The Analysis of Phenylbutazone and Its Active Metabolite, Oxyphenbutazone, in Equine Tissues (Muscle, Kidney, and Liver), Urine, and Serum by LC-MS/MS.

This study reports the use of two validated LC with tandem MS (MS/MS) methods to study the residue depletion profile of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OXPBZ) from equine serum, urine, and muscle, kidney, and liver tissues. One LC-MS/MS method, with an LOQ of 1.0 ng/mL for PBZ and 2.0 ng/mL for OXPBZ, was used for the analysis of the two drugs in the biological fluids (equine urine and serum); the other LC-MS/MS method, with an LOQ of 0.5 ng/g for PBZ and OXPBZ, was used for the analysis of the drugs in the equine tissue samples. PBZ was administered intravenously to two horses dosed with 8.8 mg/kg PBZ once daily for 4 days and sacrificed humanely at a slaughter plant 7 days after the last drug administration. Urine, serum, and kidney, liver, and muscle tissues were collected from the two horses and shipped on ice to the laboratory and stored at -20°C until analysis. The concentrations of PBZ and OXPBZ residues in the biological fluid and tissue samples collected at slaughter were measured with the two validated LC-MS/MS methods using deuterated internal standards. The results demonstrate that the validated methods are fit for studying the depletion kinetics of PBZ residues in equine tissues and biological fluids.

Analysis of phenylbutazone residues in horse tissues with and without enzyme-hydrolysis by LC-MS/MS.

Phenylbutazone (PBZ) is permitted to be used for the treatment of musculoskeletal pain and inflammation in race horses but it is not approved for use in horses destined for human consumption. In a recent study initiated in our laboratory to study the disposition of PBZ and its oxyphenbutazone (OXPBZ) metabolite in equine tissues, we compared the effect of an additional enzymatic hydrolysis step with ß-glucuronidase on the results of the analysis for PBZ without enzymatic hydrolysis. Incurred tissue samples obtained from a female horse dosed with PBZ at 8.8 mg/kg for 3 days and sacrificed 6 days following the last administration were used for this study. Liver, kidney, and muscle tissues were collected, extracted, cleaned up on a silica-based solid-phase extraction (SPE) preceded by a weak-anion exchange SPE and analyzed with our in-house validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for PBZ and OXPBZ. Addition of the hydrolysis step resulted in a significant increase in recovery of both PBZ and OXPBZ residues. © 2016 Her Majesty the Queen in Right of Canada. Drug Testing and Analysis © 2016 John Wiley & Sons, Ltd.

Determination of non-steroidal anti-inflammatory drugs residues in animal muscles by liquid chromatography-tandem mass spectrometry.

A confirmatory method for the determination of residues of nine non-steroidal anti-inflammatory drugs and one metabolite in animal muscles has been developed. After enzymatic hydrolysis samples were extracted with acetonitrile and cleaned up using alumina and C(18) SPE cartridges. Liquid chromatography-tandem mass spectrometry was used for the separation and determination of analytes. The method was validated in bovine muscles, according to the Commission Decision 2002/657/EC criteria. Applicability of the method in the analysis of swine, horse and chicken muscles was checked by precision and recovery experiment. The influence of matrix effect on the quantification of non-steroidal anti-inflammatory drugs residues was investigated. The method was used for the confirmation of phenylbutazone and oxyphenbutazone in horse muscle sample.

Analysis of adulterants in a traditional herbal medicinal product using liquid chromatography-mass spectrometry-mass spectrometry.

Adulterations with synthetic drugs are common problems with herbal medicine and this can potentially cause serious adverse effects. It is therefore important to determine the presence of synthetic drugs in herbal medicine to ensure patients' safety. The objective of this study was to develop sensitive and specific methods to analyse phenylbutazone, caffeine and oxyphenbutazone present in a traditional Indonesian herbal product. Liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) methods in the selected reaction-monitoring (SRM) mode were developed. It was found that the sample contained 0.53% w/w (n=3, RSD=7.56%) phenylbutazone and 0.04% w/w (n=3, RSD=8.39%) caffeine. This corresponded to 43.17 mg phenylbutazone and 3.23 mg caffeine in each sachet of powder. The methods were validated for linearity, precision, accuracy, LOD and LOQ. LOD and LOQ were found to be 3.69 and 12.29 ng/ml, respectively for phenylbutazone. For caffeine, the LOD and LOQ were 0.84 and 2.80 ng/ml, respectively. Oxyphenbutazone in the sample was found to be present at a level below the quantification level of 10.2 ng/ml. With better methods developed for analysis of adulterants in herbal medicine, the quality and safety of these medicines can be better controlled and regulated to ensure patients' safety.

