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Objective In the management of patients with suspected acute drug poisoning, a screening test using the patient's urine is usually performed. The Triage DOA ® and INSTANT-VIEW M-1 ® kits are two commonly used point-of-care screening kits in Japan. However, the relationship between the results of these screening kits and the blood concentration of the poisoning drug is not clear. In this study, we evaluated which kit is more useful for acute drug poisoning screening based on a comparison of their results with the results of a serum drug analysis. Methods This prospective cross-sectional study investigated all patients with acute drug poisoning admitted to a general hospital in Tokyo, Japan, over a nine-month period. The Triage DOA ® and INSTANT-VIEW M-1 ® screening kits were used, and a qualitative serum analysis was conducted simultaneously in all cases. We compared the kits for use in screening patients with acute drug poisoning and evaluated the utility of the kits. Results For the 117 patients enrolled in this study, the 2 kits showed different sensitivities to benzodiazepines (Triage ® , 78.6%; INSTANT-VIEW ® , 90.5%). Both kits showed high sensitivity to barbiturates (Triage ® , 87.0%; INSTANT-VIEW ® , 91.3%) but low sensitivity to tricyclic antidepressants (Triage ® , 25.0%; INSTANT-VIEW ® , 45.8%). Conclusion Because the sensitivity varies depending on the kind of drug, it is difficult to discuss the superiority of these kits. However, this study compared the results of two types of urinary drug screening kits with the results of qualitative analysis of drugs in serum as a gold standard, providing important reference data.

Zoliflodacin is a novel spiropyrimidinetrione with activity against bacterial type II topoisomerases that inhibits DNA biosynthesis and results in accumulation of double-strand cleavages in bacteria. We report results from two phase 1 studies that investigated the safety, tolerability, and pharmacokinetics (PK) of zoliflodacin and absorption, distribution, metabolism, and excretion (ADME) after single doses in healthy volunteers. In the single ascending dose study, zoliflodacin was rapidly absorbed, with a time to maximum concentration of drug in serum ( T max ) between 1.5 and 2.3 h. Exposure increased dose proportionally up to 800 mg and less than dose proportionally between 800 and 4,000 mg. Urinary excretion of unchanged zoliflodacin was <5.0% of the total dose. In the fed state, absorption was delayed ( T max , 4 h), accompanied by an increase in the area under the concentration-time curve (AUC) at 1,500- and 3,000-mg doses. In the ADME study (3,000 mg orally), the PK profile of zoliflodacin had exposure (AUC and maximum concentration of drug in serum [ C max ]) similar to that of the ascending dose study and a median T max of 2.5 h. A total of 97.8% of the administered radioactivity was recovered in excreta, with urine and fecal elimination accounting for approximately 18.2% and 79.6% of the dose, respectively. The major clearance pathway was via metabolism and elimination in feces with low urinary recovery of unchanged drug (approximately 2.5%) and metabolites accounting for 56% of the dose excreted in the feces. Zoliflodacin represented 72.3% and metabolite M3 accounted for 16.4% of total circulating radioactivity in human plasma. Along with the results from these studies and based upon safety, PK, and PK/pharmacodynamics targets, a dosage regimen was selected for evaluation in a phase 2 study in urogenital gonorrhea. (The studies discussed in this paper have been registered at ClinicalTrials.gov under identifiers NCT01929629 and NCT02298920.)., (Copyright © 2018 American Society for Microbiology.)

