Determination of Barbiturates by Femtosecond Ionization Mass Spectrometry.

Barbiturates are highly susceptible to dissociation in mass spectrometry (MS) because of their long side chains combined with a nonaromatic ring consisting of several carbonyl and amine groups. As a result, they exhibit extensive α-cleavage and subsequent rearrangement, making the identification of these compounds difficult. Although a library of electron ionization MS (EIMS) is available, most barbiturates have very similar fragment patterns. Accordingly, it would be desirable to develop a technique for soft ionization, providing a molecular ion and large fragment ions as well. In this study, a molecular ion was clearly observed, in addition to large fragment ions, for a variety of barbiturates based on multiphoton ionization MS (MPIMS) using a tunable ultraviolet femtosecond laser as the ionization source (fs-LIMS). This favorable result was achieved when the optimal laser wavelength for minimizing the excess energy remaining in the ionic state was used. An examination of the photofragmentation pathways suggested that an H atom in the side chain was abstracted by an oxygen atom in the carbonyl group in the ring structure thus initiating fragmentation and subsequent rearrangement. Barbiturates that are substituted with alkyl groups (amobarbital and pentobarbital) had narrower spectral regions for optimal ionization than the other barbiturates with alkyl and alkenyl groups (butalbital and secobarbital) and more with alkyl and phenyl groups (phenobarbital). All of the barbiturates studied provided unique mass spectral patterns in fs-LIMS, which was useful for the reliable identification of these compounds in practical trace analysis.

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.

Postdischarge secobarbital after ED migraine treatment decreases pain and improves resolution.

OBJECTIVE: The objective of the study was to determine whether the addition of postdischarge oral secobarbital to standard emergency department (ED) migraine headache therapy improves pain relief and headache resolution compared with placebo. SETTING: The setting is an urban ED with 70 000 yearly visits. METHODS: This is an Institutional Review Board-approved, randomized, nonconsecutive, double-blinded, concealed, and placebo-controlled clinical trial. Patients with a clinical diagnosis of migraine underwent standard ED treatment and were discharged with 2 tablets of either secobarbital 100 mg or placebo. At home arrival, subjects recorded headache pain on a visual analog scale (VAS), took 1 tablet, and went to bed, taking the second tablet after 1 hour if not asleep. Upon awakening, subjects completed a second VAS and survey. STATISTICAL ANALYSIS: The VAS data were analyzed using 2-tailed t test with unequal variance. Headache resolution data were analyzed using Fisher exact test. RESULTS: Fifty subjects were enrolled. Complete data and follow-up were available for 30 subjects (60%). Fourteen subjects received placebo; 16 received secobarbital. Secobarbital subjects reported an average headache pain decrease of 25 mm (-13 to -38) compared with an average increase of 3 mm (-13 to 19) in the placebo group (P = .01). Ninety-four percent of the secobarbital group vs 50% of the placebo group had complete or partial headache resolution (P < .02). All subjects in the secobarbital group reported some relief. CONCLUSIONS: Addition of postdischarge oral secobarbital to a standard ED migraine treatment regimen decreased headache pain at 24 hours after discharge and improved the rate of headache resolution compared with placebo.

Wada test using secobarbital sodium (Ional) to determine language dominance.

The intracarotid sodium amobarbital (Amytal) test, the Wada test, remains an efficient test for evaluation of language and memory function. However, due to a world shortage of amobarbital, it has become necessary to investigate the use of alternatives. We report the efficacy of the Wada test using secobarbital sodium (Ional) in determining language dominance. An accurate determination of language dominance was required in 43 patients preoperatively at our institution. Patients underwent the Wada test using secobarbital sodium, effectiveness and safety were assessed. Patients were monitored for vital signs (blood pressure, respiratory rates, heart rates and saturation of oxygen). Ten patients were further monitored for continuous intra-arterial blood pressure and monitored with scalp electroencephalography (EEG). Language dominance was determined by the Wada test with secobarbital sodium in all patients. Total volume of secobarbital sodium injected was 10-25 mg (mean 16.5 ± 3.2 mg). Changes in vital signs were minimal and any induced neurological deficits completely disappeared within 8 min. On EEG records, induced theta waves immediately appeared on the ipsilateral side of the intra-arterial injection and disappeared within 6 min. One patient described a scintillating scotoma (sensation of shimmering light in his eyes) at the moment of injection; another experienced an epileptic episode during the test and recovered after 6 min. No adverse events were observed in the remaining 41 cases. We propose secobarbital sodium as a safe and reliable alternative to sodium amobarbital used in the Wada test to determine language dominance.