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry.

A method is described for the qualitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 microliters were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 microgram/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 microgram/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.

Physical and structural comparison of oxyphenbutazone monohydrate and anhydrate.

Two crystal forms of oxyphenbutazone (a monohydrate and an anhydrate) were prepared by recrystallization. The forms were characterized by means of differential scanning calorimetry, thermogravimetry, infrared spectrophotometry, X-ray powder diffraction patterns, thermomicroscopy, scanning electron microscopy, as well as powder and intrinsic dissolution rates. The crystal structure of the anhydrate has been elucidated and compared with that of the monohydrate.

Physical characterization of two oxyphenbutazone pseudopolymorphs.

Two pseudopolymorphic forms of oxyphenbutazone, a benzene solvate (Solvate B) and a cyclohexane solvate (Solvate C), were prepared by recrystallization from benzene and cyclohexane, respectively. The forms were characterized by means of differential scanning calorimetry, thermogravimetry, infrared spectrophotometry, X-ray powder diffraction, and thermomicroscopy, as well as powder and intrinsic dissolution rates. The dissolution rates of the two pseudopolymorphs were shown to be superior to those of the anhydrate, hemihydrate, and monohydrate which were previously reported. A brief stability report is included.

Proton magnetic resonance spectroscopic method for determination of phenylbutazone and oxyphenbutazone in tablets.

A simple, specific, and rapid 1H nuclear magnetic resonance spectroscopic method for the assay of phenylbutazone and oxyphenbutazone is described. Spectra are recorded in CDCI3 containing 1,3-dichloro-5-nitrobenzene as an internal standard. The aromatic proton resonances for the standard, at delta 7.7 and 8.2, are well separated from those of phenylbutazone and oxyphenbutazone, which are in the region of delta 6.5-7.3 ppm. Average percent recoveries of phenylbutazone and oxyphenbutazone were 98.9 and 98.6 with standard deviations of 0.6 and 0.7, respectively. Commercial formulations were analyzed and the results obtained by the proposed method closely agreed with those found by the British Pharmacopoeia method.

Rapid and simple determination of oxyphenbutazone in dosage forms by biamperometric titration.

Oxyphenbutazone was determined in the presence of glacial acetic acid, hydrochloric acid, and potassium bromide by bromometric titration with biamperometric end point indication. The course of the titration was followed with double platinum electrode with 100 mV of polarizing voltage.

Influence of phenylbutazone on eicosanoid levels in equine acute inflammatory exudate.

In a two part cross-over experiment, acute inflammatory exudates were induced in 7 ponies by subcutaneous implantation of 3 sterile carrageenin-soaked polyester sponge strips. Treatment comprised a single therapeutic geenin-soaked polyester sponge strips. Treatment comprised a single therapeutic dose of 4.4 mg/kg phenylbutazone (PBZ) administered intravenously at the time of sponge implantation. Exudates were harvested at 6, 12 and 24 hours and examined for leukocyte and erythrocyte numbers using the improved Neubauer technique; for eicosanoids by radioimmunoassay and by high performance liquid chromatography for concentrations of PBZ and its principal metabolite oxyphenbutazone. Plasma PBZ and oxyphenbutazone levels were measured in treated animals at 6, 12 and 24 hours. The administration of PBZ produced, at 6 hours, highly significant (P less than 0.001) reductions in exudate levels of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha (the stable breakdown product of prostacyclin, PGI2). Significant (P less than 0.01) reductions in these eicosanoids were maintained in treated animals at 12 and 24 hours. Levels of thromboxane B2 (TXB2), the catabolite of TXA2, were reduced in treated animals at 6 and 12 hours but these changes were not significant. Leukocyte numbers were significantly (P less than 0.001) increased from 6-hour values at 12 and 24 hours in both control and PBZ-treated animals but differences between control and treated ponies were not significant. This is the first report in ponies of eicosanoid inhibition following the administration of a non-steroid anti-inflammatory drug (NSAID). It is proposed that the model of inflammation used in this study might provide a means of assessing the efficacy and duration of action of NSAIDs in the horse.

Colorimetric analytical study of oxyphenbutazone.

A procedure was developed for the colorimetric determination of oxyphenbutazone by treatment with dilute nitrite and dilute hydrochloric acid solutions for 10 min, then rendering alkaline with potassium hydroxide. By this simple colorimetric measurement, quantities of oxyphenbutazone from 2.5 to 10 mg/100 ml are determined with an accuracy of 98.80 +/- 1.33%. Application of the suggested method to different pharmaceutical preparations has shown no significant difference compared with the Brit. Ph. 1980 method. A colour reaction mechanism is discussed.