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., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Drug candidates, which have the potential of enhancing athletic performance represent a risk of being misused in elite sport. Therefore, there is a need for early consideration by anti-doping authorities and implementation into sports drug testing programmes. The hypoxia-inducible factor (HIF) or prolyl hydroxylase inhibitor (PHI) GSK1278863 represents an advanced candidate of an emerging class of therapeutics that possess substantial potential for abuse in sport due to their capability to stimulate the biogenesis of erythrocytes and, consequently, the individual's oxygen transport capacity. A thorough characterization of such analytes by technologies predominantly used for doping control purposes and the subsequent implementation of the active drug and/or its main urinary metabolite(s) are vital for comprehensive, preventive, and efficient anti-doping work. In the present study, the HIF PHI drug candidate GSK1278863 (comprising a 6-hydroxypyrimidine-2,4-dione nucleus) and its bishydroxylated metabolite M2 (GSK2391220A) were studied regarding their mass spectrometric behaviour under electrospray ionization (ESI-MS/MS) conditions. Synthesized reference materials were used to elucidate dissociation pathways by means of quadrupole/time-of-flight high resolution/high accuracy tandem mass spectrometry, and their detection from spiked urine and elimination study urine samples under routine doping control conditions was established using liquid chromatography-electrospray ionization-tandem mass spectrometry with direct injection. Dissociation pathways to diagnostic product ions of GSK1278863 (e.g. m/z 291, 223, and 122) were proposed as substantiated by determined elemental compositions and MS(n) experiments as well as comparison to spectra of the bishydroxylated analogue M2. An analytical assay based on direct urine injection using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of GSK1278863 in combination with its bishydroxylated metabolite M2. Validation parameters including limit of detection (0.5-1 ng/mL), linearity, specificity, ion suppression/enhancement (<10%), intra- and inter-day precision (6-22%) were determined, demonstrating the fitness-for-purpose of the assay for doping control screening of urine samples for the presence of the drug candidate and its main metabolite and for expanding current anti-doping efforts to this new class of therapeutics. However, administration study urine sample analysis suggested the use of M2 rather than the intact drug due to extensive metabolic conversion. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Date-rape drugs have the potential to be used in drug-facilitated sexual assault, organ theft and property theft. Since they are colorless, tasteless and odorless, victims can drink without noticing, when added to the beverages. These drugs must be detected in time, before they are cleared up from the biofluids. A simultaneous extraction and determination method in urine for GHB, ketamine, norketamine, phenobarbital, thiopental, zolpidem, zopiclone and phenytoin (an anticonvulsant and antiepileptic drug) with LC-MS/MS was developed for the first time with analytically acceptable recoveries and validated. A 4 steps liquid-liquid extraction was applied, using only 1.000mL urine. A new age commercial C18 poroshell column with high column efficiency was used for LC-MS/MS analysis with a fast isocratic elution as 5.5min. A new MS transition were introduced for barbital. 222.7>179.8 with the effect of acetonitrile. Recoveries (%) were between 80.98-99.27 for all analytes, except for GHB which was 71.46. LOD and LOQ values were found in the ranges of 0.59-49.50 and 9.20-80.80ngmL(-1) for all the analytes (except for GHB:3.44 and 6.00μgmL(-1)). HorRat values calculated (between 0.25-1.21), revealed that the inter-day and interanalist precisions (RSD%≤14.54%) acceptable. The simultaneous extraction and determination of these 8 analytes in urine is challenging because of the difficulty arising from the different chemical properties of some. Since the procedure can extract drugs from a wide range of polarity and pKa, it increases the window of detection. Group representatives from barbiturates, z-drugs, ketamine, phenytoin and polar acidic drugs (GHB) have been successfully analyzed in this study with low detection limits. The method is important from the point of determining the combined or single use of these drugs in crimes and finding out the reasons of deaths related to these drugs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Butalbital, a barbiturate, is present in analgesic combinations used by headache sufferers. Overuse/abuse of these combinations may cause dependence, chronic migraine, and medication-overuse headache (MOH). MOH is difficult to manage: it improves interrupting analgesic overuse, but requires monitoring, because relapses are frequent. A gas chromatography-mass spectrometry (GC-MS) method for hair analysis has been developed and validated to document abuse of an analgesic combination containing butalbital and propyphenazone by a patient with MOH. For over ten years the patient managed her headache using eight suppositories/day of an analgesic combination containing butalbital 150mg, caffeine 75mg, and propyphenazone 375mg per suppository. An outpatient detoxification treatment was carried out. After three weeks, the patient reduced the consumption to one suppository/day. At the first control visit, after three months from the beginning of detoxification, the patient increased the use of the combination to four suppositories/day and at the second control visit, after seven months from the beginning of detoxification, she was back to eight suppositories/day. At the two control visits, a hair sample was taken for determination of butalbital and propyphenazone. Moreover blood and urine samples for determination of butalbital were drawn at the beginning of detoxification treatment and at the two control visits. With the segmental analysis of two hair samples the medication history of ten months could be estimated. In the first hair sample, collected at the first control visit, in the distal segment, butalbital and propyphenazone concentrations were, respectively, 17.5ng/mg and 56.0ng/mg, confirming the prolonged abuse; in the proximal segment, concurrently with the detoxification treatment, butalbital and propyphenazone concentrations had reduced respectively to 5.45ng/mg and 11.1ng/mg. The second hair sample, collected at the second control visit, proved the fair course of the detoxification treatment in the distal segment and signalled relapse in the abuse of the analgesic combination in the proximal segment. In the clinical context, hair analysis can be advantageously used to monitor the abuse of analgesic combinations with butalbital, common among headache patients. The validation data showed that GC-MS method developed for determination of butalbital and propyphenazone was rapid, highly sensitive, specific and selective., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The abuse of barbiturate drugs is widespread, and the development of methods for their efficient separation and quantification is needed. Three barbiturate drugs were preconcentrated and determined by in-line solid-phase extraction (SPE) capillary electrophoresis (CE) in urine samples. Different parameters affecting preconcentration were evaluated, such as the sample pH, the volume of the elution plug and the sample injection time. This strategy enhanced the detection sensitivity in the range of 170- to 1840-fold, compared with normal hydrodynamic injection. The method provides limits of detection (LODs) for standard samples in the range of 0.5 to 5 ng/mL with good repeatability and reproducibility values. The LODs obtained for urine samples were in the range of 5 to 60 ng/mL. The validation with human urine samples spiked with the studied compounds demonstrated the applicability of the optimized method. This method provides a reproducible and sensitive analysis of urine samples in the determination of barbiturates drugs.

Three barbiturate drugs, barbital, phenobarbital, and secobarbital were separated and analyzed by electrokinetic supercharging. The influence of different parameters on electrokinetic supercharging performance was evaluated using both univariated and multivariated optimization processes. The parameters studied were sample pH, concentration, and length of the leading and terminating electrolytes, electrokinetic injection of the sample and composition and hydrodynamic injection of the solvent plug. The leading electrolyte (50 mM NaCl) was hydrodynamically injected (50 mbar × 120 s) prior to the sample that was adjusted to pH 9.6 and electrokinetically injected at -8.5 kV for 300 s. The terminating electrolyte (100 mM of 2-(cyclohexylamino) ethanesulphonic acid) was then hydrodynamically injected (50 mbar × 140 s). The results showed that this strategy enhanced detection sensitivity around 1050-fold compared with normal hydrodynamic injection, providing detection limits ranging between 1.5 and 2.1 ng/mL for standard samples with good repeatability in terms of peak area (values of relative standard deviation, %RSD < 3). The applicability of the optimized method was demonstrated by the analysis of human urine samples spiked with the studied compounds at different concentration levels and further liquid-liquid extraction step. The estimated detection limits obtained in the urine samples extract ranged between 8 and 15 ng/mL., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Urine drug testing is an important tool that is commonly used to assess patient compliance with prescription regimens. Point-of-collection immunoassay devices allow for timely availability of laboratory test results to guide therapy during the same office visit. Two waived immunoassay-based urine drug screen cups were evaluated in this study. The NexScreen cup and the DrugCheck Waive RT cup claim to detect 10-12 drug classes of commonly used and/or abused drugs. This study included a sensitivity and precision challenge with 4-6 replicates at concentrations 0-150% of the manufacture's claimed cutoff, using drug-free urine spiked with purified reference standards. The stability of test results was evaluated by reading the results at intervals between five and 1,440 min. Specificity was evaluated by parallel comparison of pooled patients' specimens, representing 56 patients and 41 known drug compounds. When comparing results to validated liquid chromatography-mass spectrometry results, false positives were observed in the NexScreen cups for benzodiazepine, methamphetamine, methadone, opiates and tricyclic antidepressant tests, but there were no false negatives. The DrugCheck Waive RT cups showed false negative results for barbiturates and opiates, but no false positives. Overall, the NexScreen cup demonstrated better sensitivity than claimed, whereas the sensitivity of the DrugCheck Waive RT cup did not meet claims.

