Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

doi:10.1016/S0378-4347(99)00478-8

Journal of Chromatography B: Biomedical Sciences and Applications

Volume 738, Issue 1, 28 January 2000, Pages 17-25

https://doi.org/10.1016/S0378-4347(99)00478-8 Get rights and content

Abstract

Ethyl acetate extracts of equine serum, containing 0–5 μg/ml of hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), indomethacin (IND), phenylbutazone (PB) and probenecid as internal standard, were evaporated with nitrogen, resuspended in methanol and analyzed by HPLC, using a C-18 column equilibrated with 51:49 acetonitrile–water, 0.1% trifluoroacetic acid, at 1 ml/min. The eluate was monitored at 254 nm. The selectivity (inter-assay C.V.<4%), sensitivity (limits of quantitation of 0.25 μg/ml for HYD, DEX and IND, 0.5 μg/ml for PB and 1 μg/ml for OPB, despite the occurrence of significant degradation of OPB and PB during the analysis) and precision (intra-assay and inter-assay C.V.’s of about 3–6 and 9–15%, respectively) of the method appeared appropriate for anti-doping control of racehorses.

Introduction

Steroidal and non-steroidal anti-inflammatory drugs (NSAID), such as hydrocortisone (HYD), dexamethasone (DEX), and phenylbutazone (PB), are commonly used in numerous musculo-skeletal inflammatory conditions of the horse [1], [2], [3]. However, the competition of treated racehorses is prohibited by several regulations in order to prevent health problems which can lead to the premature withdrawal of the animal from athletic life [4], [5]. Actually, the use of PB is allowed by some racing jurisdictions but serum levels should not exceed 2–8 μg/ml [5], [6], [7], [8].

To the best of our knowledge, there is no HPLC method capable of determining simultaneously HYD, DEX, oxyphenbutazone (OPB), indomethacin (IND) and PB in equine serum. Therefore, we decided to develop a method for the analysis of these anti-inflammatory drugs during anti-doping controls.

Section snippets

Chemicals and reagents

Hydrocortisone (HYD, 98% pure), dexamethasone (DEX, 98% pure), oxyphenbutazone (OPB, 99% pure), probenecid (PRB, 99.8% pure), indomethacin (IND, 99% pure), phenylbutazone (PB, 99.7% pure), sodium citrate (99% pure), sodium phosphate (99% pure) and normal equine serum (NES) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade methanol, acetonitrile and water, were purchased from Lab-Scan (Dublin, Ireland). Trifluoroacetic acid (99.8% pure) was from Merck (Darmstadt, Germany).

Effect of degradation on the UV spectrum of phenylbutazone

A 50 μl volume of a methanolic solution of PB (10 μg/ml) were added to 950 μl of:

•
methanol;
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ethyl acetate, and the sample was evaporated under a nitrogen stream at room temperature and resuspended in 1 ml of methanol;
•
ethyl acetate, and the sample was evaporated as in (b), kept at 100°C for 30 min, cooled in an ice bath for 30 s, and resuspended in 1 ml of methanol;

and the UV spectra were obtained using a UV/Vis spectrophotometer (model Lambda Bio 20, from Perkin-Elmer Co., Norwalk, USA).

Influence of degradation on the overall recovery of phenylbutazone

A 10 μl

Selectivity

When 50 μl of methanolic solutions (5 μg/ml) containing hydrocortisone (HYD), dexamethasone (DEX), oxyphenbutazone (OPB), probenecid (PRB), indomethacin (IND) and phenylbutazone (PB) were analyzed by reversed-phase HPLC, using a C-18 column with a mobile phase containing about 50% acetonitrile in water, the chromatogram shown in Fig. 1 was obtained. It was noted that the peaks were well separated even following extraction of the drugs from equine serum without significant interference of

Discussion

A quantitative, reversed-phase HPLC method, for the simultaneous analysis of selected steroidal and non-steroidal anti-inflammatory drugs in equine serum, with limits of quantitation [0.25 μg/ml for HYD, DEX and IND, 0.5 μg/ml for PB and 1 μg/ml for OPB (Table 1)] suitable for anti-doping control, was described.

Ethyl acetate, a non-toxic solvent, was preferred over others, such as acetonitrile, ethanol, trichloroacetic and perchloric acid because of the better yield and selectivity (data not

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Simultaneous determination of hydrocortisone, dexamethasone, indomethacin, phenylbutazone and oxyphenbutazone in equine serum by high-performance liquid chromatography

Abstract

Introduction

Section snippets

Chemicals and reagents

Effect of degradation on the UV spectrum of phenylbutazone

Influence of degradation on the overall recovery of phenylbutazone

Selectivity

Discussion

J. Chromatogr. B

J. Pharm. Sci.

J. Pharm. Sci.

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J. Pharm. Sci.

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