Chlorpromazine: direct measurement of differential behavioral effect.

A retarded child with a high stereotyped rocking rate was conditioned to pull a ball on a reinforcement schedule in which the fixed ratio aof rewarded to nonrewarded responses was 100. Results show no rocking movements during ball-pulling; but when ball-pulling was on extinction, rocking returned to its original rate. Chlorpromazine blocked rocking movements during extinction, but had no effect on ball-pulling. Delivery of one free reinforcer was sufficient to reinstate ball-pulling after extinction, but the stimulus properties of the free reinforcer were not affected by the drug.

Destruction of cytochrome P 450 by secobarbital and other barbiturates containing allyl groups.

Administration of certain commonly used barbiturates containing allyl groups, such as secobarbital, allobarbital, or aprobarbital to rats treated chronically with a microsomal enzyme inducer causes a rapid destruction of the liver microsomal hemoprotein that serves as the terminal oxidase for drug metabolism. In contrast, barbiturates without an allyl group do not have this effect. The decrease in this hemoprotein, cytochrome P(450), by the barbiturates containing an allyl group could also be demonstrated in an in vitro liver microsomal system requiring reduced nicotinamide adenine dinucleotide phosphate. These results suggest that the barbiturates containing an allyl group are converted to a metabolite that leads to the destruction of cytochrome P(450).

Absence of REM rebound after barbiturate withdrawal.

Administration of three different barbiturates reduced rapid eye movement (REM) sleep. Drug withdrawal led to a return to baseline REM] values without significant overshoot. Similar results are observed with administration of benzodiazepines in pharmacologically equivalent dosages; therefore, a distinction between these two drug classes on the basis of withdrawal effects on the sleep electroencephalogram appears unwarranted. Further investigation is required determine why high REM levels are sometimes associated with the withdrawal of sedative-hypnotic agents.

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.

Barbiturates and lung cancer in humans.

In a hypothesis-generating study looking for possible carcinogenic effects of drugs in humans, each of three barbiturates (pentobarbital sodium, phenobarbital, and secobarbital sodium) showed a statistically significant association with the subsequent development of lung cancer, with relative risks ranging from 1.5 to 2.8. Further analysis showed that the association was not explained by the increased prescription of barbiturate drugs shortly before the diagnosis of lung cancer or by an association of barbiturate use with cigarette smoking. Much of the data did not support a causal relationship. Neither a relation of lung cancer to duration or intensity of use not one between barbiturate use and a specific histologic type could be demonstrated.

Screening prescription drugs for possible carcinogenicity: eleven to fifteen years of follow-up.

Using computerized pharmacy records from 1969 to 1973 for a cohort of 143,574 members of the Kaiser Permanente Medical Care Program, we have been testing associations of 215 drugs or drug groups with subsequent incidence of cancer at 56 sites. This paper presents findings with follow-up through 1984. There were 227 statistically significant (P less than 0.05, two-tailed) associations: 170 positive, 57 negative. Some were undoubtedly chance findings; others were likely due to confounding by unmeasured covariables. However, several associations suggested hypotheses for further studies and/or the need for continued observation. Most notable among findings not previously reported were associations of several antibiotics, both oral and topical, with lung cancer. These associations could not be explained by indications for drug use or by differences in smoking habits between users and nonusers, and suggest a possible link between the occurrence of bacterial infections and risk for cancer. In general, our results continue to suggest that most medications used during that period did not affect cancer incidence substantially. However, for less frequently prescribed medications, our power to detect moderate increases in cancer risk was quite low.

Inhibition of the acute toxicity and adrenocorticolytic effect of 7,12-dimethylbenz(a)anthracene by isopropylvaleramide and allylisopropylacetamide in the rat.