In this paper we describe the synthesis of a molecularly imprinted polymer (MIP) by precipitation polymerisation, with barbital as the template molecule, and the application of the barbital MIP as a molecularly selective sorbent in the solid-phase extraction (SPE) of barbiturates from human urine samples. The MIP was synthesised by precipitation polymerisation using 2,6-bis-acrylamidopyridine as the functional monomer and DVB-80 as the cross-linking agent. The spherical MIP particles produced were 4.2 ± 0.4 μm in diameter; a non-imprinted control polymer (NIP) in bead form was 4.8 ± 0.4 μm (mean±standard deviation) in diameter. The particles were packed into a solid-phase extraction cartridge and employed as a novel sorbent in a molecularly imprinted solid-phase extraction (MISPE) protocol. The MIP showed high selectivity for the template molecule, barbital, a feature which can be ascribed to the high-fidelity binding sites present in the MIP which arose from the use of 2,6-bis-acrylamidopyridine as the functional monomer. However, the MIP also displayed useful cross-selectivity for other barbiturates besides barbital. For real samples, the MIP was applied for the extraction of four barbiturates from human urine. However, due to the high urea concentration in this sample which interfere the proper interaction of barbiturates onto the MIP, a tandem system using a commercially available sorbent was developed., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this paper, a novel and simple method for the determination of trace amounts of barbituric acid in water and biological samples was developed by using dispersive liquid-liquid microextraction (DLLME) techniques combined with spectrophotometric analysis. The procedure is based on color reaction of barbituric acid with p-dimethylaminobenzaldehyde and extraction of the color product using the DLLME technique. Some important parameters such as reaction conditions and the type and volume of extraction and dispersive solvents as well as the extraction time were investigated and optimized in detail. Under the optimum conditions, the calibration graphs were linear over the range of 5.0 to 200 ng ml(-1) with limit of detection of 2.0 ng ml(-1). Relative standard deviation for five replicate determinations of barbituric acid at 50 ng ml(-1) concentration level was calculated to be 1.64%. Average recoveries for spiked samples were determined to be between 94% and 105%. The proposed method was applied for the determination of barbituric acid in pharmaceutical formulation and biological samples., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

The availability of point-of-care (POC) medical devices for drug testing has surged. Reduction in turnaround time, and hence, rapid results are attractive, particularly to acute care facilities, rehabilitation facilities and specialized clinics such as pain management clinics. Here we describe our validation results for the Integrated E-Z Split Key Cup II, a low-cost, rapid urine test that utilizes competitive immunoassay technology to detect 12 drugs or drug classes of commonly abused drugs. Positivity is based on the absence of a colored band at a labeled portion of the detection strip; a negative result produces a distinct, colored band. Using reagent-grade standards, the apparent cut-off for each of the drugs was challenged. The stability of the results was monitored over time. Five urine samples known to be negative for all drug categories and 24 patient samples confirmed positive for a total of 95 drugs and/or drug metabolites claimed to be detected by the device were tested. Adulterants and potential cross-reacting compounds were also evaluated. One false-positive result for benzodizepines was observed. One false-negative result for barbiturates was observed, but was resolved. Overall, the cups demonstrated excellent sensitivity, specificity, and diagnostic efficiency for all drugs represented.

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.

Two dogs were presented within 24 hours to the Department of Small Animal Medicine and Surgery at the University of Veterinary Medicine Hannover for investigation of the sudden onset of neurological abnormalities following a walk in the same park. One dog was observed ingesting a piece of meat. Analysis of urine by gas chromatography-mass spectrometry from each of the dogs identified the presence of barbiturates. Both dogs recovered with supportive treatment. This is the first report to describe the use of toxicological urinalysis with gas chromatography-mass spectrometry for the diagnosis of barbiturate intoxication in dogs.

In recent years, there has been observed an increasing number of traffic users being under influence of psychoactive substances that affect the central nervous system. A total of 198 blood samples and 23 urine samples collected from traffic users (drivers, passengers and pedestrians) suspected of having ingested psychoactive substances were examined. The analysis included blood samples collected from living individuals and blood or urine from the deceased. Ethyl alcohol levels were determined by gas chromatography, while body fluids were examined by Elisa tests for determination of cannabinoids, amphetamines, opium narcotics, cocaine (benzoiloecgonine), benzodiazepines, barbiturates and tricyclic antidepressants. The confirmation of positive results was carried out by gas chromatography with mass detector. Twenty-nine blood samples were positive, what constituted 14.6% of the total number of investigated cases, including 12 (7.8%) of samples originating from living individuals and 17 (37.8)--from the fatalities. In both groups, the most commonly detected substances were cannabinoids (THC and its metabolite carboxy-THC) and amphetamines and its analogues.

