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.

High-throughput determination of valproate in human samples by modified QuEChERS extraction and GC-MS/MS.

A new high-throughput method was developed for analysis of valproate in human plasma samples by QuEChERS extraction and gas chromatography-tandem mass spectrometry (GC-MS/MS). Plasma samples (0.2 ml) spiked with valproate and secobarbital-d5 (internal standard) were diluted with 1.3 ml of distilled water. Acetonitrile (1 ml) was added followed by 0.4 g MgSO4 and 0.1 g NaOAC. After a centrifugation step (2000 g for 10 min), 1 ml of the supernatant was transferred to a dispersive-solid phase extraction (dSPE) tube containing 150 mg MgSO4 and 50 mg C18. This mixture was vortexed and centrifuged at 3000 g for 5 min, and then the upper layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 40 µl ethyl acetate, and a 1-µl aliquot was injected into the GC-MS/MS. The GC separation of the compounds was achieved on a fused-silica capillary column Rxi-5Sil MS (30 m × 0.25 mm i.d.; 0.25-µm film thickness) and detected by MS/MS operating in electron ionization ion source mode. The regression equations showed excellent linearity (r > 0.9997) from 50 to 5000 ng/ml for plasma, with limit of detection of 10 ng/ml. The extraction efficiency of valproate for plasma ranged between 71.2%-103.5%. The coefficient of variation was <18.5%. The method was successfully applied to actual analyses of an autopsy case. This method can be useful for simple and reliable measurements of valproate in clinical and toxicological analyses; it can be integrated in screening and simultaneous determination methods for multiple drugs and poisons in the further studies.

Influence of cannabidiol on secobarbital effects and plasma kinetics.

To investigate the possible metabolic interaction between cannabidiol (CBD) and secobarbital, 6 male volunteers received 150 mg/70 kg sodium secobarbital orally immediately after smoking a marihuana cigarette prepared to deliver 0, 150, or 500 mug/kg CBD. The study was performed in a double-blind manner with each of the three treatments being assigned to every subject. Clinical effects and plasma secobarbital concentrations were recorded at periodic intervals. CBD did not alter the summary parameters which describe the secobarbital plasma concentration time curve. Secobarbital half-life, peak concentration, time of peak concentration, and area under the curve were unchanged by the coadministration of CBD. Clinical effects of secobarbital were also unaltered by CBD pretreatment. Thus at the doses administered, CBD does not appear to inhibit secobarbital metabolism in man.

Decreased enkephalinase activity following postnatal treatment with phenobarbital.

The effect of postnatal administration of phenobarbital on enzymes degrading enkephalin was examined. Daily subcutaneous injections (45 mg/kg) of phenobarbital were given to male and female rats from postnatal day 1 to 19. Brains from rats treated with saline and phenobarbital were used to prepare aminopeptidases (high speed supernatant) and enkephalinase A (synaptosomal membrane preparation). Incubation of methionine enkephaline (ME) with aminopeptidases from rat brain liberated tyrosine (T), while incubating with enkephalinase A resulted in the formation of tyrosylglycylglycin (TGG). Separation and quantification of tyrosine, tyrosylglycylglycin and methionine enkephalin was performed using a high performance liquid chromatograph, coupled to electrochemical and ultraviolet detectors in series. The treatment of the rats with phenobarbital resulted in a significant inhibition of enkephalinase A when measured in vitro, using methionine enkephalin as substrate. Preliminary studies with secobarbital show similar results to those obtained with phenobarbital.

A rapid micro-method for the screening and measurement of barbiturates and related compounds in plasma by gas-liquid chromatography.

A rapid gas-chromatographic method has been developed for the determination of butobarbitone, amylobarbitone, hexabarbitone, pentobarbitone, quinalbarbitone, phenobarbitone glutethimide, and methaqualone in ;finger-prick' samples of plasma. This has been applied to the analysis of some of these drugs in plasma taken from patients after therapeutic dosage and over-dosage.

Starch and talc emboli in drug addicts' lungs.

The lungs of eight drug addicts dying as a consequence of their habit have been examined. All showed the presence of small amounts of talc emboli and five the presence of starch emboli. Talc was invariably associated with a marked foreign body reaction which was insignificant in association with starch. Animal experiments showed very rapid (90% in 24 hours) removal of maize starch emboli; such rapid removal in man would explain the lack of a foreign body response. Quantitation of the amount of starch present in lungs from two of the cases gave values of 1.5 and 5.2 g. The higher amount could have been a contributory factor in the sudden death of the addict. The amounts of talc seen were not sufficient to be of clinical significance.

Overdosage with pentobarbital and secobarbital: assessment of factors related to outcome.