Isopropylvaleramide (IVA) and allylisopropylacetamide (AIA) inhibit hemorrhagic adrenocortical necrosis and mortality caused by 7,12-dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats. Unlike their effect on hepatic microsomal cytochrome P-450, the anti-DMBA action of these compounds does not depend on the presence of the reactive allyl group in the molecule. Similarly, related barbiturates, regardless of whether they contain, like AIA, an allyl group and consequently destroy cytochrome P-450 (secobarbital and aprobarbital) or have, like IVA, saturated side chains and therefore do not effect the microsomal hemoprotein (pentobarbital and phenobarbital), proved ineffective in preventing both adrenal damage and death caused by DMBA. Hence, the protective action of IVA and AIA cannot be attributed to the destruction of the microsomal enzyme system responsible for the activation of DMBA. The toxicity of another carcinogen, dimethylnitrosamine, which also requires metabolic activation by microsomal enzymes, is not influenced by either IVA or AIA. IVA, which counteracts the adrenocorticolytic action of DMBA when given prior to, simultaneously with, or even after this carcinogen, has no discernible effect on hydrocarbon metabolism in vivo or in vitro. IVA is one of the most powerful inhibitors of the acute toxicity of DMBA. It has the simplest aliphatic structure and the smallest molecule among protectors of the adrenals against hydrocarbon-induced damage; its mechanism of action awaits further elucidation.

Barbiturates induce mitochondrial depolarization and potentiate excitotoxic neuronal death.

Barbiturates are widely used as anesthetics, anticonvulsants, and neuroprotective agents. However, barbiturates may also inhibit mitochondrial respiration, and mitochondrial inhibitors are known to potentiate NMDA receptor-mediated neurotoxicity. Here we used rat cortical cultures to examine the effect of barbiturates on neuronal mitochondria and responses to NMDA receptor stimulation. The barbiturates tested, secobarbital, amobarbital, and thiamylal, each potentiated NMDA-induced neuron death at barbiturate concentrations relevant to clinical and experimental use (100-300 microm). By using rhodamine-123 under quenching conditions, barbiturates in this concentration range were shown to depolarize neuronal mitochondria and greatly amplify NMDA-induced mitochondrial depolarization. Barbiturate-induced mitochondrial depolarization was increased by the ATP synthase inhibitor oligomycin, indicating that barbiturates act by inhibiting electron transport sufficiently to cause ATP synthase reversal. Barbiturates similarly amplified the effects of NMDA on cytoplasmic free calcium concentrations. The cell-impermeant barbiturate N-glucoside amobarbital did not influence mitochondrial potential or potentiate NMDA neurotoxicity or calcium responses. However, all of the barbiturates attenuated NMDA-induced calcium elevations and cell death when present at millimolar concentrations. Whole-cell patch-clamp studies showed that these effects may be attributable to actions at the cell membrane, resulting in a block of NMDA-induced current flux at millimolar barbiturate concentrations. Together, these findings reconcile previous reports of opposing effects on barbiturates on NMDA neurotoxicity and show that barbiturate effects on neuronal mitochondria can be functionally significant. Effects of barbiturates on neuronal mitochondria should be considered in experimental and clinical application of these drugs.

Human wakefulness and biological rhythms after birth.

Both a sleep-wakefulness cycle and a basic rest-activity cycle were observed in 20 normal infants left undisturbed for ten hours following birth. Behavioral wakefulness occurred immediately following delivery and in between sleep periods despite the lack of feeding and other intervention. Medication given to mothers during labor resulted in decreased amounts of infant wakefulness and increased amounts of quiet (non rapid eye movement) sleep.

Resin hemoperfusion for acute drug intoxication.

Eight patients with drug intoxication were hemoperfused on ten occasions at a blood-flow rate of 300 ml/min with a 650gm column of Amberlite XAD-4 resin, which is a macroreticular resin with a specific adsorptive attraction for lipid-soluble organic molecules. Column clearances of glutethimide and a variety of barbiturates ranged from 207 to 300 ml/min for treatment sessions extending from 2 1/2 to ten hours. After ingestion of 75 gm of glutethimide, one patient received hemoperfusion on three successive days for nine, ten, and eight hours, respectively. She recovered after the column removed over 30 gm of drug. The patients demonstrated dramatic clinical responses with no evidence of meaningful toxic reactions. Column hemoperfusion with Amberlite XAD-4 resin was simpler and more effective than any known method of removing barbiturates and glutethimide from the blood of patients with drug overdoses.

Barbiturates inhibit intracellular Ca2+ rise induced by thrombin in rat platelets.

