Determination of Barbiturates in Biological Specimens by Flat Membrane-Based Liquid-Phase Microextraction and Liquid Chromatography-Mass Spectrometry.

The wide abuse of barbiturates has aroused extensive public concern. Therefore, the determination of such drugs is becoming essential in therapeutic drug monitoring and forensic science. Herein, a simple, efficient, and inexpensive sample preparation technique, namely, flat membrane-based liquid-phase microextraction (FM-LPME) followed by liquid chromatography-mass spectrometry (LC-MS), was used to determine barbiturates in biological specimens. Factors that may influence the efficiency including organic extraction solvent, pH, and composition of donor and acceptor phases, extraction time, and salt addition to the sample (donor phase) were investigated and optimized. Under the optimized extraction conditions, the linear ranges of the proposed FM-LPME/LC-MS method (with correlation coefficient factors ≥ 0.99) were 7.5-750 ng mL-1 for whole blood, 5.0-500 ng mL-1 for urine, and 25-2500 ng g-1 for liver. Repeatability between 5.0 and 13.7% was obtained and the limit of detection (LOD) values ranged from 1.5 to 3.1 ng mL-1, from 0.6 to 3.6 ng mL-1, and from 5.2 to 10.0 ng g-1 for whole blood, urine, and liver samples, respectively. This method was successfully applied for the analysis of barbiturates in blood and liver from rats treated with these drugs, and excellent sample cleanup was achieved.

[The identification of barbituric acid derivatives in the old blood stains on textiles]. / Issledovanie proizvodnykh barbiturovoi kisloty v zastarelykh piatnakh krovi na tkaniakh.

Thus article was designed to report a few cases of the identification of barbituric acid derivatives in the old blood stains on the clothes and other textiles. The data presented give evidence that barbiturates are capable of persisting in dry blood stains during rather a long period. The authors emphasize the necessity of mandatory control investigations to avoid obtaining the false positive results.

[The development of the method for enzymatic hydrolysis for the extraction of toxic substances from the hair samples]. / Razrabotka metodiki fermentativnogo gidroliza dlia izolirovaniia toksichnykh veshchestv iz obraztsov volos.

The objective of the present study was to develop and validate the method for the extraction of toxic substances from the hair samples as exemplified by enzymatic hydrolysis of barbituric acid derivatives. The experiments were carried out with the use of laboratory animals (white female rats and albino guinea pigs) that had been daily given a phenobarbital solution per os during 4 months preceding the study. The hairs obtained from the experimental animals were subjected to acid hydrolysis with a 6 mole hydrochloric acid and enzymatic hydrolysis with the use of chymopsin, trypsin, chymotrypsin, and papain solutions. The analysis of the extracted materials was performed by means of gas chromatography with mass-selective detection. The application of the proposed method for enzymatic hydrolysis produced the better results than acid hydrolysis. This technique was validated. The results of the study made possible the comparative characteristic of the effectiveness of acid and enzymatic hydrolysis.

New approach for barbiturates, phenytoin, methyprylon and glutethimide determination and fragmentation (UHPLC-MS/MS).

Barbiturates which are old pharmaceutical drugs are still widely used in medical treatment of epilepsy and for general anesthesia. To date, more than 2500 different barbituric acid analogs have been synthesized, and 50 of them were introduced into medical use over the last century. Due to their highly addictive properties, pharmaceuticals containing barbiturates are under strict control in many countries. However, by considering the worldwide problem with new psychoactive substances (NPS) the introduction of new designer barbiturate analogs into the dark market might serve a serious public health problem in the near future. For this reason there is an increasing need for application methods for barbiturates monitoring in biological samples. The UHPLC-QqQ-MS/MS method for determination of 15 barbiturates, phenytoin, methyprylon and glutethimide was developed and fully validated. The biological sample volume was reduced to only 50 µL. A simple LLE (pH 3 with ethyl acetate) was successfully applied. The lower LOQ was 10 ng/mL. The method enables differentiation of structural isomers: hexobarbital and cyclobarbital; as well as amobarbital and pentobarbital. Chromatographic separation was achieved with the use of the alkaline mobile phase (pH 9) and Acquity UPLC BEH C18 column. Furthermore, the novel fragmentation mechanism of barbiturates was proposed, which may have a great impact in identification of novel barbiturates analogs introduced to illegal marketplaces. The presented technique has a great potential to be applied in forensic, clinical and veterinary toxicological laboratories, as was evidenced by the positive results of international proficiency tests.