A voltammetric sensor based on a molecularly imprinted polymer (MIP) brush grafted to sol-gel film on graphite electrode is reported for the selective and sensitive analysis of barbituric acid (BA) in aqueous, blood plasma, and urine samples. The modified electrode was preanodised at +1.6 V (vs. saturated calomel electrode), where encapsulated BA involved hydrophobically induced hydrogen bondings, in MIP cavities exposed at the film/solution interface, at pH 7.0. Scanning electron microscopy (SEM) was employed to characterise the surface morphology of the resultant imprinted film of MIP brush. The differential pulse, cathodic stripping voltammetry (DPCSV) technique was employed to investigate the binding performance of the sol-gel-modified imprinted polymer brush, which yielded a linear response in the range of 4.95-100.00 microg mL(-1) of BA with a detection limit of 1.6 microg mL(-1) (S/N=3).

A rapid, human on-site urine multidrug test was used to screen canine urine samples for the presence of five illegal drugs and drugs from three commonly abused drug classes. Each sample was sent to a toxicology laboratory for gas chromatography/mass spectrometry (GC/MS) validation. On-site test results and GC/MS assays confirmed that the human on-site test kit did identify barbiturates, opiates, benzodiazepines, and amphetamines/methamphetamines in urine from dogs that had received these common illicit drugs/drug classes either intravenously and/or orally. However, neither the on-site test kit nor the GC/MS individual assays for marijuana or methadone, a synthetic opiate, were effective in identifying marijuana and methadone in urine from dogs with suspected or known exposure. No index of suspicion was seen for exposure to phencyclidines or cocaine during the study period, and no exposures were indicated by the on-site test results. Overall, the test is a rapid, readily available, affordable, and useful complement to the veterinarian's clinical consideration and professional judgment.

A method was developed for the quantitative analysis of 30 drugs of abuse and their metabolites in urine, including opiates, barbiturates, amphetamines, cocaine, cannabinoids, phencyclidine, methadone, and benzodiazepines. This method uses solid-phase extraction (SPE) on an Oasis HLB column followed by liquid chromatography-tandem mass spectrometry. Analytes were quantified by multiple reaction monitoring with the deuterated analogues as internal standards, using an atmospheric pressure ionization-electrospray interface. The method was validated by examining specificity, precision, accuracy, linearity, recovery, reproducibility, and detection limits. The limits of detection ranged from 9 pg/mL to 2.29 ng/mL in urine depending on the analyte. The SPE procedure was automated on a RapidTrace workstation to increase analytical throughput, and the results obtained via automated SPE were compared to those obtained by manual SPE to examine carryover effect, precision, accuracy, recovery, and reproducibility. To evaluate method performance, 108 urine samples were collected anonymously and tested for the presence of these drugs.

Two commercially available drug-screening assays were evaluated: the Roche kinetic interaction of microparticles in solution (KIMS) assay and the Microgenics cloned enzyme donor immunoassay (CEDIA). Urine samples from known drug-abuse patients were analyzed for amphetamines, barbiturates, benzodiazepines, benzoylecgonine, cannabinoids, LSD, methadone and opiates. Samples with discordant findings for the two assays were analyzed by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/electron capture detection (GC/ECD). Amphetamines showed 96.0% concordant results, with two false positive findings by CEDIA, three by KIMS and a further two false negatives by KIMS. Barbiturates showed 99.4% concordant results, with one false negative by KIMS. Benzodiazepines showed 97.4% concordant results, with two false negatives by KIMS (cutoff 100 microg/L, CEDIA cutoff 300 microg/L). Benzoylecgonine showed 17.8% concordant positive and 82.2% concordant negative results and no false finding by either assay. Cannabinoids showed 99.3% concordant results, with one sample negative by KIMS at a cutoff of 50 microg/L and positive by CEDIA (cutoff 25 microg/L). For LSD, 6.7% of findings were not in agreement. Methadone showed 97.5% concordant results, with two false positives by CEDIA, and one false positive and one false negative by KIMS. Opiates showed 96.9% concordant results, with no false KIMS results, but four false positives by CEDIA. The results indicate that the agreement of the CEDIA and KIMS results for the eight drugs is rather good (93.3-100%).

A method was suggested for identifying reladorm and donormil in pharmaceutical drugs and biological objects. The above substances are isolated by 96% ethanol or by mixture of chloroform and isopropanol. 7 color reactions, 3 microcrystalloscopic reactions and chromatography in thin sorbent layer are suggested for identification.

Simultaneous determination of seven barbiturates in human whole blood and urine by combining direct immersion solid-phase microextraction (DI-SPME) with gas chromatography-mass spectrometry (GC-MS) is presented. The main parameters affecting the DI-SPME process, such as SPME fibers, salt additives, pHs, extraction temperatures and immersion times were optimized for simultaneous determination of the drugs. The extraction efficiencies were 0.0180-0.988 and 0.0156-2.76% for whole blood and urine, respectively. The regression equations of the drugs showed excellent linearity for both samples; the correlation coefficients (r(2)) were 0.994-0.999. The detection limits for whole blood were 0.05-1 microg x ml(-1), and those for urine 0.01-0.6 microg x ml(-1). Actual quantitation could be made for pentobarbital in whole blood and urine obtained from volunteers, who had been orally administered a therapeutic dose of the drug. The DI-SPME/GC-MS procedure for barbiturates established in this study is simple and sensitive enough to be adopted in the fields of clinical and forensic toxicology.

