Quantitative analysis of thiamylal and its metabolite secobarbital using liquid chromatography-tandem mass spectrometry in adipose tissue, serum, and liver.

Thiamylal is an ultrashort-acting barbiturate used for intravenous administration or general anesthesia induction. However, some cases of poisoning and suicide with thiamylal administration have been reported. Additionally, there are few reports on its analysis in the organs and adipose tissue, which requires purification by column chromatography and evaporation. A rapid and sensitive method was developed for quantifying thiamylal and its metabolite, secobarbital, in the adipose tissue, serum, and liver using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared using modified QuEChERS extraction. For adipose tissue samples, an acetonitrile-hexane partitioning step was added to the extraction. This method was applied to investigate a suspected self-poisoning autopsy case. The quantitation accuracy for thiamylal added to porcine pericardial fat (0.18 µg/g), human serum (0.015 µg/mL), and porcine liver (0.18 µg/g) was 103%, 113%, and 95.3%, respectively. The quantitation limits calculated for porcine pericardial fat, human serum, and porcine liver at a signal-to-noise ratio of 10 were 0.06 µg/g, 0.005 µg/mL, and 0.06 µg/g, respectively. In addition, the thiamylal and secobarbital levels in the forensic autopsy case were 140 and 1.5 µg/g, respectively, in myocardial fat; 3.5-4.9 and 0.12-0.20 µg/mL, respectively, in serum; and 6.2-42 and 0.58-1.1 µg/g, respectively, in liver tissue. Thiamylal is especially distributed in the adipose tissue. The thiamylal-to-fat ratio may help estimate the time from administration to death. The developed modified QuEChERS extraction method with acetonitrile-hexane partitioning is suitable for analyzing hydrophobic compounds, such as thiamylal, in the adipose tissue.

Barbiturate detection in oral fluid, plasma, and urine.

BACKGROUND: Although current abuse of barbiturates is low compared with other classes of abused drugs, their narrow margin of safety, risk of dependence, and abuse liability remain a health concern. Limited information is available on the disposition of barbiturates in different biologic matrices. OBJECTIVE: The authors conducted a clinical study of the disposition of barbiturates in oral fluid, plasma, and urine after single-dose administration to healthy subjects. METHODS: Three parallel groups of 15 subjects were administered a single oral dose of one barbiturate: butalbital (50 mg), Phenobarbital (30 mg), or sodium secobarbital (100 mg). Subjects remained at the clinic for two confinement periods; the first was -1 to 36 hours postdose and again at 48 to 52 hours. Oral fluid specimens were collected by bilateral collection (Intercept; one on each side of the mouth simultaneously). Blood specimens were obtained by venipuncture and urine specimens were collected through separate collection pools of varying periods. Oral fluid specimens were analyzed for barbiturates by liquid chromatography-tandem mass spectroscopy with a limit of quantitation of 8 ng/mL. Plasma and urine specimens were analyzed by gas chromatography-mass spectroscopy with a limit of quantitation of 100 ng/mL. RESULTS: Barbiturate side effects included dizziness, drowsiness, and somnolence. All effects resolved spontaneously without medical intervention. The three barbiturates were detectable in oral fluid and plasma within 15 to 60 minutes of administration and in the first urine pooled collection at 2 hours. Butalbital and Phenobarbital remained detectable in all specimens through 48 to 52 hours, whereas secobarbital was frequently negative in the last collection. Oral fluid to plasma ratios appeared stable over the 1- to 48-hour collection period. CONCLUSION: This study demonstrated that single, oral therapeutic doses of butalbital, Phenobarbital, and secobarbital were excreted in readily detectable concentrations in oral fluid over a period of approximately 2 days. Oral fluid patterns of appearance and elimination were similar to that observed for plasma and urine.

Isotopic analogs as internal standards for quantitative analyses by GC/MS--evaluation of cross-contribution to ions designated for the analyte and the isotopic internal standard.

