Quantitative determination of dihydroetorphine in rat plasma and brain by liquid chromatography-tandem mass spectrometry.

The extraordinarily strong analgesic dihydroetorphine (DHE) was registered as one of the most strictly controlled narcotic drugs by the United Nations in 1999. However, an effective detection method for DHE in biological samples has not yet been established. We developed a quantitative method for assay of DHE in rat plasma and brain by liquid chromatography-tandem mass spectrometry equipped with an ionspray interface. A 0.5-ml volume of plasma and brain homogenate spiked with buprenorphine (internal standard) was purified by the solid-phase extraction column Bond Elute Certify. DHE produced numerous weak fragment ions by collision induced dissociation. Therefore, collision energy was utilized to decompose the interferences, and the protonated molecular ion was used for both precursor and product ion monitoring. As a result of the method validation, the dynamic concentration range was determined as 0.05-10 ng/ml. DHE in these samples was stable for 2 months at -4 degrees C and for 24 h at ambient temperatures. Using the present method, DHE was detected in rat plasma and brain tissue after intravenous injection (0.5 microg/kg).

Determination of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry using selected-ion monitoring.

A method for the determination of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids by GC-MS with selected-ion monitoring using etorphine as internal standard was established. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after concentration to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed which showed good behavior on GC-MS with electronic-impact ionization. The main fragment, m/z 522, which is the base peak, was selected as the ion for quantitation and the corresponding ion, m/z 520, was selected for monitoring the internal standard, etorphine. The recoveries and coefficients of variation of the whole procedure were determined with five controlled dihydroetorphine-free urine and plasma samples spiked with different concentrations of dihydroetorphine. The concentration of dihydroetorphine for quantitation was in the range 1-20 ng/ml for urine and 2.5-250 ng/ml for plasma. The correlation coefficients of the standard curves are sufficient to determine the dihydroetorphine. The accuracy for quantitation of dihydroetorphine in urine and plasma is less than 10.6%.

Identification of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry.

A method for the monitoring of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids was developed, involving GC-MS with multiple selected-ion monitoring. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after having been concentrated to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed, which showed good behaviour in GC-MS with electron impact ionization. Its molecular ion, m/z 609, and its main fragments, m/z 576, 534, 522 and 508, were selected as the ions for identification owing to their relative peak intensities and characteristics. The target drug was identified based on its retention time, its selected multiple ions and their relative intensities. This method was successfully used for the detection of dihydroetorphine in blood and urine from a dihydroetorphine addict and a poisoned patient, respectively.

[Monitoring of dihydroetorphine hydrochloride in biological fluid].

A method for the monitoring of dihydroetorphine hydrochloride, a powerful anesthetic and analgesic drug, in biological fluid was developed by means of GC-MS with selected ion monitoring. Dihydroetorphine was extracted from human blood and urine with dichloromethane, and then was derivatized with N-heptafluorobutyrylimidazole after it was concentrated to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed, which showed good characteristics on GC-MS with electronic impact mode. Its M+ was m/z 609. Besides this, its main fragments included m/z 576, 534, 522 and 508. These main ions were all selected as the ions of identification due to their relative peak intensity and stability. The detection limit was 1 pg. The target drug was identified based on its retention time multiple ion monitoring and relative peak intensity. This method has been successfully used for detection of dihydroetorphine hydrochloride in blood and urine from dihydroetorphine addict and poisoned patient, respectively. Good results have been obtained.

Narcotic analgesics, their detection and pain measurement in the horse: a review.

Narcotic analgesics produce pharmacological effects by interacting with specific opiate receptors. At least five major types of opiate receptors have been recognised. These include mu (morphine) and kappa (ethylketazocine) receptor types. Narcotic analgesics which interact with mu receptors produce locomotor and autonomic stimulation at doses that produce little or no analgesia. Therefore, use of these drugs as analgesics in equine medicine has not been very satisfactory. Theoretical considerations suggested that the role of kappa agonists in equine analgesia be investigated. Using a pure kappa agonist, U-50, 488H, good analgesia was produced in the horse with little or no locomotor stimulation or autonomic effects. These data suggest that kappa agonists may be superior analgesics for clinical use in the horse. On the other hand, the locomotor stimulant effects of mu agonist analgesics enable their use as illegal medications. Specifically, these agents produce a good running response, signs of central nervous stimulation and analgesia, all potentially useful effects in a racehorse. Regulatory control of most narcotic analgesics can be obtained by high performance thin layer chromatographic screening. However, effective screening for the fentanyls and small doses of etorphine can only be achieved by use of immunoassay.

Radioimmunoassay for etorphine in horses with a 125I analog of etorphine.

To improve the sensitivity and specificity of screening for etorphine in horses, an 125I-labeled etorphine analog was synthesized and an antibody to etorphine was raised in rabbits. A radioimmunoassay (RIA) for etorphine was developed, using these reagents. Bound and free 125I-labeled etorphine was separated by a double-antibody method that reduced interference from materials associated with equine urine. The 125I-labeled etorphine binding was rarely greater than 250 pg of background etorphine equivalents/ml in raw urine and was 100 pg/ml in hydrolyzed urine. The 125I-RIA was capable of detecting etorphine equivalents in urine above these background values. Etorphine equivalents were detected in equine urine samples for about 7 days after 4 mares were dosed with 0.22 microgram of etorphine/kg of body weight, IV. The stability of etorphine in urine from these mares was evaluated. Urine from these dosed mares was held in constant -20 C storage, and aliquots were repeatedly frozen and thawed. When analyzed for etorphine equivalents using an 125I-RIA, etorphine and its metabolites in urine samples were stable for less than or equal to 38 days if continuously frozen and also were resistant to repeated freezing and thawing.

Development of a radioimmunoassay for etorphine (preliminary communication).

A radioimmunoassay for etorphine has been developed using antibodies raised in a rabbit against an etorphine-3-hemisuccinate-bovine serum albumin conjugate. Using tritiated etorphine as the label, the antiserum could be used at a final dilution of 1:4 and had an avidity of 1.8 times 10-9 L.M.-1. The sensitivity of the assay was 0.88 plus or minus 0.04 ng etorphine per ml of serum and there was no cross-reactivity with any of the six opiate alkaloids examined over the range 0-100 ng/ml.