The subject of the research was the analysis of frequency and type of psychoactive substances used, basing on the determinations the blood and/or urine samples, performed in the toxicological laboratory of the Department of Clinical and Industrial Toxicology Jagiellonian University in Kraków in the period from December 2001 to November 2003. From 17,649 performed determinations--45.5% were positive. 50% of the positive determinations were psychoactive substances. The most often psychoactive substance determined was ethyl alcohol (52.86%), next benzodiazepines (17.41%), amphetamines (10.54%), opiates (8.05%), THC (6.87%), barbiturates (3.74%), and occasionally atropine and cocaine. There was observed a variety of mixed, simultaneously taking psychoactive substances, especially ethyl alcohol, opiates, amphetamine derivatives and cannabinoids. The analysis of the occurrence of psychoactive substances in biological samples from patients treated in different hospital departments, others hospitals and ordered by private persons also was performed. In the last two years 369 private patients ordered psychoactive substances determinations and 78 of them were positive.

Analyzing prescription drugs in urine is a challenge for both the lab and the agency monitoring health professionals.

There are about 60,000 chemical substances which may cause poisoning. Identifying the cause substances is, therefore, very important for patient at emergency department. Triage is an immunoassay kit for the qualitative test for the metabolites of 8 major abuse drugs in urine. We assessed the usefullness of Triage on two patient groups. The first Group consists of the patients considered having not taken substances at initial diagnosis; the second Group consists of the patients considered having taken substances. The result are as follows. 1) The rate of Triage positive patients in the first Group were: attempt-suicide 23%, coma 24%, shock 10%, trauma 7%, respectively. Except for the habitually used medicine, narcotic and stimulant drugs were detected. In the first Group, negative result of Triage was effective in diagnosing the patients as not poisoned, excluding the possitivity of 8 major drugs usage. 2) The rate of Triage positive patients in the second Group were very high: attempt-suicide 77%, coma 51%, shock 57%, trauma 30%, respectively, showing mostly any of 8 major drugs were the cause of poisoning. In the second Group, positive result of Triage was effective in diagnosing the patient as poisoning or as coexisting poisoning with other diseases. 3) The specificity of Triage diagnosis in the first Group was 80% (113/142). The specificity and the sensitivity in the second Group were 64% (50/78) and 97% (74/76), respectively. These results means that Triage is very useful for diagnosis on 8 major drugs poisoning. 4) Triage is efficient for identifying the cause substances in drug poisoning and, therefore, can save medical expense. Triage is a very useful test kit at emergency department.

Following the screening of urine samples for a panel of drugs over a period of 1 year, a peak was observed during December 1999 and January 2000 in the numbers of samples testing positive for both morphine and barbiturates. This increase may be due to the adulteration of street heroin with barbiturate during this period.

Bucolome (BCP) is a non-steroidal anti-inflammatory drug, which is used in the treatment of chronic articular rheumatism. Bucolome N-glucuronide (BCP-NG), a metabolite of BCP, is the first unique N-glucuronide of barbituric acid derivatives. First, the stability of BCP-NG in various pH aqueous solutions was studied. BCP-NG was quite unstable under neutral and acidic conditions, and is easily hydrolyzed to BCP. Based on these characteristics of BCP-NG, a simple, rapid and highly sensitive method for the simultaneous determination of BCP and BCP-NG with phenylbutazone (I.S.) in biological fluids was developed using high-performance liquid chromatography (HPLC). A reversed-phase ODS column was used for the separation of BCP, BCP-NG and I.S. A pharmacokinetic study for BCP and BCP-NG was carried out in male Wistar/ST rats following i.v. administration of BCP at a dose of 10 mg/kg body weight. The slow plasma elimination of BCP with time was shown. A major metabolite of BCP in bile was N-glucuronide. The cumulative amounts of BCP and BCP-NG in the bile over 8 h were approximately 2.4 +/- 1.4% and 12.6 +/- 2.3% of the dose, respectively. BCP and BCP-NG in the urine were 2.7 +/- 0.7% and 3.2 +/- 0.3% of the dose. Although BCP had a long half-life (over 8.5 h), the preliminary pharmacokinetic parameters (0-8 h) were determined: t 1/2, 8.52 +/- 1.96 h; AUC, 419.9 +/- 45.2 microg x h/ml; MRT, 3.29 +/- 0.11 h; CLtot, 5.93 +/- 0.54 ml/h; and Vdss, 19.5 +/- 1.3 l. These observations are the first pharmacokinetic findings for the N-glucuronide of the barbituric acid derivatives.

The pressure to reduce the cost of analytic testing makes it tempting to discontinue routine confirmation of urine specimens positive for drugs of abuse by immunoassay. Beyond the economic motivation, the requirement for confirmation should be driven by the positive predictive value of the screening tests. We have quantitated positive predictive values of our screening immunoassays in a large metropolitan Veterans Affairs Medical Center. We reviewed the confirmatory rate of urine specimens positive for drugs of abuse with Beckman Synchron reagents from June 1998 to June 1999 and tabulated the false-positive screening rate. There were 175 instances of false-positive screens during the 13 months we analyzed. Positive predictive values ranged from 0% (amphetamine) to 100% (THC). We determined that the low positive predictive value of the amphetamine assay in our laboratory was primarily due to the use of ranitidine (Zantac). Urine specimens containing greater than 43 microg/mL ranitidine were positive in our amphetamine assay. This concentration is routinely exceeded in our patients taking ranitidine. In our clinical and analytic setting, the Beckman THC assay did not require confirmation. The positive predictive values of the Beckman opiate, cocaine, barbiturate, propoxyphene, and methadone immunoassays dictate routine confirmatory testing in specimens that screen positive for these substances. Finally, because of its extreme sensitivity to ranitidine, the Beckman amphetamine assay has little utility in our laboratory setting.