Isotopic analogs of the analytes are currently preferred internal standards (IS) for quantitative analyses of drugs and their metabolites in biological matrices by GC/MS procedures. Contributions of the analyte and the IS to the intensities of ions designated for the IS and the analyte, respectively--an undesirable phenomenon termed "cross-contribution"--greatly weakens the effectiveness of this approach. The cross-contribution phenomenon has been, in the past, evaluated by a "direct measurement" approach, in which intensities of interested ions were measured in two separate experiments using equal quantities of the analyte and the IS. Alternate procedures that may generate improved results are hereby studied. For the "improved direct measurement" approach, ion intensity data derived from the previously reported direct measurement procedure are first normalized before being used to calculate the extent of cross-contribution. An "internal standard" approach is also developed, in which a set amount of a third compound is incorporated into these two separate experiments, thus allowing corrections of ion intensity data that are imbedded with variations inherent to separate experiments. Finally, a "standard addition" approach, involving a series "addition" of "standards", generates multiple data points; thus, providing a mechanism to validate the resulting cross-contribution data. Secobarbital/(2)H(5)-secobarbital and secobarbital/(13)C(4)-secobarbital pairs are adapted as the exemplar systems for this study.

Isotopic analogues as internal standards for quantitative analyses of drugs and metabolites by GC-MS--nonlinear calibration approaches.

In order to achieve accurate quantitation of drugs and metabolites (analytes) in complex matrices, 2H- (and less commonly 13C-) labeled analogues of the analytes are now routinely adapted as the internal standards (IS) using linear calibration models to fit data generated by selected ion monitoring gas chromatography-mass spectrometry (GC-MS) protocols. In this study, the effects of cross-contribution (contribution of the IS to the intensity of the ion designated for the analyte and vice versa) on the linearity of the calibration data are examined. Nonlinear approaches that may address this problem are also studied. Two ion pairs (one with least and one with significant cross-contribution) from each of the following analyte/IS pairs are used as the exemplar systems for this study: butalbital/13C4-butalbital, butalbital/2H5-butalbital, secobarbital/13C4-secobarbital, and secobarbital/2H5-secobarbital. Analyte/IS ion intensity ratios of a series of standard solutions are correlated with the analyte/IS concentration ratios using one-point, multiple-point (unweighted and weighted) linear, and hyperbolic functions. The one-point calibration approach produces excellent calibration results in treating data derived from ion pairs with no significant cross contribution. In cases where significant cross-contribution exists, results derived from the one-point approach show, as expected, significant deviations at both ends of the concentration range. With the cross-contribution phenomenon accounted for, the hyperbolic calibration model is clearly more effective in fitting calibration data at both the lower and higher analyte concentration ends, thus significantly lowering the detection limit and extending the calibration range to a higher level. However, the calibration range cannot be extended indefinitely. At the low concentration end, noise-to-signal ratio and the cross-contribution of the IS to the intensity of the ion designated for the analyte, however insignificant, will incrementally reduce the quality of the observed ion intensity and intensity ratio data. At the high concentration end, detection saturation and the cross-contribution of the analyte to the intensity of the ion designated for the IS, however insignificant, will incrementally decrease the "slope" of the calibration curve. Thus, acceptable sensitivity (increase in analyte/IS ion-pair intensity ratio per unit increase in analyte concentration) of the calibration curve will become the limiting factor.

An unusual drug death involving maggots.

Toxicologic analysis of decomposed specimens provides greater analytical challenges than those encountered with fresh postmortem specimens. Despite the difficulties involved, in cases in which the cause of death is not determined at autopsy or when there is a strong indication of drug intoxication, all reasonable steps must be undertaken to perform as comprehensive a drug screen as possible. An unidentified white male was found in a field near a river. The body was decomposed and skeletonized, and 3- to 4-mm maggots were present on the body. Near the body was an empty bottle of secobarbital that had been prescribed to a female. There was no evidence of injury. Calf muscle and maggots were sent for toxicologic analysis. No volatile substances or drugs were detected in the calf muscle. Because intoxication due to secobarbital was strongly suggested from the scene investigation, the only other specimen available, the maggots, were tested for acid-neutral drugs. Secobarbital was identified by retention time and was confirmed by full-scan electron ionization gas chromatography/mass spectrometry. Based on the available information, the medical examiner ruled that the cause of death was secobarbital intoxication and the manner of death was suicide.

