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.

Determination of barbiturates in urine by micellar liquid chromatography and direct injection of sample.

A liquid chromatographic procedure for the determination of six barbiturates (barbital, diallyl barbituric acid, phenobarbital, butabarbital, amobarbital and pentobarbital) in urine samples is described. The proposed system uses a Spherisorb octadecyl-silane ODS-2 C18 analytical column and a guard column of similar characteristics. The UV detector was set at 240 nm. A study to select adequate composition of the micellar mobile phase for the separation of these compounds in urine samples is performed. Maximum resolution was achieved with a 0.07 M sodium dodecylsulphate-0.3% propanol at pH 7.4 eluent. Limits of detection at 240 nm were ranged between 0.13 microg ml(-1) for diallyl barbituric acid and 2.7 microg ml(-1) for amobarbital. The procedure allows for the determination of these compounds in 20 minutes, it does not require prior a sample preparation step and it can be very useful to the investigation of intoxication.

Improved gas chromatography/mass spectrometry analysis of barbiturates in urine using centrifuge-based solid-phase extraction, methylation, with d5-pentobarbital as internal standard.

Effective solid-phase extraction, derivatization, and GC/MS procedures are developed for the simultaneous determinations of butalbital, amobarbital, pentobarbital, and secobarbital, using a deuterated pentobarbital (d5-pentobarbital) as the internal standard. Buffered (pH 7) urine samples were extracted with Bond Elute Certify II cartridge. Iodomethane/tetramethylammonium hydroxide in dimethylsulfoxide was used for methylation, while a HP 5970 MSD equipped with a 13 m J & W DB-5 column (5% phenyl polysiloxane phase) and the Thru-Put Target software package were used for GC/MS analysis and data processing. This protocol was found to be superior, in both chromatographic performance characteristics and quantitation results, over a liquid-liquid extraction procedure without derivatization using hexobarbital as the internal standard. Extraction recoveries observed from control samples containing four barbiturates range from 80% to 90%. Good one-point calibration data are obtained for all four barbiturates in the 50 to 3200 ng/mL range. Interestingly, the one-point calibration data for pentobarbital are inferior to the other three barbiturates--due to interference from the internal standard (d5-pentobarbital). The calibration data of pentobarbital are best described by a hyperbolic curve regression model. Precision data (% CV) for GC/MS analysis, over-all procedure, and day-to-day performance are approximately 2.0%, 6.0%, and 8.0%, respectively. With the use of a 2 mL sample size, the attainable detection limit is approximately 20 ng/mL.

Identification of the diastereomers of pentobarbital N-glucosides excreted in human urine.

A study was undertaken to determine if humans excreted pentobarbital N-glucosides as urinary metabolites following oral administration of pentobarbital. (1'RS,5RS)-1-(beta-D-Glucopyranosyl)pentobarbital ((1'RS,5RS)-PTBG) was isolated from the urine of one subject. The two diastereomers, (1'RS,5R)-PTBG and (1'RS,5S)-PTBG were separated and found to be identical to synthetic standards when compared using HPLC retention times coupled with UV (with and without post-column ionization) and mass spectrometry (HPLC/MS). A HPLC method was developed for detecting and quantifying (1'RS,5R)-PTBG, (1'RS,5S)-PTBG and pentobarbital in urine. Following a single oral dose of sodium pentobarbital to male subjects (n = 6), 1.6-6.2% of the pentobarbital dose was excreted as (1'RS,5S)-PTBG over 60 hours. (1'RS,5R)-PTBG was also detected in one subject and accounted for 0.3% of the pentobarbital dose. Using a modified HPLC system, the four pentobarbital N-glucosides were resolved and analysis of a partially purified pentobarbital N-glucoside extract from one subject indicated that only (1'R,5R)-PTBG and (1'S,5S)-PTBG could be detected as urinary excretion products. These results indicate that the side chain chirality of pentobarbital may influence the observed enantioselectivity for the formation and/or urinary excretion of the pentobarbital N-glucosides.

Solid-phase extraction and GC/MS confirmation of barbiturates from human urine.

