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.

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.

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.

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.

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.

Comparison of the serum barbiturate fluorescence polarization immunoassay by the COBAS INTEGRA to a GC/MS method.

The authors evaluated a new Cassette Serum Barbiturates fluorescence polarization immunoassay (FPIA) on the COBAS INTEGRA. The assay was calibrated with secobarbital standards at 0, 0.5, and 4.0 microg/mL. The assay range was 0.030 to 80 microg/mL using an automatic 1/20 postdilution feature. Precision was established for two COBAS INTEGRA instruments for ten days by assaying secobarbital target concentrations ranging from 0.125 to 2.2 microg/mL. The coefficients of variation (CV) for the above target concentrations for the first instrument ranged from 2.7% to 8.3%, and for a second instrument, 3.8 to 8.3%. Seven clinically elevated bilirubin samples were spiked with 0.46 and 1.77 microg/mL secobarbital. Bilirubin interference was less than 10.9 and less than 7.9%, respectively. The average recovery ranged from 85% to 94%. The mean difference in recovery in serum versus plasma was < or = 3%. Fifty-two clinical samples were analyzed for butalbital, pentobarbital, secobarbital, and phenobarbital by GC/MS, and the results were compared to the new Cassette Serum Barbiturates FPIA. The diagnostic sensitivity and specificity were 95% and 100%, respectively. Both FPIA and GC/MS assays are clinically efficacious for monitoring serum barbiturates.

Automated procedure for determination of barbiturates in serum using the combined system of PrepStation and gas chromatography-mass spectrometry.

A system of an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatograph-mass spectrometer (GC-MS) was developed for the simultaneous analysis of seven barbiturates in human serum. A sample clean-up was performed by a solid-phase extraction (SPE) on a C18 disposable cartridge. A SPE cartridge was preconditioned with methanol and 0.1 M phosphate buffer. After loading 1.5 ml of a diluted serum sample into the SPE cartridge, the cartridge was washed with 2.5 ml of methanol-water (1:9, v/v). Barbiturates were eluted with 1.0 ml of chloroform-isopropanol (3:1, v/v) from the cartridge. The eluate (1 microl) was injected into the GC-MS. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.1 to 10 microg ml(-1) for all barbiturates extracted. The proposed method was applied to 27 clinical serum samples from three patients who were administrated secobarbital.

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.

[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.

[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 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.

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.

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.

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.

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.

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.

Mice tolerant to nitrous oxide are not tolerant to barbiturates.

Mice continuously exposed to 50% nitrous oxide for 2 weeks or more become tolerant to nitrous oxide, as measured by an 8% to 19% increase in their nitrous oxide ED50 (the dose required to abolish the righting reflex in 50% of the animals). Control mice tolerant to nitrous oxide were given intraperitoneal injections of secobarbital (50 mg/kg), phenobarbital (120 mg/kg), or pentobarbital (55 or 65 mg/kg). Sleep onset times, sleep times, and serum levels of barbiturates upon awakening did not differ between the two groups. These results suggest that nitrous oxide and barbiturates do not share a common mechanism of action.