Development of the Chromatographic Method for Simultaneous Determination of Azaperone and Azaperol in Animal Kidneys and Livers.

A precise and accurate method for the simultaneous determination of azaperone and azaperol in meat tissues has been developed. This paper describes the first method to be so fast, simple, and useful, especially for many laboratories that do not have sophisticated equipment. This method is based on LC separation and UV-Vis detection. During the sample preparation, the meat tissue was homogenized in acetonitrile at a ratio of 1:4 (tissue weight:acetonitrile volume). The homogenate was centrifuged, the supernatant was evaporated in a lyophilizator, and then the evaporation residue was dissolved in 20 µL of ethanol. For deproteinization, 15 µL of perchloric acid was added, and the sample prepared in this way was injected into a chromatographic column and analyzed using reversed-phased HPLC. The mobile phase consisted of 0.05 mol/L phosphate buffer pH 3.00 (component A) and acetonitrile (component B). UV detection was conducted at 245 nm. The experimentally determined LOQs were 0.25 µg/kg for azaperone and 0.12 µg/kg for azaperol. For both analytes, the calibration curves showed linearity in the tested concentration range from 50 to 300 µg/kg of tissue. The accuracy of the presented method did not exceed 15%, and the recovery was in the range of 85-115%. A validated analytical procedure was implemented for the analysis of various animal tissues for their content of azaperone and azaperol.

Depleción tisular de azaperona y su metabolito azaperol tras administración oral de azaperona en cerdo / Tissue depletion of azaperone and its metabolite azaperol after oral administration of azaperone in food-producing pigs

Azaperona es un tranquilizante de tipo butirofenona usado en ganado porcino. Los cerdos son particularmente sensibles al estrés durante el manejo y transporte al matadero. La azaperona es parcialmente metabolizada in vivo a azaperol, un metabolito con actividad farmacológica. Las concentraciones altas y persistentes de azaperona y azaperol en el lugar de inyección contraindican el uso de azaperona por vía intramuscular para el transporte de cerdos de producción de carne al matadero; el uso oral podría ser una alternativa para evitar residuos en el lugar de inyección. El presente estudio determinó la depleción en los tejidos de azaperona y su metabolito azaperol después de la administración oral de la formulación Stresnil®. Cerdos machos (30-45 kg de peso corporal) fueron tratados con Stresnil® (dosis oral única de 4 mg azaperona/kg de peso corporal) y se sacrificaron 6, 24 y 48 horas después de la administración. De cada animal se obtuvo músculo, piel + grasa, hígado y riñón. Azaperona y azaperol se analizaron por HPLC tras la extracción en fase sólida. Las concentraciones de azaperona más azaperol en todos los tejidos analizados no superaron el Límite Máximo de Residuos (LMR) establecidos por la Unión Europea (100 mg / kg en el músculo, el hígado, los riñones y la piel + grasa) en ningún momento del muestreo. Como consecuencia, según los resultados obtenidos en el presente estudio, los tejidos comestibles de los cerdos tratados por vía oral con 4 mg/kg de azaperona, 6 horas antes al sacrificio, podrían ser aceptables para garantizar la seguridad de los consumidores. Sin embargo, se estimó un tiempo de espera de cero horas por análisis de regresión lineal (AU)

Azaperone is a butyrophenone tranquilizer for swine. Food producing pigs are particularly sensitive to stress during handling and transport to the abattoir. In vivo, azaperone is partially metabolised to azaperol, a metabolite with pharmacological activity. The high and persistent concentrations of azaperone and azaperol in the injection site contra-indicates the use of azaperone using the intramuscular route for the transport of the food producing animals, pigs, to the slaughterhouse; the oral use could be an alternative to avoid residues at the injection site. The present study determined the tissue depletion of azaperone and its metabolite azaperol after oral administration of the formulation Stresnil®. Male pigs (30-45 kg of body weight) were treated with Stresnil® (single oral dose of 4 mg azaperone/kg body weight) and were sacrificed 6, 24 and 48 hours after the administration. Muscle, skin + fat, liver and kidney were collected from each animal. Azaperone and azaperol were assayed by HPLC after solid phase extraction. The concentrations of the azaperone plus azaperol in all analysed tissues did not exceed to the Maximum Residue Limit (MRL) established by the European Union (100 μg/kg in muscle, liver, kidney and skin plus fat) at any sampling time. As a consequence, from the results obtained in the present study, edible tissues of pigs treated orally with 4 mg/kg azaperone, 6 hours before to the sacrifice, might be acceptable to guarantee safety for the consumers. Nevertheless a withdrawal time of cero hours was estimated by linear regression analysis (AU)