The performance of the new fluorescence polarization immunoassay reagents Cassette COBAS INTEGRA Serum Benzodiazepines assay (SBENZ) and Cassette Serum Barbiturates assay (SBARB) was evaluated as compared to other immunoassays (Abbott TDx Serum Benzodiazepines, Abbott TDx Urine Benzodiazepines, Behring EMIT Serum Benzodiazepines, Abbott ADx Serum Barbiturates, Behring EMIT Serum Barbiturates, and the COBAS INTEGRA Barbiturates (BARB) urine assay) and gas chromatography-mass spectrometry (GC-MS). Recoveries of nordiazepam and secobarbital using the SBENZ and SBARB assays, respectively, were equivalent for serum, plasma, and urine. Cross-reactivities of structurally related benzodiazepines, barbiturates, and their metabolites were very similar in serum and urine for the SBENZ and SBARB assays. Precision was within 5.4% for SBENZ serum and within 11% from 10 to 100 ng/mL for urine. Precision was within 5% for SBARB serum and within 7% from 136 to 277 ng/mL for the urine application. The standard curves for SBENZ and SBARB were stable for at least 16 weeks with the reagents stored open on the COBAS INTEGRA analyzer. Clinical comparison of the SBENZ serum assay indicated an increased pickup rate, as confirmed by GC-MS, compared to TDx and EMIT. The diagnostic sensitivities of the SBENZ serum application, TDx, and EMIT versus GC-MS were 100%, 89%, and 36%, respectively. The diagnostic specificities were 71%, 79%, and 100%, respectively. The diagnostic sensitivities of the SBENZ urine application and TDx versus GC-MS were 100% and the diagnostic specificities were 88%. The increased positive pick-up of the SBENZ assay compared to the other immunoassays is most probably due to the difference in the limit of detection (LOD) and the increased cross-reactivity for the low-dose benzodiazepines. Clinical comparison of the SBARB serum assay indicated an increased positive pick-up rate, as confirmed by GC-MS. The diagnostic sensitivities of the SBARB serum application, ADx, and EMIT versus GC-MS were 96%, 65%, and 35%, respectively. The diagnostic specificities were all 100%. The diagnostic sensitivities for the SBARB urine application and BARB versus GC-MS were all 100%, and the diagnostic specificities were all 91%. The SBENZ and SBARB kits demonstrated increased sensitivity for the detection of benzodiazepines and barbiturates in both serum and urine compared to the other immunoassays.

Broad-spectrum drug screening requires that all relevant substances be isolated, detected, and identified, regardless of their structure and/or polarity. To this end, systematic solid-phase extraction (SPE) approaches for drug isolation from biological fluids are required. Because speed and cost effectiveness are key issues in analytical toxicology, we have evaluated a disc-format extraction device for this purpose and compared the latter with an existing packed-bed column-format method. The discs were SPEC.PLUS.C18AR/MP3 cartridges with 10-mL solvent reservoirs, providing hydrophobic and cation exchange interactions. Blank human urine was spiked at 2 microg/mL with a selection of acidic, neutral, and basic drugs representing a variety of relevant drug classes. Urine specimens (2 mL) were diluted with 2 mL 0.1 M phosphate buffer (pH 5.0) and then applied to the preconditioned disc. Washing was done with 1 mL water. Acidic and neutral drugs were eluted with 1 mL ethyl acetate/acetone (1:1), and basic drugs were eluted with 1 mL ammoniated ethyl acetate. The eluates were collected separately, evaporated down to about 0.1 mL, and analyzed by gas chromatography-flame-ionization detection to check cleanliness, recoveries, and reproducibilities. The discs showed good extraction properties for all drugs and were easy to handle. Recoveries were 75-100% with coefficients of variation of around 5%. The resulting eluates showed only a few matrix interferences. As compared to our standard SPE method with packed-bed columns, the disc procedure allowed reductions in elution volumes and total processing time of approximately 60-65%.

Abuscreen OnLine assays for drugs of abuse screening in urine have recently been developed for use on Hitachi 917 analyzers (Roche Diagnostics GmbH). The assays are based on the kinetic interaction of microparticles as measured by changes in light transmission. Drug in a sample inhibits the formation of particle aggregates and diminishes absorbance change increases. It was the goal of this study to evaluate precision and comparability of the new asssys with CEDIA drugs of abuse tests on Hitachi 917 in different laboratories (three European and three US). The assays were calibrated in the nonlinear mode with four to six standards (semiquantitative application). Initial within-run (21 replicates, four labs) and between-day (10 days, two labs) imprecision studies using Abuscreen OnLine tests and commercial negative (0.5 x cut-off) and positive (1.5 x cut-off) controls revealed the following median CVs [withinrun neg./pos. control/between-day neg./pos. control]: amphetamines 1.9/1.3/3.4/2.4, barbiturates 3.0/1.6/3.9/3.1, benzodiazepines 4.7/1.5/6.3/3.0, cocaine metabolite 1.8/0.9/2.4/1.7, methadone 5.4/1.6/5.5/2.2, opiates 5.5/2.8/5.3/2.7, THC 8.9/4.8/21.8/12.1. CVs < 10% were obtained for the THC test using controls with concentrations closer to the cut-off. An identical set of 170 GC/MS analyzed urine samples was distributed to the six laboratories and measured with Abuscreen OnLine tests on Hitachi 917. The median values for each individual sample were calculated and compared with the results obtained on individual Hitachi 917 analyzers by Passing-Bablok regression analysis. A good agreement between the laboratories was found with less than +/- 11% slope deviation and intercepts below 7% of the cut-off except for benzodiazepines (one slope 17%, one slope--26%) and THC (one slope 34%, one slope--18%). The comparability with CEDIA tests was analyzed by concordance plots using randomized routine samples in three laboratories. The following results were obtained in one of the participating laboratories [cut-off ng/mL] (No. of positive/negative/discrepant samples): amphetamines [500] 2/147/0, barbiturates [200] 1/148/0, benzodiazepines [100] 52/91/7, cocaine metabolite [300] 17/129/3, methadone [300] 113/34/2, opiates [300] 31/114/4, THC [50] 66/81/2. GC/MS was performed for clarification of the discrepant results. In summary, Abuscreen OnLine tests on Hitachi 917 give precise results which compare well when analyzed in different laboratories. They can be rated as convenient and flexible methods for drugs of abuse screening in the routine.