A highly selective and sensitive procedure has been developed for isolating and identifying barbiturates in human urine. With a new disposable bonded silica gel solid-phase extraction (SPE) column and hexobarbital as an internal standard (IS), amobarbital, butabarbital, pentobarbital, phenobarbital, secobarbital, and methaqualone were selectively isolated from endogenous urine components. Capillary gas chromatography/ion trap mass spectrometry (GC/MS) analysis of the extracts generated a full mass spectrum for the detection, identification, and quantitation of barbiturates. Linear quantitative response curves for the drugs have been generated over a concentration range of 20-500 ng/mL. Overall extraction efficiencies for drugs averaged greater than 90%, and the quantitative response curves exhibited correlation coefficients of 0.996 to 0.999.

4'-Hydroxylated derivatives as urinary metabolites of two barbiturates.

4'-Hydroxybutobarbitone and 4'-hydroxypentobarbitone have been synthesized. These 4'-hydroxy derivatives have been quantified in the urine of volunteers following single doses of butobarbitone, and single and multiple doses of pentobarbitone. The corresponding aldehydes have also been synthesized, and shown to be excreted in minor quantities. The excretion of an ingested dose of 4'-hydroxybutobarbitone, and its oxidation products, has also been followed over 24 h.

Multidose studies in the human metabolism of pentobarbitone.

The metabolism of pentobarbitone has been investigated in two healthy volunteers, and the urinary excretion of unchanged drug, and the products of omega- and (omega-1)-oxidation, quantified for four days after each of three doses to each volunteer. The rates of excretion are discussed in terms which include enzyme induction effects.

N-Hydroxylation of pentobarbital in man.

After oral administration of 14C-labeled pentobarbital to healthy subjects, most ot the radioactivity was recovered in urine over a period of 6 days. Only a minute amount (approximately 1%) of unchanged pentobarbital was found in the urine. Four major metabolites were found and isolated. One was 3'-hydroxypentobarbital, which has been previously identified by Maynert. The second could be identified as N-hydroxypentobarbital on the basis of its spectral and chemical properties. The other two metabolites were not identified.

Screening for drug abuse: use of NaCl to increase drug recovery from papers coated with ion-exchange resin.

Use of papers loaded with ion-exchange resins to adsorb drugs from urine specimens resulted in large losses during the procedure. The first step, removal of drugs from urine specimens, was 25-85% efficient. The second step, elution of drugs from paper for further processing, was approximately 40-70% complete. The efficiency of the first step was decreased and the efficiency of the second step was increased by addition of NaCl, except in the case of barbiturates. Presence of salt during elution increased the yield of dihydromorphine by 20%, of methadone by 16%, of amphetamine by 34%, and of chlorpromazine by 40%, but did not enhance the yield of pentobarbital. Overall recovery rates were: 51% for the opiates, 57% for methadone, 72% for a phenothiazine tranquillizer, but only 35% for amphetamine and 15% for a barbiturate.

Simple, highly selective screening method for barbiturates in urine.

I present a new, simple colorimetric method for detecting and estimating barbiturates in urine. After the barbiturates are extracted with ether, an aliquot of the washed ether phase is added to the color reagent (a bivalent mercury/dithizone chelate in chloroform). On addition of diluted pyridine and shaking, a pinkish-violet color appears if a barbiturate is present. The overall sensitivity of the above method was evaluated by probit analysis in the case of sodium phenobarbital. The concentration of sodium phenobarbital in urine detectable at least 99% of the time was 6.72 mg/liter, with 95% confidence limits of 5.37 to 10.36 mg/liter. Sodium phenobarbital, 10 mg/liter, can be detected in the presence of phenytoin (50 mg/liter), glutethimide (100 mg/liter), or bemegride (100 mg/liter). The whole procedure requires less than 10 min. An excretion study illustrates application of the procedure.

Comparison of radioimmunoassay with thin-layer chromatographic and gas-liquid chromatographic methods of barbiturate detection in human urine.

A radioimmunoassay (I) for barbiturates was compared with thin-layer chromatographic (II) and gas-liquid chromatographic (III) methods for barbiturate detection in human urine. Timed urine samples were obtained from volunteers who had ingested 100 mg of a barbiturate. I detected barbiturate in all urines tested up to 76 h after the dose, and III in all up to 52 h and in 90% up to 76 h. II detected barbiturates in 90% of all urine samples for only 30 h, after which is reliability declined. Glutethimide interfered with radioimmunoassay of barbiturate, producing false positives. I is sensitive, reliable, and fast, and lends itself to screening large numbers of urine samples for barbiturates. For routine urine surveillance, however, we found I to be less useful than II, which is still the method of choice. I has, however, proved to be an excellent method for confirming results of II.