First evidence of the incorporation of daprodustat and other hypoxia-inducible factor stabilizers into equine hair by passive transfer based on segmental quantitative analysis.

Daprodustat is a hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) inhibitor and is used as an erythropoiesis stimulant for the treatment of anemia in humans. In general, administering daprodustat to horses will result in a lifetime ban from both equestrian sports and horseracing by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. To control the misuse/abuse of daprodustat, we conducted nasoesophageal administration of daprodustat (100 mg/day for 3 days) to three thoroughbred mares and the post-administration hair samples collected from the three horses over 6 months were analyzed to demonstrate the potential longer-term detection of daprodustat and its metabolites in hair compared with the detection times of daprodustat of 1 and 2 weeks in plasma and urine respectively. The results of the quantitative 2-cm segmental analysis showed that daprodustat was primarily localized in the proximal region (0-2 cm) at 0.375-0.463 pg/mg at 1 month post-administration. These drug bands were gradually spread out along the hair shaft at a rate consistent with the reported growth rate of horse mane hair (approximately 2.5 cm/month) over the following 6 months. In addition, to attain deeper insight into the mechanism of drug incorporation into hair, a total of 11 relevant parameters, including the actual PK parameters and simulated physicochemical and biopharmaceutical parameters for three HIF stabilizers (i.e., daprodustat, vadadustat, and IOX4), were investigated after normalization of the z-scores of all these parameters. Multiple regression analysis indicated that the major factors contributing to the incorporation of the three drugs into hair were their maximum plasma concentrations and lipophilicities, strongly suggesting that the three HIF stabilizers permeated from the bloodstream into the hair bulb via passive transfer with concentration gradients. This work is the first reported evidence showing the incorporation of HIF stabilizers into hair via passive transfer. In addition, cross-species comparison of drug incorporations into hair between daprodustat in horse and roxadustat in human was made in order to have a better understanding of the interactive interpretations about the analysis results obtained from different species. The above findings are not only useful and beneficial for the purpose of doping control but also provide a better understanding of the mechanism of drug incorporation into horse hair.

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.

[Investigation vesicle cycle in nerve formations in somatic muscle of the earthworm Lumbricus terrestris].

Luminous spots with a diameter of 1-2 microm, which are clusters of "synaptic buds", were revealed in the muscular wall of the earthworm using endocytotic fluorescent dyes FM1-43, FM2-10 and FM4-64. Application of the membrane probe Dil that is capable of being subjected to anterograde axonal transport to abdominal ganglia of the nervous chain, and subsequent (in a day) staining of nerve formations by endocytotic dye FM4-64 showed complete imposition of the emission data of the dyes that fluoresce in different parts of the spectrum. Using fluorescent marker DiBAC4(3) showed an increased emission of neural elements with increasing concentration of K+ in the extracellular environment. Application of FM2-10 showed that the higher concentration of K+ in solution, and hence the depolarization of the nerve cells, the faster the upload of the dye, and vice versa, the process slowed down in the absence of K+ in the medium. The seizure and removal of FM2-10 were blocked in calcium-free solutions in the presence of Ca2+ buffers, BABTA or BABTA-AM, but only after a preliminary 40 min incubation. The processes of exo- and endocytosis occurred in the clusters of synaptic "buds" and were preserved in conditions of "rest". This vesicle cycle depends on membrane potential and concentration of K+ and Ca2+, and, it is very likely that the calcium sensor operates on the principle "all or nothing".

[Micellar liquid chromatography and its application in toxicological analysis].

Micellar liquid chromatography (MLC) is a reversed phase liquid chromatography with mobile phases containing surfactant above its critical micellar concentration (CMC). The basic mechanism and advantages of MLC in physicochemical analysis were reviewed, and its applications in analysis of drugs, barbiturates, benzodiazepines were chiefly introduced in this paper. MLC is a potential method to toxicological analysis due to strong selectivity, wide application scope and easy biological samples, etc.