[Determination of azaperone and carazolol residues in animals kidney using LC-MS/MS method]. / Oznaczanie pozostalosci azaperonu i karazololu w nerkach zwierzat metoda LC-MS/MS.

The method is presented to analyze azaperone and carazolol in pigs kidney. Samples were extracted with acetonitryle. The determination was performed by LC-ESI-MS/MS. The LC was equipped with column Luna C18 Phenomenex. Haloperidol was used as internal standards. The method was validation according to the criteria of Decision Commission No 2002/657/EC. Recoveries for the level 100 microg/kg azaperone and 25 microg/kg carazolol were in the range 91.2-107.0% and 70.8-93.2%. The limit of decision (CCalpha) and detection capability (CCbeta) was respectively 125.9 microg/kg, 160.0 microg/kg for azaperone and 29.3 microg/kg, 33.3 microg/kg. LC-MS/MS technique fulfill the requirements of Decision Commission No 2002/657/EC.

Simultaneous determination of azaperone and azaperol in animal tissues by HPLC with confirmation by electrospray ionization mass spectrometry.

A simple method is described for the determination of azaperone and its metabolite, azaperol, in animal tissues by high-performance liquid chromatography with ultraviolet detection (HPLC/UV). Chromatography was performed using an ODS column, an acetonitrile-0.025% aqueous diethylamine mixture (2:3, v/v) as a mobile phase and UV detection at 250 nm. Peak heights were found linearly related to the concentrations injected from 0.05 to 2 microg/mL (r>0.999). Azaperone and azaperol spiked into several animal tissues were solubilized in 1 mol/L NaOH, extracted with hexane, transferred to 0.1 mol/L H(2)SO(4) and re-extracted with hexane in a mild basic condition. Recoveries of both compounds from 12 types of samples (swine muscle, swine adipose tissue, swine liver, bovine muscle, bovine adipose tissue, bovine liver, poultry muscle, poultry adipose tissue, poultry liver, bovine milk, poultry egg, and salmon muscle) were more than 72%. The lower limit of quantification of was 0.025 microg/g. Azaperone and azaperol at 0.1 microg/g were confirmed by LC/MS. In conclusion, we found this method is both simple and useful for the determination of azaperone and azaperol in a variety of animal tissues for food safety and veterinary applications.

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 a method for detecting and quantifying azaperone, azaperol and carazolol in pig tissues by liquid chromatography-tandem mass spectrometry.

A quick, simple method for quantifying carazolol, azaperol and azaperone is described. Liquid extraction was followed by a clean-up on an Oasis SPE cartridge. The analytes were separated by HPLC and analysed by MS-MS with atmospheric pressure chemical ionisation in the positive mode. The method was applied to muscle and kidney from untreated pigs, the samples being spiked with the three molecules of interest. Recovery was between 70 and 106%. Quantification parameters were also good: the accuracy was between 80 and 110% and the coefficient of variation did not exceed 16%, being below 8% for 90% of the samples. Linearity was good from MRL/4 to 2MRL. For unequivocal identification of each analyte, four ions were detected. The method proved very suitable for routine analysis.

Confirmation of azaperone and its metabolically reduced form, azaperol, in swine liver by gas chromatography/mass spectrometry.