In order to study the effect of steric hindrance on competition between two kinds of beta-hydroxylation, a compound bearing on a pyrimidinetrione nucleus both a branched side chain with a tertiary carbon atom in position beta (isobutyl group) and a linear side chain (ethyl group), was selected and administered to rats. Urine and faeces were collected and extracted. Hydroxymetabolites and their derivatives were isolated and then identified. The beta-hydroxylation of the linear chain was more important than the beta-hydroxylation of the branched chain. Steric hindrance plays a decisive role in this regioselectivity.

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.

It is now accepted that drug misuse is a large and growing problem in the United Kingdom, some estimates of the number of regular illicit drug users being as high as three million. The aim of this paper is to provide insight into the methods used to detect drug misuse. The strategy adopted by one laboratory is described and methods of screening for, and identification of, a wide range of compounds are provided. No claim is made that this is the best approach or that the list of drugs detected is comprehensive; the range of drugs encountered is always increasing and the lists are constantly updated. It is hoped that users of toxicology laboratory services will gain an appreciation of the capabilities and limitations of the techniques available; and that those who may wish to provide such a service will find the necessary information in a readily accessible format.

The availability of automated, rapid and reliable methods for the systematic toxicological analysis (STA) of drugs and poisons in biosamples is of great importance in clinical and forensic toxicology laboratories. Gas chromatography-continuous scan mass spectrometry (GC-MS) possesses a high potential in STA because of its selectivity and identification power. However, in order to develop a fully automated STA method based on GC-MS two main obstacles have to be overcome: (a) sample preparation is rather sophisticated owing to the need to isolate analytes from the aqueous matrix and to allow a correct GC repartition of polar analytes; (b) the large amount of information collected within a single analysis makes it difficult to isolate relevant analytical information (mass spectra of analytes) from the chemical noise. Using a bench-top GC-MS system equipped with a laboratory robot for sample preparation (the Hewlett-Packard 7686 PrepStation) and an original method for mass spectral purification, a fully automated STA procedure was developed involving isolation of drugs from the sample (whole blood with minimal pretreatment, plasma, urine) by means of solid-phase extraction, derivatization (trimethylsilylation) of the acidic-neutral and of the basic extracts, GC-MS analysis, processing of data, and reporting of results. Each step of the procedure, and the method for data analysis in particular, can be easily integrated with other existing STA methods based on GC-MS.

A system for an automatic sample preparation procedure followed by on-line injection of the sample extract into a gas chromatography-mass spectrometry (GC-MS) system was developed for the simultaneous analysis of seven barbiturates in human urine. 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 a 1.5 ml volume of a urine 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 a GC-MS system. The calibration curves, using an internal standard method, demonstrated a good linearity throughout the concentration range from 0.02 to 10 microg/ml for all barbiturates extracted. The proposed method was applied to several clinical cases. The total analysis time for 20 samples was approximately 14 h.

A gas chromatography-mass spectrometric method is described for the quantitative measurement of 6 commonly used barbiturates in blood and urine specimens. The targeted barbiturates are butalbital, amobarbital, pentobarbital, secobarbital, mephobarbital and phenobarbital. They are recovered along with the internal standard, tolybarb, from blood and urine using liquid extraction then alkalated to form the N-ethyl derivatives. The ethylated barbiturates have symmetrical peaks which are well separated from each other on a non-polar methylsilicone capillary column. The derivatives on a non-polar methylsilicone capillary column. The derivatives facilitate quantitations between 50 and 10,000 ng/mL. The day-to-day CVs for all 6 barbiturates were between 4 and 9% at 200 and 5000 ng/mL. The method has been extended for identifying other acidic drugs and drug metabolites. They are mainly non-steroidal anti-inflammatory drugs, diuretics, and anticonvulsants. An additional 83 compounds can be qualitatively identified.

