Extraction of thiamylal in serum using hydrophilic acetonitrile with subzero-temperature and salting-out methods.

For high-performance liquid chromatography (HPLC) of thiamylal, one of the barbiturates, the drug in serum samples was extracted by two alternative liquid-liquid extraction techniques using hydrophilic acetonitrile as a solvent and subzero-temperature and salting-out methods. Acetonitrile was mixed with the sample, separated by cooling at -20 degrees C or addition of sodium chloride, and injected directly into the HPLC apparatus. In both the methods, thiamylal was extracted effectively in the acetonitrile phase and pH adjustment of the sample was not required. The salting-out extraction method is rapid and would be suitable for quantitation of drugs in many samples. To avoid coextraction of added salt, the subzero-temperature extraction method was applied to identification of thiamylal by gas chromatography/mass spectrometry and liquid chromatography-tandem mass spectrometry.

[Quantitative analysis of plasma concentration of barbiturate for diagnosis of brain death].

Organ transplantation from brain death patients started in Japan in 1997. However it is difficult to diagnose brain death in patients treated with barbiturate therapy. In this study, the influence of long continuous administration of barbiturate on diagnosis of brain death was investigated by measuring plasma concentration of barbiturate. In 15 patients treated with barbiturate therapy, plasma concentrations of thiamylal were measured by liquid chromatographic apparatus every day until it's level decreased below 0.1 microgram/ml after cessation of continuous administration. At the same time, plasma thiamylal levels were checked on the day when burst-suppression (b-s) pattern had disappeared in 9 cases, light reflex of pupil appeared in 7 cases and spontaneous respiration had been detected by trigger lamp in 11 cases. The plasma concentrations of thiamylal on the day when b-s pattern had disappeared differed clearly among the cases in the range of 8.8 to 37.9 micrograms/ml. Those cases in which light reflex of the pupil had been recognized were also different in the range of 17.8 to 57.8 micrograms/ml. The cases in which spontaneous respiration had been detected were in the range of 4.4 to 23.0 micrograms/ml. These concentrations varied about 4, 3 and 5 times among the cases examined. The intervals between cessation of continuous administration of thiamylal and the decrease of plasma concentration to below 0.1 microgram/ml also varied from 2 to 14 days from case to case. The minimum concentration of thiamylal on the day when b-s pattern had disappeared, light reflex of the pupil had been recognized and spontaneous respiration had been detected was 8.8, 17.8 and 4.4 micrograms/ml respectively. These results suggest that diagnosis of brain death in patients treated with barbiturate therapy is able to be made when the plasma thiamylal level is below 4.4 micrograms/ml.

Protein binding and the metabolism of thiamylal enantiomers in vitro.

UNLABELLED: Thiamylal, a chiral thiobarbiturate, is marketed as a racemic product. We studied the serum protein binding and microsomal metabolism of thiamylal enantiomers in vitro. The unbound fraction of R(+)-thiamylal was greater than that of S(-)-thiamylal. The analysis of binding data revealed that both enantiomers bound to human serum albumin through only one site. In displacement studies with site-specific probes, dansylsarcosine, but not warfarin, significantly decreased the binding of both enantiomers. The bindings of enantiomers were also decreased by octanoate and a large concentration of oleate. These findings suggest that both enantiomers bind to Site II of albumin with higher affinity for S(-)-enantiomer. R(+)-thiamylal was metabolized more rapidly than S(-)-enantiomer by human liver microsomes. An experiment with isoform-selective inhibitors and cytochrome P-450 (CYP) isoforms showed that CYP2C9 had the highest activity for the metabolism of both enantiomers, the activity being 7 to 10 times that of CYP2E1 and CYP3A4. CYP2C9 showed a significantly rapid metabolism of R(+)-enantiomer, suggesting that CYP2C9 is mainly involved in the enantioselective metabolism of thiamylal. IMPLICATIONS: Because clinically marketed thiamylal is a racemic compound, a pharmacokinetic study of each enantiomer may be beneficial. We found that the enantioselectivity of thiamylal existed in protein binding and metabolism. This may be caused by the differences in the affinities of enantiomers for albumin and cytochrome P-450 isoform.