The method described confirms the use of the tranquilizer azaperone by detecting the parent compound and the metabolically reduced form, azaperol. Both are confirmed in swine liver at a target concentration of 10 ppb by gas chromatography/mass spectrometry (GC/MS) with electron ionization in the selected-ion-monitoring mode. Swine liver tissue is ground with dry ice. Acetonitrile is added to extract the drug from the tissue. Sodium chloride buffer is added to the extract in preparation for solid-phase extraction (SPE). The aqueous extract is loaded onto an SPE cartridge designed to extract acidic and neutral drug residues from biological matrixes. The cartridge is washed with methanol and conditioned with sodium phosphate buffer. Azaperone and azaperol residues are eluted with a 2% ammonium hydroxide in ethyl acetate. The extracts are evaporated to dryness under a stream of nitrogen and reconstituted in ethyl acetate for GC/MS analysis. A DB-1 analytical column is used to separate the compounds prior to electron ionization. The parent ion, the base peak ion, and one diagnostic fragment ion are monitored for both compounds. The method was validated with fortified tissue samples containing both azaperone and azaperol. Azaperone-incurred tissues also were analyzed, and the presence of the parent drug and the metabolically reduced form, azaperol, was confirmed.

Determination of drugs in hair using GC/MS/MS.

An important task for the forensic toxicologist and expert witness is the detection of the noxa in biological matrices. Because of this, the identification and quantification of residues of illegal drugs in human hair is still of growing interest. Utilizing the advantages of GC/MS/MS testing human hair is performed for most common drugs of abuse like heroin and other opioides, cocaine, cannabis and amphetamine derivatives. Analyzing hair specimens for substances that present a toxicological risk is another challenge. Several quality control parameters must be observed to avoid false positive or false negative results and to gain additional information. Blank sample, blank hair as well as the combined wash extracts are tested for the presence of the relevant compounds within every series. Careful evaluation of the findings can provide an approximate measure of the intensity of drug use in the majority of cases.

[Analysis of some neuroleptics and carazolol in pork kidneys]. / Analytik einiger Neuroleptika sowie von Carazolol in Schweinenieren.

A method is described for the analytical determination of the neuroleptics azaperone (plus its metabolite azaperol), acetylpromazine, propionylpromazine, chlorpromazine and the beta-blocking agent carazolol in pork kidneys. These compounds may be used illegally to calm pigs during their transport to the abattoir. The kidney samples (plus atosil as an internal standard) are incubated with NaOH (90 degrees C, 60 min); the rather fluid samples are extracted with diethylether. The separation of interfering compounds in the extracts is achieved on a silica gel column. The remaining interfering compounds are removed with ether after acidification of the eluted material. After alkalization of the aqueous solution, the drugs are extracted with either and applied to a Symmetry RP18 column (mobile phase: acetate buffer solution pH 4.5/acetonitrile/tetrahydrofurane, 65/30/10 by volume by HPLC. Recovery in spiked samples of pork kidneys (recovery of 50 micrograms/kg, carazolol 5 micrograms/kg) was between 71.5% (for propionylpromazine) and 88% (for azaperol). The method was verified on samples of treated pigs.

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.

Fluorometric determination of some butyrophenones after permanganate treatment.

A fluorometric method is described for direct and sensitive determinations of azaperone, haloperidol and bromoperidol in pharmaceutical preparations. These compounds can be converted into strongly fluorescing derivatives by the action of potassium permanganate on their alcoholic solutions in acid medium, the limits of detection being respectively 8.10(-3), 5.10(-2) microgram/ml. The fluorescence phenomenon is very stable. Preliminary results are reported on the isolation and structure determination of one of the main fluorophors, produced in the case of azaperone.

Fluorescence characteristics of azaperone and of an azaperone mononitrogen oxide.

The structure elucidation of the compound isolated after peroxide treatment of azaperone is described. A mononitrogen oxide was formed at the piperazine N1 atom after reaction with excess hydrogen peroxide. The fluorescence characteristics of derivative were examined and compared with the native fluorescence capacities of the azaperone base; both were identical, depending on the solvent nature. The phenomenon is explained by the fact that the fluorescent properties of the azaperone molecule are principally produced by its ortho-nitrogen substituted pyridine nucleus.