Simultaneous determination of barbiturates, phenytoin and topiramate in hair by LC-MS/MS and application to real samples.

INTRODUCTION: Hair analysis is useful for monitoring exposure to drugs such as antiepileptics owing to long-term therapy and a high possibility of abuse of drugs, which could be fatal. An effective and rapid analytical method for the simultaneous determination of six barbiturates, as well as phenytoin and topiramate in hair samples was developed and validated by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). METHODS: Three different extraction methods were investigated for the development of an appropriate analytical method. Hair was finely cut and then extracted with methanol, methanol containing 1% hydrochloric acid, and liquid-liquid extraction in acidic condition. RESULTS: There was no significant difference in the matrix effects among these three methods. Recoveries clearly declined in the extraction involving both acidic methanol extraction and a LLE in acidic condition. Methanol incubation was chosen as the appropriate extraction method with acceptable matrix effects and recoveries. After validating the methanol incubation, the limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.01 and 0.02 ng/mg for topiramate and 0.25-0.5 and 0.5-1 ng/mg for the others in hair. The LC-MS/MS method was precise and accurate with a dynamic linear range of 0.02-5 ng/mg for topiramate and 0.5 or 1-50 ng/mg for others. This method was applied to authentic hair samples of two drug users. The hair concentrations of phenobarbital were 0.2-17.1 ng/mg in segmental analysis in one female subject and those of topiramate were 0.19-0.93 ng/mg in another female subject. DISCUSSION: The quantitative method was developed to determine 8 antiepileptics using LC-MS/MS. This method performed hair segmental analysis to provide useful informative and chronological data in both of the forensic and clinical toxicology fields.

Barbiturates: radioimmunoassay.

The development of a radioimmunoassay for barbiturate is described. The barbiturate is made antigenic by coupling it to a protein, bovine gamma globulin. The radioimmunoassay can measure as little as 5 nanograms of barbiturate.

Comparison of drugs of abuse detection in meconium by EMIT II and ELISA.

The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme multiplied immunoassay technique (EMIT((R) )II) and enzyme-linked immunosorbent assay (ELISA) methods were compared. The sample preparation for the ELISA screen was a simple buffer extraction versus a lengthy and more laborious sample preparation procedure for the EMIT II screen. The ELISA method was automated using a TECAN Genesis. The EMIT II analysis was automated with an Olympus AU400e. The opioid screen was calibrated with hydromorphone and the benzodiazepine screen was calibrated with clonazepam to maximize detection for these analytes. Previously validated gas chromatography-mass spectrometry (GC-MS), two-dimensional GC-MS, or liquid chromatography-tandem MS methods were used for confirmation. Results from the two techniques compared well. Agreement of the ELISA assay was greater than 90% when compared to EMIT II for all drug classes except barbiturates and benzodiazepines. ELISA appears to be more sensitive than EMIT II for the detection of amphetamines, methadone, propoxyphene, and cocaine. ELISA compared well to EMIT II for cannabinoids, opioids, and PCP. Specificity of the ELISA assay was slightly better for PCP and opioids. EMIT II appears to be more sensitive for the detection of barbiturates and benzodiazepines. The ELISA method reduced turnaround time by 50% compared to the EMIT II method.

Fungicidal effect and the mode of action of piscidin 2 derived from hybrid striped bass.

Piscidin 2 (P2), a 22-residue cationic peptide isolated from the mast cells of hybrid striped bass, has potent antibacterial activities. However, its antifungal properties are not completely understood. In the current study, we investigated the antifungal effects and mode of action of P2. P2 exhibited potent antifungal activity against human pathogenic fungi. To understand the fungicidal properties of P2, we focused on a membrane-active mechanism of the peptide by in vivo and in vitro testing. Flow cytometric analysis using bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)] and protoplast regeneration experiments showed that P2 caused fungal membrane damage. Furthermore, fluorescence analysis using 1,6-diphenyl-1,3,5-hexatriene (DPH) revealed that P2 created pores in fungal membranes. These results were confirmed with dye leakage tests by using liposomes composed of phosphatidylcholine/phosphatidylserine (3:1, w/w), which mimicked fungal membranes. The present study indicated that P2 exerts its fungicidal effects by perturbing membrane activities.

