Modified dispersive liquid-phase microextraction based on sequential injection solidified floating organic drop combined with HPLC for the determination of phenobarbital and phenytoin.

A modified dispersive liquid phase microextraction based on sequential injection solidified floating organic drop was developed for simultaneous separation/preconcentration of trace amounts of phenobarbital and phenytoin. The important factors affecting on the extraction recovery including pH, the volume of extraction solvent, ionic strength, and the number of injections were investigated and optimized by Box-Behnken design and desirability function. Under the optimum experimental conditions, the calibration graph was linear in the concentration range of 1.0-300.0 µg/L (r2  = 0.997) for phenobarbital and 2.0-400.0 µg/L (r2  = 0.996) for phenytoin. The limit of detection and limit of quantification were 0.35 and 1.2 µg/L for phenobarbital and 0.65 and 2.2 µg/L for phenytoin, respectively. The relative standard deviation for six replicate determinations at 10 µg/L was 3.3 and 4.1% for phenobarbital and phenytoin, respectively. The developed method was successfully applied to the determination of phenobarbital and phenytoin in urine and plasma samples.

A dilute-and-shoot flow-injection tandem mass spectrometry method for quantification of phenobarbital in urine.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the gold standard of urine drug testing. However, current LC-based methods are time consuming, limiting the throughput of MS-based testing and increasing the cost. This is particularly problematic for quantification of drugs such as phenobarbital, which is often analyzed in a separate run because they must be negatively ionized. METHODS: This study examined the feasibility of using a dilute-and-shoot flow-injection method without LC separation to quantify drugs with phenobarbital as a model system. Briefly, a urine sample containing phenobarbital was first diluted by 10 times, followed by flow injection of the diluted sample to mass spectrometer. Quantification and detection of phenobarbital were achieved by an electrospray negative ionization MS/MS system operated in the multiple reaction monitoring (MRM) mode with the stable-isotope-labeled drug as internal standard. RESULTS: The dilute-and-shoot flow-injection method developed was linear with a dynamic range of 50-2000 ng/mL of phenobarbital and correlation coefficient > 0.9996. The coefficients of variation and relative errors for intra- and inter-assays at four quality control (QC) levels (50, 125, 445 and 1600 ng/mL) were 3.0% and 5.0%, respectively. The total run time to quantify one sample was 2 min, and the sensitivity and specificity of the method did not deteriorate even after 1200 consecutive injections. CONCLUSIONS: Our method can accurately and robustly quantify phenobarbital in urine without LC separation. Because of its 2 min run time, the method can process 720 samples per day. This feasibility study shows that the dilute-and-shoot flow-injection method can be a general way for fast analysis of drugs in urine. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous extraction and quantification of lamotrigine, phenobarbital, and phenytoin in human plasma and urine samples using solidified floating organic drop microextraction and high-performance liquid chromatography.

A novel and simple method based on solidified floating organic drop microextraction followed by high-performance liquid chromatography with ultraviolet detection has been developed for simultaneous preconcentration and determination of phenobarbital, lamotrigine, and phenytoin in human plasma and urine samples. Factors affecting microextraction efficiency such as the type and volume of the extraction solvent, sample pH, extraction time, stirring rate, extraction temperature, ionic strength, and sample volume were optimized. Under the optimum conditions (i.e. extraction solvent, 1-undecanol (40 µL); sample pH, 8.0; temperature, 25°C; stirring rate, 500 rpm; sample volume, 7 mL; potassium chloride concentration, 5% and extraction time, 50 min), the limits of detection for phenobarbital, lamotrigine, and phenytoin were 1.0, 0.1, and 0.3 µg/L, respectively. Also, the calibration curves for phenobarbital, lamotrigine, and phenytoin were linear in the concentration range of 2.0-300.0, 0.3-200.0, and 1.0-200.0 µg/L, respectively. The relative standard deviations for six replicate extractions and determinations of phenobarbital, lamotrigine, and phenytoin at 50 µg/L level were less than 4.6%. The method was successfully applied to determine phenobarbital, lamotrigine, and phenytoin in plasma and urine samples.