Factors related to clinical outcome following acute overdosage with pentobarbital or secobarbital were assessed in a series of 162 patients hospitalized during the period 1962 to 1975. The mean ingested dose was 2 Gm (range 0.2 to 10.0 Gm), and plasma barbiturate concentrations ranged from 2.0 to 72.0 microgram/ml. Serious intoxication was common. Intubation and assisted ventilation were required in 59 per cent of patients, and 23 per cent developed clinically important hypotension. Four patients died, all relatively young females. Multiple regression and discriminant function analyses, performed on a subset of 88 patients for whom complete data were available, indicated that plasma barbiturate concentration and/or ingested dose were the most important correlates of serious intoxication among identifiable variables available on admission. Coingestion of other central nervous system depressants, such as ethanol, had no obvious effect on outcome. The present study suggests that measurement of plasma barbiturate concentrations is of value in identifying patients at risk of developing serious intoxication after overdosage with pentobarbital or secobarbital.

Routine identification and determination of 11 barbiturates in biological samples.

A very easy, reliable and specific gas-chromatographic method for identification and dosage of 11 barbiturates in plasma is presented. Methylene unit values determined on two types of column (one polar, one non-polar) allows very accurate identification with a minimum risk of error due to fluctuations in operating conditions. Sensitivity of the method is 0.5 microgram/ml plasma with a flame ionization detector. Derivatization procedure is complete, without any degradation phenomenon as tested by mass spectrometry. Such a method can be easily used as routine procedure for toxicological or pharmacological applications.

An improved high-pressure liquid chromatographic assay for secobarbital in serum.

A high-pressure liquid chromatographic method for the analysis of secobarbital in serum was developed. Secobarbital was extracted from buffered serum (pH 5.5) with a solvent mix of hexane-ether-n-propanol. 5-(4-Methylphenyl)-5-phenylhydantoin was added as an internal standard. Separation of secobarbital and internal standard from serum constituents and other drugs was achieved on a 5-mum C-18 reversed-phase column using an acetonitrile-phosphate buffer (pH 4.4) mobile phase. The eluent was monitored at 195 nm. The sensitivity limit of the assay was approximately 0.02 microgram/ml with 0.5 ml of serum sample. The application of this method to pharmacokinetic studies in pediatric patients was demonstrated.

Therapeutic drug monitoring: measurements of antiepileptic and barbiturate drug levels in blood by gas chromatography with nitrogen - selective detector.

The nitrogen-specific detector for gas chromatography consists of a non-volatile rubidium silicate bead, around which nitrogen-containing compounds are pyrolyzed. Speed, sensitivity, specificity, accuracy, small sample size and minumum sample handling are characteristics of the nitrogen detector that render it superior to conventional gas chromatographic detectors. The detector has been utilized to effect a simple and rapid quantitation of allobarbital, amobarbital, butabarbital, heptabarbital, pentabarbital, phenobarbital and secobarbital, plus the anticonvulsants diphenylhydantoin and primidone. Extraction of the drugs from acidified serum into organic solvent containing internal standard is followed by oncolumn methylation with methanolic trimethylphenyl ammonium hydroxide. The drugs, separated on a column of 3 percent OV-101 on Gas-Chrom Q, 100-120 mesh are readily quantitated by simple calculations based on peak-height ratios. Therapeutic drug monitoring is discussed in relation to recent concepts of drug-protein binding, drug-drug interactions, drug biotransformation and problems of multiple drug therapy.

Death through injection of barbiturates.

The authors report two cases of lethal intoxication due to barbiturates in two male individuals, respectively 24 and 35 years old. They stress the comparatively rare mode of administration of such drugs in the absence of another party, i.e. the parenteral way.

Interpretations of the TDxFLx calibration data of the abused drugs.

The useful TDxFLx calibration data was obtained for the interpretation of the interactions of the abused drugs to sheep antiserum protein. The antibody of TDxFLx calibrators was prepared from sheep antiserum. Furthermore these data can be used to interpret the abused drug-protein binding phenomena in human body and the TDxFLx screening results of the abused drugs in urine samples. TDxFLx system uses fluorescence polarization immunoassay technique that is a competitive binding immunoassay methodology to allow tracer-labeled antigen (*Drug) and patient antigen (Drug) to compete for the same binding sites on the antibody molecules of sheep antiserum. To obtain the binding parameters, binding constant (K) and number of independent binding site (n), generally, Scatchard equation is used. This Scatchard equation is expressed in the concentration terms of free drug, bound drug, and protein (antibody). The binding parameters can not be obtained by applying the TDxFLx calibration data to the Scatchard equation directly because the TDxFLx calibration data are composed of the fluorescence polarization and the total drug concentrations. To obtain the binding parameters from the TDxFLx calibration data the new useful equation which was expressed in the total concentrations of drug and fluorescence polarization should be derived. Derivation of new equation was based on the Scatchard equation. The TDxFLx calibration data was curve fitted to the derived equation using KaleidaGraph program and Macintosh computer. The binding constant (K) and the number (n(P(t))) of binding site of 11-nor-delta(9)-tetrahydrocannabinol-9-carboxylic acid (COOH.THC) on the antibody were 1.14 x 10(8)l/mole and 4.04 x 10(-7)M, respectively. The binding constant and the number (n(P(t))) of binding site of amphetamine were 5.15 x 10(5)l/mole and 2.05 x 10(6)M, respectively. In case of COOH.THC the fluorescence polarization decreased linearly with the concentration. However, in case of amphetamine or the other three abused drugs the fluorescence polarizations decreased exponentially with their concentrations.