Improved method of determining thiamylal enantiomers in human serum by high-performance liquid chromatography.

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a simple and rapid method for measuring the thiamylal enantiomers in human serum. The method involves a liquid-liquid extraction procedure followed by chiral resolution using a 5 microm silica-bonded alpha1-acid glycoprotein column (Chiral-AGP). The thiamylal enantiomers and internal standard were eluted within 15 min and were well-resolved. At concentrations of 1, 5 and 20 microg ml(-1), the relative standard deviations of R(+)- and S(-)-thiamylal were 1.35-2.88% and 1.37-3.01%, respectively, for the intra-day assay, and 2.93-4.46% and 2.46-4.84%, respectively, for the inter-day assay. This method facilitates the routine monitoring and pharmacokinetic studies of thiamylal enantiomers.

Pharmacokinetics of thiamylal enantiomers in humans.

Thiamylal, a chiral thiobarbiturate, is marketed as the racemate. The pharmacokinetic behavior of thiamylal enantiomers was studied in patients undergoing thiamylal treatment. The percentage of R(+)-thiamylal unbound to serum protein was 1.5 times greater than that of S(-)-enantiomer (17.5 +/- 2.6% and 11.7 +/- 2.0% mean +/- SD, p < 0.001, n = 7). The pharmacokinetic parameters of enantiomers were estimated in 6 patients. S(-)-thiamylal serum concentration was higher than R(+)-enantiomer in all patients at all time points examined. Total clearance of R(+)-thiamylal (0.27 +/- 0.23 1/hr/kg) was 1.8 times greater (p < 0.05) than that of S(-)-thiamylal (0.15 +/- 0.13). The volume of distribution at steady state of R(+)-thiamylal (3.66 +/- 1.99 l/kg) was 1.4 times higher (p < 0.05) than that of S(-)-enantiomer (2.60 +/- 1.35). The differences in these parameters may be due mainly to enantioselective binding to serum protein.

Enantioselective determination of thiamylal in human serum by high-performance liquid chromatography.

Thiamylal, a widely used anesthetic drug, has two enantiomers. We developed a novel and simple method for measuring thiamylal enantiomers in human serum using reversed-phase high-performance liquid chromatography. R(+)- and S(-)-Thiamylal were separated using a chiral mobile phase containing beta-cyclodextrin, and detected at the range of 50 ng/ml-25 micrograms/ml in serum. The relative standard deviations of R(+)- and S(-)-thiamylal were 3.4-8.7% and 2.8-8.7% for the intra-day assay, and 2.8-12.0% and 2.8-13.0% for the inter-day assay. This method may be applied to enantioselective pharmacokinetic studies of thiamylal.

Effects of phenylbutazone on thiamylal disposition and anaesthesia in ponies.

Phenylbutazone given during the perisurgical period has been reported to increase the intensity and duration of thiamylal anaesthesia in horses. A possible mechanism of competitive plasma protein binding has been suggested. The purpose of the present study was to experimentally reproduce the phenomenon of increased intensity and/or duration of thiamylal anaesthesia and to determine if there is competitive displacement of plasma protein bound thiamylal by phenylbutazone. Six ponies each received one of three treatments, 11 mg/kg intravenous (i.v.) thiamylal; 8.8 mg/kg i.v. phenylbutazone; and 11 mg/kg i.v. thiamylal with 8.8 mg/kg i.v. phenylbutazone given 9 min later. Thirteen blood samples were collected from 0 time through 600 min following drug administration and plasma drug concentrations quantified by high performance liquid chromatography. The pharmacokinetics of thiamylal and phenylbutazone were best described by three- and two-compartment models, respectively. There were no significant differences in pharmacokinetic parameters for thiamylal in the presence of phenylbutazone. However, there were differences in phenylbutazone pharmacokinetics when preceded by thiamylal administration. Unbound phenylbutazone concentrations were increased at 171, 231 and 351 min when given with thiamylal, accompanied by decreases in per cent bound phenylbutazone (P < 0.05). There were also significant (P < 0.05) changes in per cent plasma protein binding of thiamylal and phenylbutazone between 120 and 360 min, when in combination. No changes in intensity or duration of anaesthesia were observed.