Novel micro-extraction by packed sorbent procedure for the liquid chromatographic analysis of antiepileptic drugs in human plasma and urine.

A method for the simultaneous determination of the antiepileptic drugs, phenobarbital (PHB), phenytoin (PTN), carbamazepine (CBZ), primidone (PRM) and oxcarbazepine (OXC) in human plasma and urine samples by using micro-extraction in a packed syringe as the sample preparation method connected with LC/UV (MEPS/LC/UV) is described. Micro-extraction in a packed syringe (MEPS) is a new miniaturized, solid-phase extraction technique that can be connected online to gas or liquid chromatography without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100-250 µL) as a plug. Sample preparation takes place on the packed bed. The bed can be coated to provide selective and suitable sampling conditions. The new method is very promising, easy to use, fully automated, inexpensive and quick. The standard curves were obtained within the concentration range 1-500 ng/mL in both plasma and urine samples. The results showed high correlation coefficients (R(2) >0.988) for all of the analytes within the calibration range. The extraction recovery was found to be between 88.56 and 99.38%. The limit of quantification was found to be between 0.132 and 1.956 ng/mL. The precision (RSD) values of quality control samples (QC) had a maximum deviation of 4.9%. A comparison of the detection limits with similar methods indicates high sensitivity of the present method. The method is applied for the analysis of these drugs in real urine and plasma samples of epileptic patients.

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine.

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 µm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1 : 1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.

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.

False negative result for amphetamines on the Triage Drug of Abuse panel? The cause of the unusual phenomenon with experimental analyses.

On-site drug screening devices are widely used today for their simple test procedures and instantaneous results. Among other devices, a Triage Drug of Abuse panel is considered to be highly reliable for its high specificity and sensitivity of abused drugs. Although it is known that a false positive amphetamine (AMP) result may be obtained from the urine samples containing putrefactive amines or ephedrine-related compounds, no clinical false negative methamphetamine results have been reported to date. However, a false negative Triage result was obtained from the urine of a fatal methamphetamine poisoning victim taking Vegetamine tablets. Further experimental analyses revealed that the cross-reactivity of methamphetamine and chlorpromazine metabolites, including nor-2-chlorpromazine sulfoxide, was the cause for a false negative Triage reaction for AMP. Forensic scientists and clinicians must be aware of the limitations of on-site drug testing devices and the need for the confirmatory laboratory tests for the precise identification and quantification of drugs in suspicious intoxication cases, as also recommended by the manufacturers.

Mass spectrometry-based metabolomics: accelerating the characterization of discriminating signals by combining statistical correlations and ultrahigh resolution.

A strategy combining autocorrelation matrices and ultrahigh resolution mass spectrometry (MS) was developed to optimize the characterization of discriminating ions highlighted by metabolomics. As an example, urine samples from rats treated with phenobarbital (PB) were analyzed by ultrahigh-pressure chromatography with two different eluting conditions coupled to time-of-flight mass spectrometric detection in both the positive and negative electrospray ionization modes. Multivariate data analyses were performed to highlight discriminating variables from several thousand detected signals: a few hundred signals were found to be affected by PB, whereas a few tenths of them were linked to its metabolism. Autocorrelation matrices were then applied to eliminate adduct and fragment ions. Finally, the characterization of the ions of interest was performed with ultrahigh-resolution mass spectrometry and sequential MS(n) experiments, by using a LC-LTQ-Orbitrap system. The use of different eluting conditions was shown to drastically impact on the chromatographic retention and ionization of compounds, thus providing a way to obtain more exhaustive metabolic fingerprints, whereas autocorrelation matrices allowed one to focus the identification work on the most relevant ions. By using such an approach, 14 PB metabolites were characterized in rat urines, some of which have not been reported in the literature.

Effects of urine pH modification on pharmacokinetics of phenobarbital in healthy dogs.