Development of a Quantitative Gas Chromatography-Tandem Mass Spectrometry Method for the Determination of Pentobarbital in Dog Food.

In 2017, a commercially available dog food was found by our laboratory to be adulterated with the euthanasia drug pentobarbital. An FDA class 1 voluntary recall by the company ensued. Since there is no set tolerance for pentobarbital in food or feed, a sensitive method for its detection was required. We describe a simple, yet efficient, method for the extraction and quantitative analysis of the barbiturate in dog food. The procedure relies on a combined food emergency response network (FERN) and QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach to sample extraction followed by quantitative analysis by gas chromatography-tandem mass spectrometry (GC/MS/MS) using pentobarbital- d5 as an internal standard. This procedure improves upon other GC/MS methodologies in that derivatization of pentobarbital or its deuterated internal standard is unnecessary, and sensitivity to a calculated limit of detection (LOD) of 0.6 ppb and a limit of quantitation (LOQ) of 2 ppb is achieved.

Quantitation of Cocaine and Metabolites, Phencyclidine, Butalbital and Phenobarbital in Meconium by Liquid Chromatography-Tandem Mass Spectrometry.

In this study, a quantitative polarity switching liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS) method was developed to detect and quantify cocaine and metabolites (cocaethylene, benzoylecgonine and meta-hydroxybenzoylecgonine), phencyclidine (PCP) and barbiturates (phenobarbital and butalbital) in meconium. Accuracy and precision samples at 0.0125% and 75% of the upper limit of quantitation (ULOQ) were analyzed in triplicate over 5 days with accuracy above 84% and average %CV values below 11%. Within-run (n = 15) and between-run (n = 15) %CV values were ≤5%. Analytical measurement ranges were reproducible and linear (R ≥ 0.995) for cocaine and metabolites (20-2,000 ng/g), PCP (10-1,000 ng/g) and barbiturates (50-5,000 ng/g). Accuracy of 100 ± 20% was observed at (the limits of detection) 10 ng/g for cocaine and metabolites, 2.5 ng/g for PCP and 25 ng/g for barbiturates. No carryover was observed at 2X ULOQ and no interfering substances were identified. Sample preparation recoveries were 53-83%. Fifty-one authentic patient samples previously characterized correlated with the newly developed test having R2 values ≥0.996. This combined method allows accurate quantitation of the targeted drugs in a complex matrix while decreasing sample preparation and analysis time with reduced sample volume. Clinical data and positivity rates were similar to previously published positivity rates. Validation data and positivity rate agreement signifies a reliable and robust assay.

Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells.

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.

Comparison of umbilical cord tissue and meconium for the confirmation of in utero drug exposure.

OBJECTIVES: Drug screening in neonates is traditionally performed using meconium, but cord tissue has been proposed as an alternative specimen. This study compares the detection of drugs in a large number of paired meconium and umbilical cord tissue samples from subjects at risk of in utero drug exposure. DESIGN AND METHODS: Physician-ordered toxicology results and clinical information were collected in a retrospective review of subject medical records. All toxicology testing was performed by a national reference laboratory using a combination of immunoassays and chromatography-mass spectrometry. The comparison was limited to drugs present in both cord and meconium panels. RESULTS: Overall agreement between cord and meconium ranged from 76% (cannabinoids) to 100% (barbiturates), but Cohen's kappa was <65% for 5 of the 6 drug classes we studied. Considering meconium as the gold standard, cord was less sensitive for the detection of 5 of the 6 drug classes, and for the detection of all 5 individual opioids. For 3 of the 5 individual opioids, the concentration of drug measured in meconium did not correlate well with qualitative detection in cord. CONCLUSIONS: This study reveals different sensitivities of drug detection in umbilical cord tissue and meconium. For the drugs studied here, meconium provides greater sensitivity, and is likely to remain the specimen of choice when sensitivity is of greatest importance. These results can help clinicians, laboratorians, and epidemiologists to (1) select the most appropriate test to confirm a suspected drug exposure and (2) interpret discordant results when testing is performed in multiple matrices.

