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.

[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.

Method for screening and quantitative determination of serum levels of salicylic Acid, acetaminophen, theophylline, phenobarbital, bromvalerylurea, pentobarbital, and amobarbital using liquid chromatography/electrospray mass spectrometry.

We investigated a method for the simultaneous screening, identification, and quantitative determination of salicylic acid, acetaminophen, theophylline, barbiturates, and bromvalerylurea, drugs that frequently cause acute poisoning in Japan and therefore require rapid analysis for effective treatment in the clinical setting. The method employs liquid chromatography/electrospray mass spectrometry (LC/MS) of solid-phase extracted serum samples. For LC/MS ionization, the electrospray-ionization method was used, with acetaminophen in the positive-ion mode, and salicylic acid, theophylline, phenobarbital, bromvalerylurea, pentobarbital, amobarbital, and o-acetamidophenol (internal standard) in the negative-ion mode, the base ions were used in each case for quantitative analysis. Quantitation was possible for the following sample concentration ranges: salicylic acid and acetaminophen, 100 to 5 microg/ml; theophylline, 100 to 0.5 microg/ml; and phenobarbital, bromvalerylurea, pentobarbital, and amobarbital, 100 to 1 microg/ml. Using full-scan mass spectrometry, the lower detection limits of 1 microg/ml for salicylic acid and acetaminophen, 0.1 microg/ml for theophylline, and 0.5 microg/ml for phenobarbital, bromvalerylurea, pentobarbital, and amobarbital were adequate for identifying acute poisoning. When each compound was added to serum to a final concentration of 5 microg/ml and solid-phase extraction was performed using Oasis HLB 1-cc (30-mg), the mean recovery rate of each compound was 89.2 to 96.1% (n=5), and the coefficients of variation of the intraday and interday assays were 3.55 to 6.05% (n=5) and 3.68 to 6.38% (n=5), respectively, which are acceptable. When this method of analysis was applied in testing the sera of a female patient who had consumed a large amount of an unknown commercial drug, salicylic acid and bromvalerylurea were identified, and the treatment strategy could be determined in accordance with the serum concentration of those drugs.

Determination of amobarbital and secobarbital in plasma samples using micellar liquid chromatography.

A new liquid chromatographic procedure for the determination of amobarbital and secobarbital in plasma samples is proposed. The method uses a Spherisorb octadecylsilane ODS-2 C(18) analytical column, a guard column of similar characteristics and 0.04 M CTAB solution buffered at pH 7.5 containing 3% 1-propanol as micellar mobile phase. The UV detection was carried out at 250 nm. Butabarbital was used as internal standard. Plasma samples preparation only required adequate dilution with the mobile phase before injection into the chromatographic system. The limits of detection were 0.2 and 0.4 mg/L for amobarbital and secobarbital, respectively. The proposed method allows the determination of amobarbital and secobarbital in plasma at therapeutic levels.

[Simultaneous analysis of theophylline, phenobarbital, amobarbital and carbamazepine in serum by high performance liquid chromatography].

Theophylline, phenobarbital, amobarbital and carbamazepine are the chief therapeutic drugs in clinical practice. Because of the differences among inter-individuals in the metabolic clearance of these drugs and their toxicity at certain levels of concentration in serum, the dosage should be regulated to maintain a therapeutic blood drugs level. To achieve this a rapid and accurate assay method for drugs in blood is necessary. This paper reports that a reversed phase high performance liquid chromatographic (HPLC) method had been established for the simultaneous separation and quantitative determination of the four drugs in serum. The separation was performed on RP-C18 column using methanol:water (1:1) as mobile phase and 4-aminoantipyrime as internal standard. The eluted substances were detected at 210nm.

Estimation of amobarbital plasma-effect site equilibration kinetics. Relevance of polyexponential conductance functions.

The time delay between drug plasma concentrations and effect has been modeled most commonly by the effect compartment approach, assuming first-order monoexponential equilibrium kinetics between plasma and effect site. So far this assumption has not been rigorously probed. The purpose of the present investigation was to model the delay between amobarbital plasma concentrations and EEG effect using a new approach based on system analysis principles. This approach models the equilibrium between plasma and effect site without assuming a specific kinetic structure. Assuming linear distribution kinetics between plasma and effect site, the relationship between the two variables may be described by a convolution type of linear operation, involving a conductance function phi(t), which is approximated by a sum of exponentials. Six male Wistar-derived rats received an iv infusion of amobarbital at a rate of 10 mg/kg per min until isoelectric periods of 5 sec or longer appeared on the EEG. Frequent arterial blood samples were obtained and EEG was continuously quantified using aperiodic analysis. The amplitudes in the 2.5-30 Hz frequency band were used as EEG effect measure. The delay between plasma concentrations and EEG effect was best modeled by a biexponential conductance function. The use of a biexponential conductance function resulted in a significant further reduction (41 +/- 10%) in hysteresis when compared to a monoexponential function, indicating that the assumption of simple first-order monoexponential equilibration kinetics is inadequate. The use of a biexponential conductance function also resulted in a significantly different shape of the effect site concentration-EEG effect relationship and hence the estimated pharmacodynamic parameters, when compared with a monoexponential function. This relationship showed a biphasic behavior, with EEG effects being maximal at amobarbital concentrations of 29.6 +/- 1.3 mg/L. At 80.2 +/- 2.0 mg/L the EEG effect was reduced 50% below baseline values. A comparison was made with the equilibration between amobarbital plasma and cerebrospinal fluid (CSF) concentrations. Six male Wistar-derived rats received an iv infusion of amobarbital, 10 mg per min for 15 min. Arterial blood and CSF samples were taken simultaneously at regular intervals. The equilibration between plasma and CSF concentrations was best fitted by a monoexponential conductance function. Significant differences in equilibration profiles of CSF and effect site with the plasma site were observed. To reach 50% equilibrium the effect site requires 2.5 +/- 0.3 min and the CSF 3.5 +/- 0.2 min, to reach 95% the values were, respectively, 90 +/- 27 and 15 +/- 1 min. This suggests that CSF is kinetically distinguishable from the effect site.