The effects of a number of barbiturates (anesthetic as well as anticonvulsant) on thrombin-induced calcium mobilization were tested in rat platelets using the fluorescent Ca2+ probe Fura-2. All drugs, except barbituric acid and Na-barbital, inhibited the thrombin-induced intracellular Ca2+ rise. Both the uptake of extracellular Ca2+ and the release of calcium from intracellular organelles were affected but influx was inhibited more strongly and at lower concentrations of the drugs (e.g. IC50 of thiopental was 0.83 mM for influx and 1.2 mM for intracellular release). Inhibitory potencies of the various barbiturates were markedly different. Thiopental was the most and barbital the least potent inhibitor. The order of inhibitory potency of the drugs appeared generally to follow their lipid solubility and the order of their hypnotic efficiency, with hexobarbital as the most conspicuous exception. Therefore, barbiturate treatment of cells perturbs agonist-induced calcium mobilization. This effect may be partially linked to their previously reported inhibitory action on two kinases, protein kinase C and phosphatidylinositol 4-phosphate kinase [1, 2].

Effects of oxybarbiturates on the germination behaviour of Impatiens bolstii pollen.

Swelling of and cytoplasmic streaming within the grain, germination pore expansion, and pollen tube emergence, in that order, constitute the normal germination pattern of Impatiens holstii pollen grains. This normal sequence of events becomes manifest when the pollen grains are placed on water-agar, barbituric acid-agar, and on barbital-agar media whereas this sequence of events is interfered with when the pollen grains are incubated on amytal-agar or on seconal-agar media. Evidence is presented which demonstrates that the normal sequence of events which make up the normal germination pattern of Impatiens holstii pollen grains can be separated from each other by varying the concentrations of the oxybarbiturates amytal and seconal used. Evidence is also presented which indicates that the actions of barbituric acid and the oxybarbiturates barbital, amytal, and seconal on Impatiens holstii pollen germination behaviour correlate well with certain aspects of the pharmacological literature concerning the actions of these organic compounds on the central nervous system (CNS).

Secobarbital attenuates excitotoxicity but potentiates oxygen-glucose deprivation neuronal injury in cortical cell culture.

We examined the effects of secobarbital and other sedative-hypnotic barbiturates on the neuronal death induced by exposure to excitatory amino acids or deprivation of oxygen or glucose in mouse cortical cell cultures. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionate, and kainate toxicities were attenuated in a concentration-dependent fashion by high concentrations of secobarbital or thiopental. Antagonism of NMDA toxicity was not overcome by increasing NMDA concentration and not mimicked by gamma-aminobutyrate. Despite these antiexcitotoxic actions, secobarbital exacerbated the neuronal death induced by deprivation of either glucose alone or oxygen and glucose together; death induced by oxygen deprivation alone was little affected. Thiopental and methohexital also increased oxygen-glucose deprivation injury. A possible explanation for this injury potentiation was provided by the observation that secobarbital enhanced the cellular ATP depletion induced by combined oxygen-glucose deprivation. Deleterious effects on ATP production may counterbalance the protective effects of barbiturates under some conditions.

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.

Effectiveness of intermediate-term use of secobarbital.

Secobarbital, 100 mg, was evaluated in two separate sleep laboratory drug evaluation studies, each with 4 insomniac patients. In both studies, the effect of secobarbital in inducing and maintaining sleep was evaluated, as well as the effects of the drug on sleep stages. Statistical analysis demonstrated that the results of the two studies could be combined. With short-term drug administration of secobarbital (up to 3 nights), there was an improvement in both sleep induction and sleep maintenance. Total wake time was decreased 43% below baseline and was consistently decreased in each third of the night. With intermediate-term drug administration (2 wk), total wake time was decreased only 14% (not statistically significant). Following drug withdrawal, the degree of sleep difficulty returned to baseline levels. The results indicate that secobarbital 100 mg is effective for short-term use but loses much of its effectiveness with intermediate use and suggest that the drug is of limited value for insomniac patients who require nightly medication beyond a period of 1 wk. With short-term administration, secobarbital induced a slight decrease in rapid eye movement (REM) and slow-wave sleep and a significant increase in stage 2 sleep. With intermediate administration, sleep stage values were similar to baseline levels. Following withdrawal, there was only a minimal increase in REM sleep above baseline levels, a significant increase in stage 3 sleep, and a significant decrease in stage 2 sleep. The rebound increase in stage 3 sleep is similar to that reported following withdrawal of pentobarbital.