Pharmacokinetics of thiamylal in cats.

Pharmacokinetics of thiamylal were determined after 13.2 mg of thiamylal/kg of body weight was administered IV to 6 healthy cats. Blood samples were obtained for 12 hours. Disposition of thiamylal best conformed to 2 multicompartmental models, a 2-compartment (n = 1) and a 3-compartment (n = 5) open pharmacokinetic model. The pharmacokinetic values were calculated for the overall best-fitted model, a mixed 2- and 3-compartmental model. The first or rapid distribution half-life was 1.91 minutes and a second, or slower, distribution half-life was 26.51 minutes. The elimination half-life was 14.34 hours. The apparent volume of distribution was 3.61 +/- 1.8463 L/kg, whereas the apparent volume of the central compartment was 0.46 +/- 0.2034 L/kg, and the total clearance was 0.135 +/- 0.0616 L/kg/h.

Follicular fluid concentrations of thiopental and thiamylal during laparoscopy for oocyte retrieval.

Because access into ovarian tissue of drugs used during anesthesia may be potentially harmful to the oocyte and/or follicular structure, we measured concentrations of thiopental (n = 15) and thiamylal (n = 9) in follicular fluid (FF) aspirates of 24 patients who underwent laparoscopic oocyte retrieval. In both groups, measurable amounts of the respective drug were found in all FF aspirates. Within individual patients, plasma concentrations of both drugs declined during the period of sampling between initial and final follicular aspiration. The mean plasma drug concentration was 7.99 +/- 3.97 micrograms/ml in the thiamylal group and 4.13 +/- 0.90 micrograms/ml in the thiopental group. Mean drug concentrations in FF were similar in both groups (thiopental 1.62 +/- 0.61 micrograms/ml; thiamylal 1.67 +/- 0.83 micrograms/ml). The mean FF/plasma concentration ratio during the sampling period was greater in the thiopental group (0.41 +/- 0.19) as compared with the thiamylal group (0.22 +/- 0.14). Several steps in the clinical management of these patients can be taken to reduce exposure of oocytes to drugs used during anesthesia.

Comparative pharmacokinetics and anesthetic effects of methohexital, pentobarbital, thiamylal, and thiopental in Greyhound dogs and non-Greyhound, mixed-breed dogs.

Pharmacokinetics and duration of anesthesia of methohexital, pentobarbital, thiamylal, and thiopental in Greyhound and non-Greyhound, mixed-breed dogs were compared. In all dogs evaluated, pentobarbital induced the longest duration of anesthesia and methohexital induced the shortest duration. Pharmacokinetics of pentobarbital and methohexital were similar in both groups of dogs. Thiobarbiturates induced longer anesthetic effects in Greyhound dogs than in mixed-breed dogs. Plasma thiobarbiturate concentrations remained above normal longer in Greyhound dogs than in mixed-breed dogs. Disposition of thiobarbiturates in Greyhound dogs was characterized by nonlinearity from 45 minutes to 8 hours after dosing.

Measurement of thiamylal and thiopental in plasma by electron capture and flame photometric gas-liquid chromatography.

We describe an isothermal gas-liquid chromatographic procedure developed for measuring thiamylal and thiopental in plasma. The unchanged drug is extracted into ethyl acetate from acidified plasma, together with an internal standard. The solvent is removed, the residue methylated, aliquots, diluted with benzene, are injected into a 183-cm gas-liquid chromatographic column containing 3% OV-17. Sensitivity of detection is in the nanogram to picogram range.