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.

Estimating the clearance of amylobarbitone from a single plasma measurement.

Amylobarbitone sodium (200 mg) was given by intravenous injection to nine healthy, young adults (four males). Subjects were drug-free and nonsmokers. Serial blood samples were drawn for 48 h following the infusion, and multiple sample and single sample estimates of clearance were calculated. The mean (+/- s.d.) values for clearance (CL) and apparent volume of distribution (V) were 0.032 (+/- 0.007) 1 h-1 kg-1 and 1.08 (+/- 0.16) 1 kg-1, respectively. The mean (+/- s.d.) single sample estimate of clearance, CL, based on just the 48 h plasma concentrations of amylobarbitone was 0.033 (+/- 0.006) 1 h-1 kg-1. The 48 h single sample CL value was shown to reliably reflect the value of CL with little bias and good precision. Values of the 48 h CL when compared to CL exhibited a mean prediction error (mpe) of 1.2% with 95% confidence limits of -6.3% to 9.4%, and a root mean squared error (rmse) of 9.4%. It is concluded that amylobarbitone's clearance can be estimated in a single dose, single sample protocol permitting its use as a single dose, single sample probe for studying host factor influences on drug metabolism.

Screening and quantification of hypnotic sedatives in serum by capillary gas chromatography with a nitrogen-phosphorus detector, and confirmation by capillary gas chromatography-mass spectrometry.

We describe a quantitative screen for hypnotic-sedative drugs in which we use capillary gas chromatography with a nitrogen-phosphorus detector (GC/NPD) as the primary method and capillary gas chromatography-mass spectrometry (GC-MS) for confirmation. GC retention times of the acid-extracted underivatized drugs were stable (CVs less than 1%), and the detector response varied linearly over a 20-fold concentration range with a mean correlation coefficient for 11 drugs of 0.989. The limits of detection were satisfactory (0.5 mg/L in a 0.5-mL serum sample and 1-microL injection volume), as were precision (average CV 5.2% within day, 6.4% between day). The complementary use of capillary GC-MS not only unambiguously confirms presumptive peaks identified by GC, but also prevents reports of false positives and identifies compounds not included in the quantitative GC screen that may be listed in the GC-MS library.

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.

[Barbamyl distribution in human blood]. / Raspredelenie barbamila v krovi cheloveka.

Barbamil is not distributed uniformly in human blood. The highest drug concentration is recorded in the protein, the lowest in the aqueous part of plasma. Formed elements of the blood (red cells, leukocytes, platelets) are capable of binding to barbamil and transporting it to the cells and tissues.

Gas-liquid chromatography of undervatized drugs after chromatographic extraction from blood.

We have developed an integrated method that overcomes the two main procedural difficulties of gas-liquid chromatography, namely, solvent-solvent extraction and chemical derivatization. Drugs are extracted from serum by column chromatography on granular diatomaceous earth (kieselguhr). Subsequent gas-liquid chromatography of underivatized samples can be performed on either of two liquid phases. A mixed liquid phase, used for quantitative gas-chromatographic assay on patients with a known therapeutic regimen, has enabled quantitation of 12 drugs in serum. Alternatively, a single liquid phase, used with the mixed liquid phase, permits the gas-chromatographic identification of unknown drugs on the basis of the characteristic pattern of the two relative retention times; by this approach more than 40 drugs have been identified in cases of suspected intoxication, both in serum and in gastric aspirate. Besides providing ease of performance and wide applicability, the proposed procedure offers a degree of precision and accuracy that compares favorably with established methods.

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.

[Demonstration of hypnotic agents in bone tissue]. / Altatószerkimutatás csontszövetböl.

Investigating the skeletons of 25 suicide victims the authors have found that the toxic materials taken can rather comfortably and safely be detected in the compactbony substance. Especially important that the poisonous drug residues can remain in the bones for a long time, even for years after death.