Although pH modification is one of the effective strategies for dissolving or preventing uroliths, little is known about its effects on the pharmacokinetics of phenobarbital in dogs. Five spayed, female Beagles were fed with a twice-daily diet that included potassium citrate and ammonium chloride for urine alkalinization and acidification, respectively. After a stabilizing period of 7 days, a single clinical dose of phenobarbital (3 mg/kg) was orally administered, and time-course changes in its serum and urine concentrations were determined by high-performance liquid chromatography. Total amounts of unchanged phenobarbital excreted into urine for 216 h were decreased by urine acidification and increased by urine alkalinization. The elimination half-life of serum phenobarbital in dogs with urine alkalinization was shortened and Cl(R) increased when compared with dogs with urine acidification. Other pharmacokinetic parameters, including C(max), T(max), AUC(0-216), Cl/F, and A(e0-216) were not changed by modification of the urine pH. These results suggest that the pH of urine is likely to be a determinant of the pharmacokinetics, especially urine excretion rate, of a clinical dose of oral phenobarbital. It is possible that the serum concentration of phenobarbital might be altered when a pH modifying-diet is administered for the purpose of dissolving or preventing uroliths.

Pharmacogenetic roles of CYP2C19 and CYP2B6 in the metabolism of R- and S-mephobarbital in humans.

OBJECTIVES AND METHODS: We assessed the relationship between the metabolism of R- and S-mephobarbital (MPB) and genetic polymorphisms of cytochrome P450 (CYP) 2C19 and CYP2B6. Nine homozygous extensive metabolizers (homo-EMs, 2C19*1/2C19*1) of CYP2C19, ten heterozygous EMs (hetero-EMs, 2C19*1/2C19*2, 2C19*1/2C19*3) and eleven poor metabolizers (PMs, 2C19*2/2C19*2, 2C19*3/2C19*3, 2C19*2/2C19*3) recruited from a Japanese population, received an oral 200 mg-dose of racemic MPB. Blood and urine samples were collected, and R-MPB, S-MPB and the metabolites, phenobarbital (PB) and 4'-hydroxy-MPB, were measured. Each subject was also genotyped for CYP2B6 gene. RESULTS: The mean area under the plasma concentration-time curve (AUC) of R-MPB was 92-fold greater in PMs than in homo-EMs. R/S ratios for AUC of MPB were much higher in PMs than in EMs (homo- and hetero-). The cumulative urinary excretion of 4'-hydroxy-MPB up to 24 h postdose was 21-fold less in PMs than in homo-EMs. The metabolic ratio of AUCPB/(AUCS-MPB + AUCR-MPB) was higher in PMs than in EMs (homo- and hetero-). In addition, this metabolic ratio was lower in the carriers of CYP2B6*6 compared with that in its non-carriers. CONCLUSIONS: Our results indicate that the 4'-hydroxylation of R-MPB is mediated via CYP2C19 and that the rapid 4'-hydroxylation of R-MPB results in a marked difference in the pharmacokinetic profiles between R-MPB and S-MPB in the different CYP2C19 genotypic individuals. In addition, a minor fraction of the interindividual variability in PB formation from MPB may be explainable by the CYP2B6*6 allele.

Effects of felbamate on the pharmacokinetics of phenobarbital.

The effects of felbamate on the pharmacokinetics of phenobarbital and one of its main metabolites, parahydroxyphenobarbital, were assessed in a parallel-group, placebo-controlled, double-blind study, in 24 healthy volunteers. Pharmacokinetic parameters of phenobarbital and parahydroxyphenobarbital were determined from plasma and urine samples obtained after 28 days of daily administration of 100 mg phenobarbital and after a further 9 days of phenobarbital plus 2400 mg/day felbamate or placebo. Felbamate increased phenobarbital values for area under the plasma concentration-time curve from 0 to 24 hours and maximum concentration by 22% and 24%, respectively, whereas placebo had no effect. This increase was caused by a reduction in parahydroxylation of phenobarbital and possibly through effects on other metabolic pathways. Because felbamate inhibits the S-mephenytoin hydroxylase (CYP2C19) isozyme in vitro, it appears that phenobarbital hydroxylation is mediated in part by this isozyme.