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.

Rapid simultaneous capillary electrophoretic determination of (R)- and (S)-secobarbital from serum and prediction of hydroxypropyl-gamma-cyclodextrin-secobarbital stereoselective interaction using molecular mechanics simulation.

Secobarbital is a hypnotic sedative that is used in the treatment of preoperative anxiety and to manage elevated intracranial pressures and cerebral ischemia due to neurosurgical procedures. Stereoselective resolution is first predicted using hydroxypropyl-gamma-cyclodextrin (HPGCD)-(R)- and (S)-secobarbital transient complex energy calculations using SYBYL 6.01 molecular modeling software. Separation is accomplished as predicted using 40 mM HPGCD in 50 mM phosphate buffer (pH 9.0), resulting in a rapid, sensitive method for the determination of (S)- and (R)-enantiomers of secobarbital from serum using solid-phase extraction, capillary electrophoresis (CE), and ultraviolet detection. The method involves extraction of both enantiomers and the internal standard, aprobarbital, from serum using C18 Bond-Elut solid-phase cartridges. The CE system consists of fused-silica capillary (52 cm x 75-microns i.d.) maintained at a run voltage of 15 kV with detection performed at a wavelength of 254 nm. The detection and quantitation limits from serum for (S)- and (R)-secobarbital are 1 microgram/mL. Linear calibration curves from 1 to 60 micrograms of both (S)- and (R)-secobarbital show a coefficient of determination of more than 0.999. The precision and accuracy of the method, calculated as relative standard deviation (%) and percent error, are 0.35-4.48% and 0.71-8.67%, respectively, for (R)-secobarbital and 0.39-4.08% and 2.16-3.70%, respectively, for (S)-secobarbital.

[Rapid identification and quantitation of barbiturates in plasma using solid-phase extraction combined with GC-FID and GC-MS method].

A rapid screening method for the quantitation and identification of 7 barbiturates in plasma is presented. The method employs solid-phase extraction at pH 3 using X-5 resin as absorbent. Identification of each barbiturate is achieved by retention time and GC-MS. Detection threshold (0.3-0.5 micrograms.ml-1 in a 0.5 ml plasma sample and 1 microliter injection volume), precision (RSD 3.46-6.41%), and linearity (r > 0.99) were satisfactory for blood level monitoring and clinical toxicological applications.

[A double column double pH solid phase extraction and capillary GC/FID method for rapid simultaneous determination of acidic and basic drugs in human plasma].

A capillary gas chromatographic method for the rapid simultaneous identification and quantitation of acidic and basic drugs in human plasma is presented. A special double column solid phase extraction device was designed, in which two X-5 resin columns can extract drugs at different pH. The detection limits for acidic and basic drugs can reach 0.5 microgram.ml-1, while the time needed is only half of that when using commercial solid phase extraction cartridges.

Quantitation of amobarbital, butalbital, pentobarbital, phenobarbital, and secobarbital in urine, serum, and plasma using gas chromatography-mass spectrometry (GC-MS).

Barbiturates are central nervous system depressants with sedative and hypnotic properties. Some barbiturates, with longer half-lives, are used as anticonvulsants. Their mechanism of action includes activation of gamma-aminobutyric acid (GABA) mediated neuronal transmission inhibition. Clinically used barbiturates include amobarbital, butalbital, pentobarbital, phenobarbital, secobarbital, and thiopental. Besides their therapeutic use, barbiturates are commonly abused. Their analysis is useful for both clinical and forensic proposes. Gas chromatography mass spectrometry is a commonly used method for the analysis of barbiturates. In the method described here, barbiturates from serum, plasma, or urine are extracted using an acidic phosphate buffer and methylene chloride. Barbital is used as an internal standard. The organic extract is dried and reconstituted with mixture of trimethylanilinium hydroxide (TMAH) and ethylacetate. The extract is injected into a gas chromatogram mass spectrometer where it undergoes "flash methylation" in the hot injection port. Selective ion monitoring and relative retention times are used for the identification and quantitation of barbiturates.