[The use of enzymatic hydrolysis for isolation of barbituric acid derivatives from blood (as exemplified by phenobarbital and barbamyl)].

Modern isolation techniques by direct extraction with organic solvents or after protein precipitation by various sedimenting or salting-out agents are characterized by low efficiency and do not permit to liberate derivatives of barbituric acid from their complexes with blood proteins. The use of enzymatic hydrolysis makes it possible to break bonds between barbiturates and protein and thereby improve the efficiency of isolation. We performed enzymatic hydrolysis of the model phenobarbital-blood and barbamyl-blood complexes with the use of trypsin, pepsin, chymotrypsin, and papain. The degree of phenobarbital extraction with trypsin and barbamyl was estimated at 62.1 +/- 1.2% and 75.1 +/- 1.6% respectively; in other words, it was 32.7 +/- 1.0% and 51.1 +/- 1.0% higher than that achieved by traditional methods. Certain validation characteristics of the new method are presented.

Determination of amobarbital and phenobarbital in serum by gas chromatography-mass spectrometry with addition of formic acid to the solvent.

A rapid and accurate method for quantification of amobarbital and phenobarbital was developed using gas chromatography-mass spectrometry (GC-MS) without derivatization. Though the compounds measured without derivatization showed low sensitivity because of adsorption, addition of 3% formic acid to the solvent improved the sensitivity for the analytes. Taking account of matrix effect, solid-phase and liquid-liquid extraction from serum were examined. The correlation coefficients of the calibration curves were 0.9995 or better, and the accuracy and precision of intraday and interday assays were in line with Food and Drug Administration (FDA) criteria.

Amobarbital interactions with 25-hydroxycholecalciferol: effects on the extraction, quantification, and competitive protein binding in vitro.

Amobarbital has been found to coelute with 25-hydroxycholecalciferol (25OHD3) on a normal phase high performance liquid chromatographic system and cause subsequent interference in the UV detection and plasma transport competitive protein binding assay for this vitamin D metabolite. Concentrations of 25OHD3 were overestimated by 95% in the presence of 0.4 mg amobarbital in the competitive protein binding assay; as little as 0.1 mg amobarbital caused a 22% overestimation in the concentration of 25OHD3 in the assay. Separation of 25OHD3 from amobarbital on a reverse phase high performance liquid chromatographic system allowed for proper quantification without interference. Because of the similarity of chemical structures, other barbital-based compounds may cause similar interactions with 25OHD3 or other vitamin D metabolites as well.

Salivary excretion of amobarbital in man.

The concentrations of amobarbital in saliva and serum were determined in 5 normal adults following ingestion of 120 mg of sodium amobarbital. There was excellent linear relationship between amobarbital concentrations in saliva and serum (r = 0.993); salivary levels were 36.1% of serum levels. Since the pH of saliva was generally lower than that of blood in man, the degree of ionization of amobarbital in serum and saliva had to be taken into consideration. Estimation of the protein binding of amobarbital in serum from concentrations of amobarbital in saliva and serum was in good agreement with the in vitro data of equilibrium dialysis.

Genetic study of amobarbital elimination based on its kinetics in twins.

Following the intravenous administration of 125 mg amobarbital sodium in 7 pairs of dizygotic and 7 pairs of monozygotic twins, the time-course of plasma concentrations was observed. The number of detectable compartments varied from subject to subject but was consistently 1 or 2 or 3 within a given individual. The terminal slope of the semilogarithmic concentration-time plot (corresponding to biologic half-life of 22.8 hr) did not represent the elimination rate constant even in persons with apparently single-compartmental characteristics. The redistribution of amobarbital was rapid in comparison with its elimination. The rate of the latter (characterized by kel = 0.051 hr-1, plasma clearance = 37.7 ml/min) could be closely identified with the rate of metabolism. The twin data showed that genetic control was exerted on kinetic parameters characterizing the rate of amobarbital elimination and, therfore, the rate of its metabolism. Correlation analysis suggested that this control was independent of size factors, which were themselves substantially heritable. The genetic analysis of twin data included, in addition to intracelass correlations and Holzinger's H factors, newly developed lower (and upper) bounds of the broad-sense heritability. In pharmacogenetic studies, assessment of model-independent kinetic parameters, such as plasma clearance with or without adjustment for body weight, is recommended.

The kinetics of amylobarbitone metabolism in healthy men and women.