An experimental methodology for the study of postmortem changes in toxic concentrations of drugs, using secobarbital as an example.

Postmortem changes in alcohol and drug concentrations are well known today. The authors used an HPLC assay of barbiturates in postmortem tissue, and developed a rat model in order to evidence postmortem changes in toxics concentrations. Postmortem changes in secobarbital concentrations were evidenced using a rat-secobarbital model. This work emphasizes the difficulty of postmortem toxicology, as concentrations found at the time of autopsy may be different from concentrations at the time of death.

Fourier transform infrared analyses of some particulate drug mixtures using a diamond anvil cell with a beam condenser and an infrared microscope.

Although the diamond anvil cell (DAC) has been used in many forensic science laboratories for the analysis of trace evidence, few applications of this technique for the analysis of controlled substances have been reported. This may be due to both an unfamiliarity on the part of forensic drug chemists with this accessory and the nature and quality of spectra that result from use of a DAC on a dispersive instrument. Along with low energy throughput, which results in relatively high noise levels, strong broad diamond absorptions occur. With the use of a Fourier transform infrared instrument, these do not present a problem and nanogram quantities of materials can be analyzed when the DAC is used with an infrared microscope. Since single crystals can be sampled with the DAC, simple physical separations (involving particle-picking) can be used in certain cases to isolate drugs from particulate mixtures for infrared analysis. This method is especially useful for some "difficult" mixtures and residues, and several examples of such analyses involving samples of forensic science interest are presented.

A fatality involving secobarbital, nitrazepam, and codeine.

A fatal case involving the suicidal ingestion of secobarbital, nitrazepam, and codeine is presented. The drugs were quantified using gas chromatography for codeine and high-performance liquid chromatography for the two other drugs. The blood concentrations of secobarbital, nitrazepam, and codeine were found to be 11.48, 1.72, and 0.036 microgram/ml, respectively. Results are discussed in the light of the existing literature.

Postmortem stability of barbiturates in blood and tissues.

The stability of five commonly prescribed barbiturates and thiopental in blood and liver at room temperature and at 4 degrees C was studied. Gas chromatography was used for oxybarbiturate analysis while liquid chromatography was used to quantitate thiopental. In blood and liver, greater than 75% of the drugs were detected at the end of the two- to three-month period. These changes were not considered significant; therefore, barbiturates appear to be stable in blood and liver under the conditions of these experiments.

Monitoring saliva concentrations of methaqualone, codeine, secobarbital, diphenhydramine and diazepam after single oral doses.

A preliminary investigation was undertaken to determine the feasibility of monitoring saliva levels of drugs for forensic purposes. Single oral doses of the title compounds were administered to healthy volunteers. Plasma and saliva levels were measured and ratios calculated for all drugs except diazepam. Saliva/plasma ratios for methaqualone, diphenhydramine and secobarbital were all less than one and reasonably consistent between collection times and subjects. The saliva/plasma ratios for codeine were more variable, but always greater than one. Although more detailed investigation is necessary, saliva may be a useful medium for forensic monitoring of drug ingestion.

Assay for cyclo-, seco- and pentobarbital by multiple ion detection: kinetics after a single dose.

A sensitive assay for the determination of four different barbiturates: butabarbital, cyclobarbital, pentobarbital and secobarbital is described. The barbiturates are determined quantitatively by gas chromatography/mass spectrometry (GC/MS) as their dimethylated derivatives. The sensitivity of this simple procedure is limited to 10 x 10(-6) g/l for cyclo- and secobarbital and 1 x 10(-6) g/l for pentobarbital in plasma. The assay has been used to measure the kinetics of pentobarbital over 6 days after a single 50 mg dose of the sodium salt, and over 3 days for one combined preparation of 25 mg cyclobarbital-calcium and 75 mg secobarbital-sodium. The corresponding barbiturate levels in saliva were determined by the same assay.

