatographic method for the determination of atazanavir (ATV) in human plasma is developed and validated. The method involves a rapid and simple solid-phase extraction (SPE) of ATV using Bond-elut C18 3 mL cartridge. The separation of ATV from internal standard and endogenous components is achieved using an isocratic elution on an octyl column and an UV detector set at 260 nm. The method is linear from 20 to 10,000 ng/mL (mean r2 = 0.9991, n = 10). The observed intra- and inter-day assay precision ranged from 2.2% to 14.7%[at the lower limit of quantitation (LOQ)], whereas accuracy varies between 1.0% and 14% (at LOQ). Mean drug recovery is 80.5% for ATV and 78.4% for IS. The method is found to be precise and accurate, practical enough for therapeutic drug monitoring in routine clinical practice and is applied for the assessment of 24-h ATV plasma concentration-time profiles in HIV-infected pregnant women.     

Determination of amoxicillin in trout by liquid chromatography with UV detection after derivatization

A liquid chromatographic (LC) method based on solid-phase extraction was developed for determination of amoxicillin in muscle tissue of rainbow trout. The compound was extracted in an aqueous solution by precipitation of organic material with a mixture of sulfuric acid and sodium tungstate. The extract was processed by solid-phase extraction on an end-capped phenyl sorbent, and concentrated on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. The extract was derivatized and analyzed by reversed-phase gradient LC on a C18 column with UV detection at 323 nm. The method detection limit was 2.9 micrograms/kg. Mean recovery in muscle was 80.5% (range 10-200 micrograms/kg). The method was applied to fillets from trout offered feed containing amoxicillin in an aquaculture pilot plant. Amoxicillin was detected in muscle tissue shortly after administration but not 3 weeks later. The relative repeatability standard deviation for incurred residues in muscle tissue was 6.4% (range 11-143 micrograms/kg).     

Determination of undecenoic acid in dermatological preparations by liquid chromatography with direct UV detection

Separation on a ODS reversed-phase column with acetonitrile—0.1% phosphoric acid (65 + 35) as mobile phase provides a simple and rapid method for the determination of undecenoic acid in dermatological preparations. Common preservatives, bactericides and antiseptics do not interfere. Tolnaftate was employed as the internal standard throughout the assay. A linear relationship was established between the peak-area ratio of undecenoic acid to the internal standard and concentration of undecenoic acid over the range 0.01–0.1 ng ml^?1.     

Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence detection

An analytical method has been developed for the quantitative determination of residues of the tranquillizer azaperone (AZN) in the kidneys of slaughtered animals. Samples were extracted with acetonitrile, extracts were acidified and further purified with solid-phase extraction (SPE) on a polymeric mixed-mode cation-exchange sorbent, Oasis. AZN and its main metabolite azaperol (AZL) were eluted by alkaline methanol (MeOH), the eluate was evaporated, re-dissolved and analysed by gradient high performance liquid chromatography (LC) on reversed and deactivated phase LiChrospher 60-RP select B at excitation and emission wavelengths of 245 and 345 nm, respectively. The method was validated according to the requirements of European Commission Decision 2002/657/EC, using fortified porcine kidneys. The method proved to be selective, specific against carazolol (CAR) and linear over a concentration range 10-150 microg kg(-1) (r2>0.99). Over a concentration range 50-150 microg kg(-1), mean recovery of AZN and AZL was 88.2 and 91.2%, respectively, with intra-laboratory reproducibility of <11.0 and <9.0%, respectively. The decision limit (CCalpha) of AZN and AZL was 112 and 111 microg kg(-1), respectively, and the limit of quantification (LOQ) was 10 and 5 microg kg(-1), respectively. The procedure was also applied to bovine, poultry and horse kidneys, giving similar results, and has been successfully implemented in statutory residue monitoring control in food of animal origin in Slovenia.     

Determination of 10 sulfonamide residues in meat samples by liquid chromatography with ultraviolet detection

A liquid chromatography (LC) method is described for the simultaneous determination of 10 commonly used sulfonamide drug residues in meat. The 10 sulfonamide drugs of interest were sulfadiazine, sulfathiazole, sulfamerazine, sulfadimidine, sulfamethizole, sulfamonomethoxine, sulfachloropyridazine, sulfadoxine, sulfadimethoxine, and sulfaquinoxaline. The residues were extracted with acetone-chloroform (1 + 1). Sulfonamides were quantitatively retained in the extracting solution and afterwards eluted from a cation-exchanger solid-phase extraction cartridge with a solution of methanol-aqueous ammonia. The solution was dried, reconstituted with 5 mL methanol and filtered before analysis by LC-ultraviolet using a C18 column with a mobile phase gradient of potassium dihydrogen phosphate buffer, pH 2.5, and methanol-acetonitrile (30 + 70, v/v). The method was applied to cattle, swine, chicken, and sheep muscle tissues. The validation was performed with a fortified cattle meat sample at level of 100 ppb, which is the administrative maximum residue limit for sulfonamides in the European Union. The limit of quantitation for all sulfonamides was between 3 and 14 ppb. Recovery was evaluated for different meat matrixes. The mean recovery values were between 66.3% for pork meat samples and 71.5% for cattle meat samples.     

Determination of aprotinin by titration with bovine trypsin with end-point detection by high-performance liquid chromatography

A method for the determination of aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is described. The procedure involves the formation of the BPTI-trypsin complex in the presence of an excess of BPTI, quantitative separation of the residual BPTI from the mixture by affinity chromatography and identification and evaluation of the residual BPTI by reversed-phase high-performance liquid chromatography. The method is precise with a mean coefficient of variation of 4.0 and 4.3% for intra- and inter-assay runs, respectively, and has a limit of determination of 3.0 micrograms of aprotinin. The proposed method can be applied to commercial samples, even in very dilute solutions, for the standardisation of aprotinin.     