1. Sodium amylobarbitone (3.54 mg/kg) was given by intravenous injection to seven healthy men and nine healthy women who were not receiving other drugs. Serum amylobarbitone and urine hydroxyamylobarbitone concentrations were measured by gas-liquid chromatography. There was no significant difference between the groups either in the serum amylobarbitone concentration/time curves or in the urinary excretion of hydroxyamylobarbitone.2. The serum amylobarbitone concentration decayed over 48 h as a double exponential function of time; the first exponential component had a mean half-time of 0.6 h (males 0.56 +/- 0.06 h, females 0.62 +/- 0.08 h, +/- S.E.) and the second exponential component had a mean half time of 21 h (males 22.7 +/- 1.6 h, females 20.0 +/- 1.0 h, +/- S.E.).3. The urinary excretion of hydroxyamylobarbitone over 48 h accounted for 34% of the dose (males 33.8 +/- 3.2%, females 35.2 +/- 3.0%, +/- S.E.). One male and two female subjects excreted hydroxyamylobarbitone partly as a conjugate which was readily hydrolysed in acid.4. An elimination constant (k(el)) derived from the serum concentration/time curve by the application of a two compartment model was approximately proportional to beta (h(-1)), the rate constant of the second exponential component. There was a positive correlation (r=0.78, P<0.001) between beta and the mean rate of urinary excretion of hydroxyamylobarbitone during the 24 to 48 h period.

Impairment of cognitive function associated with hydroxyamylobarbitone accumulation in patients with renal insufficiency.

1. Sodium amylobarbitone was given by intravenous infusion to six patients with chronic renal insufficiency and to six healthy volunteer subjects. Serum concentrations of amylobarbitone and its major metabolite hydroxyamylobarbitone were measured by a gas chromatograph method.2. The serum concentrations of amylobarbitone were consistently lower in the patient group than in the control group and the concentration half time was shorter (0.10>P>0.05); the 48 h urinary excretion of hydroxyamylobarbitone was reduced (P<0.001) and the serum concentrations of hydroxyamylobarbitone were consistently raised.3. When two patients were given 200 mg of sodium amylobarbitone daily over five consecutive days the serum concentration of hydroxyamylobarbitone rose steadily to a maximum of about 8 mug/ml. The serum concentrations in two healthy control subjects did not exceed 0.5 mug/ml.4. Three parallel tests of cognitive function (Otis matched test forms A, B and C) were given to 16 control patients and to 12 amylobarbitone-treated patients. Significant impairment of performance was observed in test B (P<0.001) at a time when amylobarbitone only could be detected in the patients' serum, and in test C (P<0.001) when amylobarbitone concentrations were very low (0.52+/-0.08 mug/ml+/-SEM) but hydroxyamylobarbitone concentrations were still high (3.30+/-1.23, mug/ml+/-SEM).5. There was a strong (r=-0.71) and significant (P<0.01) negative correlation between the performance in test C and the serum concentration of hydroxyamylobarbitone. It is concluded that hydroxyamylobarbitone has cerebral depressant effects in man.

Metabolism of amylobarbitone in patients with chronic liver disease.

1. A single dose of amylobarbitone (3.23 mg/kg) was given by intravenous injection to each of ten healthy controls and two groups of five patients with chronic liver disease. A curve of serum amylobarbitone concentration against time was prepared for each subject and the proportion of the serum amylobarbitone bound to protein determined. The urinary excretion of the metabolite hydroxyamylobarbitone, ethyl (3 hydroxyisoamyl) barbituric acid was measured.2. The degree of protein binding of serum amylobarbitone was reduced in the five patients (group I) with abnormally low concentrations of albumin in serum (<3.5 g/100 ml) but was normal in the five patients (group II) with normal serum albumin concentrations (>3.5 g/100 ml).3. The equation for a double exponential decay was fitted to the concentration/time curves for amylobarbitone free in the serum water. The mean intercepts and rate constants were used to calculate the dimensions of mathematical models based on a two compartment open system.4. The five patients (group I) who had abnormally low concentrations of albumin in serum showed impairment of amylobarbitone metabolism; the rate constant beta(h(-1)) for the second exponential decay of serum amylobarbitone concentration was reduced (P<0.01), the urinary excretion of hydroxyamylobarbitone was reduced (P<0.001) and the mean serum water clearance (C, ml/min) representing amylobarbitone elimination by metabolism was reduced.5. The five patients (group II) who had normal concentrations of albumin in serum showed no impairment of amylobarbitone metabolism. Within the total patient group there were strong and significant positive correlations between the serum albumin concentration and each of the indices of the rate of amylobarbitone metabolism.6. Both patient groups showed an increase in the first dispositional rate constant alpha(h(-1)) and in the clearance (C(t) ml/min) representing transfer between central and peripheral compartments. The physiological basis for this observation is uncertain.7. The clinical response to the single intravenous dose of amylobarbitone was not significantly greater (P=0.11) in the patient group (I) with slow amylobarbitone metabolism than in the patient group (II) with a normal rate of amylobarbitone metabolism.