Requirements for the provision of an efficient and reliable service for drugs of abuse screening in urine have been summarized in Part I of this review. The requirements included rapid turn-around times, good communications between requesting clinicians and the laboratory, and participation in quality assessment schemes. In addition, the need for checking/confirmation of positive results obtained for preliminary screening methods was stressed. This aspect of the service has assumed even greater importance with widespread use of dip-stick technology and the increasing number of reasons for which drug screening is performed. Many of these additional uses of drug screening have possible serious legal implications, for example, screening school pupils, professional footballers, parents involved in child custody cases, persons applying for renewal of a driving licence after disqualification for a drug-related offence, doctors seeking re-registration after removal for drug abuse, and checking for compliance with terms of probation orders; as well as pre-employment screening and work-place testing. In many cases these requests will be received from a general practitioner or drug clinic with no indication of the reason for which testing has been requested. This also raises the serious problems of a chain of custody, provision of two samples, stability of samples, and secure and lengthy storage of samples in the laboratory-samples may be requested by legal authorities several months after the initial testing. The need for confirmation of positive results is now widely accepted but it may be equally important to confirm unexpected negative results. Failure to detect the presence of maintenance drugs may lead to the patient being discharged from a drug treatment clinic and, if attendance at the clinic is one of the terms of continued employment, to dismissal. It seems likely that increasing abuse of drugs and the efforts of regulatory authorities to control this, will lead to the manufacture of more designer drugs. Production of substituted phenethylamines was facilitated by the drug makers' cook book, 'PIHKAL' (Phenethylamines I Have Known And Loved) by Dr Alexander Shulgin and Ann Shulgin, and production of substituted tryptamines is promised in their next book, TIHKAL. Looking to the future, laboratories will need to ensure that they can detect and quantitate an ever-increasing number of drugs and related substances. The question of confidence in results of drugs of abuse testing raised in 1993 by Watson has assumed even greater importance as a result of attention focused on the OJ Simpson trial in Los Angeles. Toxicological investigations are likely to be challenged more frequently in the future. Even if analyses have been performed by GC-MS, there is a need to establish the level of match between the spectrum of the unknown substance and a library spectrum which is considered acceptable for legal purposes. It will also be essential to ensure that computer libraries contain spectra for all substances likely to be encountered in drugs of abuse screening.

Solid-phase microextraction (SPME) in conjunction with quadrupole ion trap GC-MS was applied to the determination of a series of barbiturates. A 65 microns Carbowax-divinylbenzene (DVB) SPME fiber was used to successfully extract a series of eight barbiturates from aqueous solution. Absorption kinetics and distribution coefficients for the 65 microns Carbowax-DVB SPME fiber were determined for the compounds. In addition the method was evaluated with respect to linearity, limit of detection, precision, desorption time, and the effect of salt. Limits of detection reached 1 ng/ml for the barbiturates. Linearity was established for the barbiturates over a concentration range of 10-1000 ng/ml, with coefficients of correlation 0.99. Overall, the precision of the method fell between 2.2%-6.5%, depending on the barbiturate. SPME was applied to the identification and quantitation of the barbiturates in a urine matrix. The method was validated by analyzing a reference standard pentobarbital-spiked urine sample. Both standard addition and internal standard with [2H5]-pentobarbital techniques were evaluated, with recoveries found to be 93% and 104%, respectively SPME was then used to rapidly screen a urine specimen tested positive for barbiturates, and butalbital was detected and quantified.

The study reported here originates from a county in California between Los Angeles and San Francisco. 658 mothers were tested over a 1-mo. period in June 1991 to ascertain the prevalence of drug misuse among pregnant women. 11% of the mothers tested positive for some type of substance. Of those substances found in the urine of the mothers who tested positive, the most prevalent were barbiturates, marijuana, methamphetamines, and amphetamines. When the multivariate analysis of logistic regression was performed with test results as the dependent variable, history of drug use was the most important factor related to mothers testing positive for drugs.

A high-performance liquid chromatographic method has been developed for the forensic analysis of 10 frequently used barbiturates (BARs) (allobarbital, amobarbital, barbital, cyclobarbital, hexobarbital, metharbital, pentobarbital, phenobarbital, secobarbital and thiopental) using a recently developed reversed-phase column packed with 2-micron particles. The results show that the new ODS column packing gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

Three cases of multiple false-positive drug tests are described. Postmortem urine specimens were screened using the enzyme-multiplied immunoassay technique. All patients had proteinuria and lactic aciduria. These false-positive reactions were due to the presence of lactate dehydrogenase (LDH), lactic acid, and protein. This finding was confirmed by creating a multiple false-positive sample with a solution of LDH and lactate in 5% bovine serum albumin at pH 6.

OnLine assays for cannabinoids, barbiturates, opiates, and cocaine from Roche Diagnostics were run on the automatic analyzer COBAS FARA II. The assay programs and the cutoff definition were modified to obtain maximum sensitivity and reagent economy. The limits of detection and curve stability data of the modified assays were compared with the original, as well as with EMIT d.a.u. Cannabinoid 20 assay, and the modified assays were found to be more favorable. Modified OnLine assays appear to be a convenient and cost-effective method for drug screening in urine.

Large numbers of specimens (5000-18,000) were screened for amphetamines, barbiturates, cocaine, marijuana, opiates, and phencyclidine by RIA (Roche), Emit II (Syva), and a new immunoassay, CEDIA (cloned enzyme donor immunoassay, Microgenics). All immunoassays performed equivalently for cocaine, opiates, and phencyclidine. All immunoassays detected the same amphetamine/methamphetamine-positive specimens, but all also detected numerous specimens containing cross-reacting sympathomimetic amines. CEDIA detected 100%, Emit II 93%, and RIA 82% of the barbiturate-positive specimens. Emit II and CEDIA detected 86-88% of the specimens found by RIA to be marijuana positive. A subset of specimens was additionally screened by OnLine (Roche) and TDx (Abbott) for amphetamines, cocaine, and marijuana. OnLine and TDx also detected all of the amphetamine-positive specimens and numerous specimens containing cross-reacting sympathomimetic amines. All immunoassays performed equivalently for cocaine, and the four nonisotopic tests detected 86-89% of the marijuana positives found by RIA. Interfering sympathomimetic amine drug compounds can be eliminated by using an oxidizing agent, thus decreasing the number of unconfirmable amphetamine presumptive positives. The CEDIAs for all of the major drugs of abuse are reliable and effective for large-volume urine screening programs.