Determination of ambroxol in dog plasma by high-performance liquid chromatography and UV detection

Summary A new sensitive HPLC-UV method has been developed and validated for the determination of amboroxol in dog plasma enabling the investigation of a newly developed 75 mg ambroxol-containing retard capsule of EGIS Pharmaceuticals Ltd., Budapest, Hungary. A gradient method was used for removing the longer retained plasma components of no interest. The separation was performed on a BDS Hypersil C18 (5 μm, 250×2.1 mm) analytical column, supplied with a 10 mm guard column containing the same packing material. The detection was performed at 210 nm. The calibration curve was linear in the range 25–2000 ng·mL⁻¹. Nerisopam (EGIS-6775) was used as internal standard. Presented at Balaton Symposium on High Performance Separation Methods, Siófok, Hungary, September 1–3, 1999     

Matrix solid-phase dispersion microextraction and determination by high-performance liquid chromatography with UV detection of pesticide residues in citrus fruit.

A multiresidue method based on matrix solid-phase dispersion (MSPD) microextraction was studied to determine the carbamate, benfuracarb, and urea insecticides, diflubenzuron, flufenoxuron hexaflumuron and hexythiazox, used in control of citrus pests. Optimisation of different parameters, such as the type of solid support for matrix dispersion, elution solvents and the clean-up step were carried out. The method used 0.5 g of orange sample, C8 bonded silica as MSPD sorbent and dichloromethane as eluting solvent. Recoveries, at spiked concentrations below the maximum residue levels established by Spanish Government, were between 74 and 84% with relative standard deviations ranging from 2 to 4%. The limits of quantification were from 0.15 to 0.25 microgram/g using high-performance liquid chromatography with UV detection at 200 nm. The method may be useful as a screening protocol for the determination of these newly developed pesticides in citrus samples.     

Determination of nicotinamide and pyridoxine in an elemental diet by column-switching high-performance liquid chromatography with UV detection.

The determination of nicotinamide and pyridoxine in an elemental diet containing 46 compounds was performed by column-switching high-performance liquid chromatography with UV detection at 260 and 290 nm, respectively. The method is simple, rapid, sensitive and reproducible. The calibration graphs for the two vitamins were linear in the ranges 0-0.2 and 0-0.015 micrograms, respectively. The recoveries of both vitamins by the standard addition method were over 95%. There was good agreement between the concentrations indicated and found for both vitamins.     

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.     

Determination of pyrimethamine in animal tissue and egg by liquid chromatography with fluorescence detection

A sensitive and selective liquid chromatographic (LC) assay was developed to determine the concentration of pyrimethamine in animal tissue and egg by fluorescent derivative. Animal samples were extracted with acetonitrile, centrifuged, and purified by hexane. Fluorescent derivatization was performed by reacting pyrimethamine with chloroacetaldehyde and subjected to LC with fluorescence detection (excitation wavelength 300 nm, emission wavelength 420 nm). The limit of detection was 10 ng/g (10 ppb) and the standard calibration curve was linear in the range of 1-100 ppb (0.01-1 ng/10 microL). Recoveries from samples fortified at levels of 0.1 and 1 ppm (microg/g) were 61.0-77.4 and 65.5-81.2%, respectively. The method was applied to the monitoring of marketed samples. Pyrimethamine was not determined in any of the 70 samples: 20 swine muscle; 20 chicken muscle; 10 chicken liver; and 20 egg.     

Determination of chlortetracycline residues in tissues using high-performance liquid chromatography with fluorescence detection

A method is described for the determination of chlortetracycline residues in tissue samples. The samples were extracted into a hydrochloric acid - glycine solution and the extracts concentrated and purified on cyclohexyl-bonded reversed-phase cartridges. Any chlortetracycline present was converted to iso-chlortetracycline at pH 12, which was then separated from interfering compounds on a reversed-phase polymeric column using high-performance liquid chromatography with fluorescence detection. The detection and determination limits of the assay were 20 and 50 ng g-1, respectively, making it suitable for statutory residue testing purposes.     

Determination of some aldehydes by using solid-phase microextraction and high-performance liquid chromatography with UV detection

A new approach has been developed for the extraction and determination of aldehydes such as veratraldehyde, m-nitrobenzaldehyde, cinnamaldehyde, benzaldehyde, and p-chlorobenzaldehyde by using solid-phase microextraction (SPME) and high-performance liquid chromatography with UV detection (HPLC/UV). The method involves adsorption of the aldehydes on polydimethylsiloxane/divinylbenzene-coated fiber, followed by desorption in the desorption chamber of the SPME-HPLC interface, using acetonitrile-water (70 + 30) as the mobile phase; UV detection was at 254 nm. A good separation of 5 aldehydes was obtained on a C18 column. The detection limits of veratraldehyde, m-nitrobenzaldehyde, cinnamaldehyde, benzaldehyde, and p-chlorobenzaldehyde are 25, 41, 13, 12, and 11 pg/mL, respectively, which are about 100 times better than the detection limits for other SPME methods using gas chromatography. The proposed method was validated by determining benzaldehyde in bitter almonds and cinnamaldehyde in cinnamon bark. The recoveries of the 5 analytes were determined by analysis of spiked drinking water.