Derivatization methodology for the analysis of butorphanol by gas chromatography-mass spectrometry.

Butorphanol is an opioid used as analgesic in humans and other species. In horses, it can cause locomotor stimulation at low doses. This drug is not well chromatographed by GC and so, it is necessary to transform it into a more suitable compound, which can be done by derivatization. The derivatization of a drug is used to impart volatility, masking polar groups to improve the results in gas chromatographic analysis. We have evaluated N,O-bis(trimethylsilyl)- trifluoracetamide (BSTFA)+ 1% trimethylchlorsilane (TMCS) and N-methyl-N-trimethylsilil-trifluoroacetamide (MSTFA) as derivatizing reagents for butorphanol at 30, 60 and 80 degrees C during 15, 30 and 60 min. The effects of dilution of these reagents with toluene and the evaporation before the derivatization were tested. Both reagents can be used for butorphanol derivatization and analysis and the dilution and evaporation steps did not alter the final results. The best derivatization conditions were 15 min at 80 degrees C, although 60 degrees C, although 60 degrees C during 60 min were also suitable.

Determination of butorphanol in horse race urine by immunoassay and gas chromatography-mass spectrometry.

An analytical procedure to screen butorphanol in horse race urine using ELISA kits and its confirmation by GC-MS is described. Urine samples (5 ml) were subjected to enzymatic hydrolysis and extracted by solid-phase extraction. The residues were then evaporated, derivatized and injected into the GC-MS system. The ELISA test (20 microl of sample) was able to detect butorphanol up to 104 h after the intramuscular administration of 8 mg of Torbugesic, and the GC-MS method detected the drug up to 24 h in FULL SCAN or 31 h in the SIM mode. Validation of the GC-MS method in the SIM mode using nalbuphine as internal standard included linearity studies (10-250 ng/ml), recovery (+/-100%), intra-assay (4.1-14.9%) and inter-assay (9.3-45.1%) precision, stability (10 days), limit of detection (10 ng/ml) and limit of quantitation (20 ng/ml).

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.

Determination of succinonitrile in horse urine by gas chromatography-nitrogen-phosphorus detector and gas chromatography-mass spectrometry.

A chromatographic method was developed to detect and confirm the presence of succinonitrile (SDN) in horse urine samples, for antidoping control. The urine samples (5 ml) were extracted with diethyl ether and screened by gas chromatography-nitrogen-phosphorus detector and the confirmation of the drug's presence was accomplished by using gas chromatography-mass selective detection. The recovery of extraction was 78 and 81% for 1.0 and 2.0 micrograms ml-1 (relative standard deviation, < 10%), respectively. Urine samples collected after the administration of Energisan were positive for SDN (1-30 h) in all samples analysed.