Age-dependent disposition of amobarbital: analog computer evaluation.

Previously published observations on 4-hour and 24-hour amobarbital blood levels in two groups of subjects (ages 20--40 and over-65) were analyzed with use of an analog computer and literature data for the rate constants of absorption, distribution and metabolism. It was found that the volume of distribution did not change with age, and the increase in biologic half-life from 22.8 hours in the young subjects to 86.62 in the elderly subjects was due to a decreased rate of metabolism. When the one-point method is used, the size of the nightly dose of amobarbital should be reduced in elderly subjects from 200 mg to 50 mg in order to maintain the same steady-state blood levels found in younger subjects.

Barbiturate blood levels found at necropsy in proven cases of acute barbiturate poisoning.

In order to determine whether blood barbiturate levels could be used to ascertain that death had been caused by barbiturate overdose, samples of blood from 128 subjects of coroners' necropsies were examined for barbiturate content. Sixty of these were well authenticated cases of barbiturate overdosage, and barbiturates were implicated, together with other factors such as alcohol and carbon monoxide, in a further 16 cases. The remaining 52 cases were of an eliminatory nature, 10 of which had low barbiturate blood levels considered to be within the therapeutic range.The results indicate that when the accepted levels producing loss of consciousness are exceeded, and maintained, death will ensue if treatment is not given. These results may be of value in assessing findings in necropsies requested by the coroner, and are in no way applicable to the living patient in whom it is well established that recovery from higher blood levels may take place if adequate treatment is available.

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.

The effects of chlordesmethyldiazepam on behavioral performance and subjective judgment in normal subjects.

Eight normal subjects were tested on a battery of subjective and psychological tests 12 hours after a hypnotic dose of chlordesmethyldiazepam (1 or 2 mg), amylobarbitone sodium (100 mg), and a placebo. The tests included self-ratings of hypnotic effects and alertness; reactiontime, card-sorting, coding and cancellation tasks; arithmetic; and tapping. Before each task, test anxiety and performance expectation was self-rated; and after the task, judgment of performance was self-rated. Residual effects were definitely detectable after the 2-mg dose of benzodiazepine with both behavioral impairment and subjective hangover. Both the 1-mg dose and the barbiturate were almost devoid of such effects. Very few drug effects on test anxiety and performance judgment were discerned. Plasma concentrations of amylobarbitone were related to decreases in test anxiety and of chlordesmethyldiazepam with ratings of sleepiness.

A system approach to pharmacodynamics. III: An algorithm and computer program, COLAPS, for pharmacodynamic modeling.

Many pharmacodynamic (PD) models may be generalized in the form E(t) = N(L[c(t)]), where E(t) is a recorded effect response, c(t) is a sampled drug level, N is a nonlinear autonomic function, and L is a linear operator that commonly is a convolution operation. The NL class of PD models includes the traditional effect compartment PD models as a subclass, but is not limited to such models. An algorithm and computer program named COLAPS, based on system analysis principles and hysteresis minimization, that enable N and L to be empirically determined for the NL class of models without addressing specific kinetic structure aspects ("model independence") are presented. The kinetic concepts of biophase conduction and transduction functions are used by COLAPS. Such an approach is more general than the effect compartment approaches because it does not assume first-order transport principles. The pitfalls of hysteresis minimization in PD modeling are discussed and the procedures taken by COLAPS to avoid these pitfalls are outlined. A transformation technique prevents improper convergence to a point. A novel reparameterization scheme is introduced that maximizes the flexibility of the kinetic functions and extends the generality of the analysis. Inequality function constraints are maintained without the need for troublesome constrained nonlinear optimization procedures. Usage of the COLAPS program is illustrated in the analysis of the PD of amobarbital. The COLAPS program resulted in an excellent minimization of the effect versus biophase level hysteresis. The biophase conduction function, the biophase drug level (normalized), and the transduction curve were determined. The transduction curve showed clear biphasic behavior.

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.

Effect of paracetamol on amylobarbitone hydroxylation in man: a gas chromatographic method for simultaneous estimation of underivatized paracetamol and barbiturates.

A rapid and specific technique for the simultaneous gas chromatographic estimation of underivatized paracetamol and barbiturates using an alkali flame ionization detector is described which is suitable for both forensic and pharmacokinetic investigations. An improved method for estimation of 3-hydroxyamylobarbitone is also detailed. These techniques were used in an investigation of the effects of oral administration of 1 g paracetamol 8 hourly on the formation of 3-hydroxyamylobarbitone from a single oral dose of 200 mg sodium amylobarbitone. No significant changes were found in the plasma concentrations and total body clearance of amylobarbitone nor was there any alteration in the urinary elimination of 3-hydroxyamylobarbitone.

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.