Acetazolamide-induced interference with primidone absorption. Case reports and metabolic studies.

Effects of acetazolamide on primidone plasma levels were studied in three patients. Apparent interaction occurred in two patients. Primidone was not detected in the plasma when given orally with acetazolamide in one patient. In another, peak serum concentration was delayed, with corresponding delays in urinary excretion of primidone and metabolites. Plasma and urine concentrations of the two metabolites, phenylethylmalonamide and phenobarbital, were also studied.

Valproic acid and plasma levels of phenobarbital.

During concurrent administration of phenobarbital and valproic acid, phenobarbital plasma concentrations often increase. This often requires a reduction of phenobarbital dosage. In normal cats and patients with epilepsy, we found no evidence of decreased renal excretion of phenobarbital. Metabolic studies in four patients revealed a decrease in the conversion of phenobarbital to hydroxyphenylphenobarbital and decreased urinary ratios of hydroxyphenylphenobarbital to phenobarbital. These data suggest that phenobarbital metabolism is inhibited by therapeutic plasma levels of valproic acid.

Pharmacokinetic equivalence of 5(ethyl(2H)5)- and unlabelled phenobarbitone.

The present study shows the absence of in vivo pharmacokinetic isotope effect on phenobarbitone (PB) C5-ethyl deuteration (PBd5) following oral administration to man of equimolar PB/PBd5 mixtures (0.40 mmol each). Plasma PB and PBd5 (17 days) and urine PB, PBd5 and parahydroxy-metabolites (PBOH, PBHOd5) levels were determined by GC-MS. Isotope effect research includes comparison of pharmacokinetic parameters, study of time-dependence of isotope ratios (IRs) in plasma and urine (linearity test), comparison of IRs between samples and administered mixtures (Mann Whitney's test) and comparison of PBOH/PBOHd5 ratios before and after urine enzymatic hydrolysis (Student's two tailed t-test). No significant isotope effect was observed on pharmacokinetic parameters, PB hydroxylation or PBOH conjugation (x less than or equal to 5%); which the absence of pentadeuteration-induced alteration in PB's HSA binding parameters (binding mode, Ka, N) corroborates (x less than or equal to 5%). These results establish bioequivalence of PB and PBd5; the latter can be used with benefit in stable-isotope clinical pharmacology (steady state pharmacokinetics, drug interactions...) investigations as well as bioavailability studies of PB preparations.

Studies with stable isotopes II: Phenobarbital pharmacokinetics during monotherapy.

Six healthy adults receiving no other medications were given tracer doses of 90 mg of stable isotope-labeled phenobarbital (PB) intravenously before, and four weeks after, and 12 weeks after beginning therapy. Serum samples were collected for 96 hours after each injection, and the concentration of stable isotope-labeled PB in each sample was determined by gas chromatographic mass spectrometry. The volume of distribution, elimination half-life, and total clearance of PB did not differ significantly on any of the three occasions measured. Phenobarbital clearance did not correlate significantly with total PB serum concentration. Clearances determined from single-dose studies before beginning PB therapy accurately predicted steady-state PB serum concentrations. Therefore, it is not necessary to adjust PB dosage for time-dependent or dose-dependent changes in clearance during monotherapy. In addition, clearance or serum concentration determined at one dosing rate directly predicts serum concentration at another dosing rate.

Simultaneous analysis of phenobarbital and p-hydroxyphenobarbital in biological fluids by GLC-chemical-ionization mass spectrometry.

A sensitive and specific GLC-chemical-ionization mass spectrometric method was developed for the simultaneous assay of phenobarbital (I) and p-hydroxyphenobarbital (II) in biological fluids (urine and plasma) using stable isotope analogs of the compounds as internal standards. After extraction, the compounds were methylated with diazomethane and quantitated by GLC-chemical-ionization mass spectrometry. The detection limit of the method was 0.1 micrograms/ml for both compounds. The intraday precision (RSD) for 0.4-2.4 micrograms/ml was < 2% for I and < 4% for II. The interday precision for 0.55 and 2.11 micrograms/ml of each compound was 5.5 and 2.9% for I and 7.3 and 5.0% for II, respectively. This method has been applied in several pharmacokinetic studies.