Simultaneous determination of three residual barbiturates in pork using accelerated solvent extraction and gas chromatography-mass spectrometry.

A new method was developed for the rapid extraction and unequivocal determination of barbital, amobarbital and phenobarbital residues in pork. The isolation of the analytes from pork samples was accomplished by utilizing an accelerated solvent extractor ASE 300. The procedure was automatically carried out in series for fat removing and extraction, respectively with n-hexane and acetonitrile pressurized constantly at 10.3 MPa for 30 min. After evaporation, the extracts were cleaned up on a C(18) solid phase extraction (SPE) cartridge and the barbiturates were eluted with hexane-ethyl acetate (7:3), evaporated on a rotary evaporator and derivatized with CH(3)I. The methylated barbiturates were separated on a HP-5MS capillary column and detected with a mass detector. Electron impact ion source (EI) operating in time program-selected ion monitoring mode (SIM) was used for identification and external standard method was used for quantification. Good linearity was obtained in the range from 0.5 microg/kg to 25 microg/kg. Average recoveries of the three barbiturates spiked in pork ranged from 84.0% to 103.0%, with relative standard deviations from 1.6% to 12%. The limit of detection (LOD) was 0.5 microg/kg for the three barbiturates (S/N>or=3). The quantification limit (LOQ) was 1 microg/kg for the three barbiturates (S/N>or=10).

Determination and stability of divicine and isouramil produced by enzymatic hydrolysis of vicine and convicine of faba bean.

The aglycones of vicine and convicine, divicine and isouramil, are the causative agents of favism and, therefore, should be analysed along with vicine and convicine in research seeking to eliminate them. This study investigated the stability of the aglycones produced by hydrolysis with ß-glucosidase. Reversed-phase, high-performance liquid chromatography (HPLC) with UV detection was shown to be able to observe both aglycone formation and further reactions in isolated fractions and extract made from faba bean and in faba bean suspension. Divicine and isouramil were unstable and degraded almost completely in extract in 60min and completely in fractions in 120min at a pH of 5 at 37°C. Adding sodium ascorbate delayed degradation of divicine. Divicine was more stable at 20°C than at 37°C. Being able to show formation and degradation of the aglycones, the proposed method allows monitoring of the vicine and convicine detoxification process.

Predictive Value of Positive Drug Screening Results in an Urban Outpatient Population.

Urine drug testing (UDT) has become an essential component in the management of patients prescribed opioid analgesics for the treatment of chronic non-malignant pain. Several laboratory methods are available to monitor adherence with the pharmacological regimen and abstinence from illicit or unauthorized medications. Immunochemical screening methods are rapid and economical, but they have limitations, including lack of specificity, and confirmatory methods are often necessary to verify presumptive positive results. We analyzed the results of confirmatory assays in an outpatient setting to determine the predictive value of presumptive positive urine drug screen results using an automated immunoassay for eight common drugs or drug classes. Positive predictive values (PPVs), in descending order, were as follows: cannabinoids (100%), cocaine (100%), opiates (86.8%), benzodiazepines (74.6%), oxycodone (67.6%), methadone (44.1%) and amphetamines (9.3%). The number of positive barbiturate results was too small to be included in the statistical analysis.

Saliva as a diagnostic matrix for drug abuse.

Scientific interest in saliva as a diagnostic matrix has greatly increased over the last decade. The Triage screening test (Biosite Diagnostics), a rapid immunological test used to detect recreational drugs in the urine, was used to compare two biological matrixes: a non-conventional one, saliva, and a traditional one, urine. Twenty-one drug abusers collected one urine and one saliva specimen, both of which were tested with the Triage kit. Data were validated by gas-chromatography-mass-spectrometry (GC-MS). Results were positive for methadone in 9 saliva and 14 urine specimens, for opiates in 2 and 10, respectively, and for barbiturates in 2 specimens. Saliva specimens were negative for cannabis, THC, benzodiazepines and tricyclic antidepressants, although the GC-MS analysis revealed low concentrations of these drugs in the saliva. The study demonstrates the possibility of using saliva as a diagnostic matrix to test for drug-taking; however, the Triage kit must be improved before being used with saliva.