Gas chromatographic properties of 1,3-dialkyl barbiturate derivatives.

The gas chromatographic properties of 1,3-dialkyl barbiturates have been studied. The retention times for 56 different 1,3-dialkyl derivatives of both aprobarbital and phenobarbital, and for 65 different 1,3-dialkyl derivatives of secobarbital were determined by gas-liquid chromatography (GLC) using a 3% SE-30 column. Retention times were found to increase with the number of carbons added. A plot of the log of the retention time versus the number of carbons in the alkyl group added for 3-alkyl derivatives of 1-alkylbarbiturates was made for each of the three barbiturates studied. A linear relationship was found to exist for derivatives having butyl or greater groups attached to the second nitrogen of the 1-alkylbarbiturates. The slopes of the graphs for the 3-alkyl derivatives of 10 different 1-alkylbarbiturates for each barbiturate studied were all found to be identical with a similar plot for straight chain hydrocarbons. This linear relationship allowed calculation of the retention times of large N,N-dialkyl barbiturates, such as N,N-eicosanylsecobarbital, which are not readily synthesized or chromatographed.

The effect of time of death on extravascular tissue/blood secobarbital concentration ratios in the rat.

Extravascular liver/blood and brain/blood ratios were found to be an average of 6% and 1% higher, respectively, in all experiments than total liver/blood and brain/blood ratios. This difference may be informative in establishing true tissue levels. There was a significant time effect (P less than 0.05) with the extravascular liver/blood ratios but not with the extravscular brain/blood ratios. Extravascular liver/blood ratios were slightly higher in phenobarbital-pretreated animals than in non-pretreated animals. Tissue secobarbital levels in pretreated and non-pretreated animals are not different at 1/4 or 1 h, even though pretreated animals received higher doses than non-pretreated animals. Tissue levels are significantly higher (P less than 0.01) in pretreated animals than in non-pretreated animals at 4 h. It is possible that, at this time period, the barbiturate-metabolizing enzymes have become saturated or exhausted.

Identification and quantification of drugs in human amniotic fluid.

Since drugs administered to gravid females are rapidly transferred to the fetus, transplacentally acquired drugs and drug metabolities should be excreted by the fetus into amniotic fluid. Analyses have been carried out on amniotic fluid obtained at the time of delivery using a gas chromatograph-mass spectrometer-computer system. The drugs that have been identified are caffeine, secobarbital and phenobarbital. Theobromine (3,7-dimethylxanthine) and smaller amounts of 1,7- and 1,3-dimethylxanthine, three metabolites of caffeine, were also found in amniotic fluid, but metabolites of secobarbital and phenobarbital were not detected. It is known that human fetal tissues have active enzyme systems for metabolizing drugs, but these results suggest that this may be a selective rather than general occurence.

Column chromatographic analysis of barbiturates in their dosage forms. II. secobarbital, amobarbital, and pentobarbital.

A general method for the analysis of barbiturates, using column parition chromatography, was extended to the assay of secobarbital, amobarbital, and pentobarbital. A solution of the barbiturate constitutes the immobile phase in the chromatographic system. It is eluted with ether-isooctane (1+9) and passed onto a K3PO4 column, which retains the barbiturate while extraneous materials are washed out. The barbiturate is removed from the column with etherisooctane (3+1), extracted from the eluate with NH3, and measured spectrophotometrically.

Correlation of brain levels of barbiturate enantiomers with reported differences in duration of sleep.

The concentrations of R(+)-and S(-)-pentobarbital and R(+)-and S(-)-secobarbital were determined in the brain stem, hypothalamus, cerebellum and cortex of mice after i.v. administration. No regional differences in enantiomer concentration or barbiturate/metabolite ratio were observed to account for the substantial potency differences reported for the enantiomeric forms of pentobarbital and secobarbital. Stereoselective differences in the rate of enantiomer metabolism and in stereochemical fit to the central nervous system receptor are discussed as possible reasons for the differences in duration of action.