GC and GC-mass spectrometric determination of p-hydroxyphenobarbital extracted from plasma, urine, and hepatic microsomes.

Analytical methodology was developed for the quantitation of p-hydroxyphenobarbital extracted from plasma, urine, and hepatic microsomes. p-Hydroxyphenobarbital was derivatized with an appropriate n-alkyl iodide in the presence of a methanolic base in aprotic solvent medium. The peralkylated derivatives were stable indefinitely and were quantitated by the sensitive and selective method of GC nitrogen-selective detection and/or selected ion monitoring. The accuracy, precision, and cross verification of all methods were good. The analysis was subsequently used to study the effects of other drugs on phenobarbital biodisposition.

Pharmacodynamics of tolerance development to the anesthetic and anticonvulsant effects of phenobarbital in rats.

In this study, the potential development of tolerance towards the anesthetic and anticonvulsant effects of phenobarbital after chronic administration to rats was investigated, differentiating between dispositional and functional tolerance. Chronic exposure to phenobarbital by daily ip injections of 20 or 100 mg/kg for 14 days caused a 30% increase in clearance within 1 week. No changes occurred in the total amounts of phenobarbital and para-hydroxyphenobarbital excreted into urine and feces. Pharmacodynamic effects were quantitated after 2 weeks of treatment. The anesthetic effect was measured by slow iv infusion of phenobarbital until onset of loss of righting reflex (LRR), followed by measurement of drug concentrations in serum (both total and free), brain, and cerebrospinal fluid (CSF). With a phenobarbital dose of 100 mg/kg daily, CSF concentrations at the onset of LRR significantly increased from 136 +/- 12 to 176 +/- 21 mg/L (p less than 0.001), indicating that functional tolerance developed for the anesthetic effect. Protection of phenobarbital against convulsions induced by pentylenetetrazol (PTZ), as measured by the elevation of the PTZ plasma threshold concentration necessary to elicit seizures at the EC50 of phenobarbital, was not altered. The discrepancy observed in the development of functional adaptation of the CNS demonstrates that different mechanisms of action are reflected in the different measures of the anesthetic and anticonvulsant effects of phenobarbital that were utilized.

Identification of phenobarbital N-glucosides as urinary metabolites of phenobarbital in mice.

Previously, the N-glucosylation of phenobarbital had been observed only in humans. The results of a species screen (mouse, rat, guinea pig, rabbit, cat, dog, pig, and monkey) found that only mice excreted the N-glucosides of phenobarbital in urine after ip administration of sodium phenobarbital. The major diastereomer excreted by the mouse had the R configuration at the C-5 position of the barbiturate ring. The N-glucoside metabolites accounted for a small percentage of the dose (approximately 0.5%). Following ip dosing of the mouse with the phenobarbital N-glucosides, free phenobarbital could be detected in the urine. Upon ip or intercerebroventricular (icv) injection of the phenobarbital N-glucosides, minimal CNS activity was observed in the mouse.

Drugs of abuse screening in the West Midlands: a 6 year retrospective survey of results.

We describe the results of urinary drugs of abuse screening performed by the West Midlands Regional Laboratory for Toxicology, Birmingham, UK, on more than 27,800 urine specimens received between January 1988 and December 1993. The number of specimens positive for amphetamine declined from 1988 to 1990, but this was followed by a doubling of specimens testing positive from 5.7% in 1990 to 12.0% in 1993. There is very little evidence of methamphetamine or Ecstasy abuse in the West Midlands. Morphine (assumed to be from heroin abuse) is the most common opiate detected, with 11.7% of all specimens received proving to be positive in 1993. The incidence of cocaine abuse is low, less than 5% when requests are based on clinical judgement, and less than 3% in the overall population monitored.