Ethyl Glucuronide as a Long-term Alcohol Biomarker in Fingernail and Hair. Matrix Comparison and Evaluation of Gender Bias.

AIMS: This work aimed to assess the performance of hair and fingernail ethyl glucuronide (EtG) measurement for use as a biomarker of alcohol consumption in persons with known drinking history across a range of drinking behaviours. METHODS: EtG concentrations were assessed from the hair and fingernails of 50 study participants. Alcohol consumption of the previous 90 days was assessed by participant interview using the alcohol timeline follow-back method. EtG concentration was determined using LC-MS-MS using a method which was validated and accredited to ISO/IEC 17025 standards. RESULTS: There was significant correlation between alcohol consumption and EtG concentrations found in hair and fingernail samples across the study group (n = 50). From participants testing positive for EtG (male n = 14, female n = 13) no significant difference was found between male and female EtG levels in either hair or fingernails. Across all participants there was no significant difference in hair or fingernail EtG concentration between male (n = 23) and females (n = 27). CONCLUSIONS: Our results support the use of EtG to indicate alcohol consumption over the previous 90 days, or ~3 months as is the normal practice in hair analysis. The results confirm that fingernails can be a useful alternative matrix where hair samples are not available.

European guidelines for workplace drug and alcohol testing in hair.

Guidelines for Legally Defensible Workplace Drug Testing have been prepared and updated by the European Workplace Drug Testing Society (EWDTS). They are based on the 2010 version published by Pascal Kintz and Ronald Agius (Guidelines for European workplace drug and alcohol testing in hair. Drug Test. Anal. 2010, 2, 367) and in concordance with the Society of Hair Testing guidelines (Society of Hair Testing guidelines for drug testing in hair. Forensic Sci. Int. 2012, 218, 20-24). The European Guidelines are designed to establish best practice procedures whilst allowing individual countries to operate within the requirements of national customs and legislation. The EWDTS recommends that all European laboratories that undertake legally defensible workplace drug testing use these guidelines as a template for accreditation. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous determination of GHB and EtG in hair using GCMS/MS.

A gas chromatographic tandem mass spectrometric (GCMS/MS) method for simultaneously determining trace concentrations of gamma-hydroxybutyrate (GHB) and ethyl glucuronide (EtG) in hair has been developed. Multiple reaction monitoring (MRM) was used to detect precursor and product ions of GHB, (233 and 147) and EtG (261 and 143) following anion exchange solid phase extraction and derivatization with N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA). Deuterated standards of GHB and EtG were used as internal standards. The assay produced excellent linearity (r(2) > 0.99) and sensitivity. The lower limit of quantitation (LLOQ) was 10 pg/mg for EtG assuming a 20 mg hair sample. The method has been used to investigate cases of suspected drug facilitated assault as well as being used to identify heavy alcohol consumption in a group of volunteers.

Gabapentin does not affect antipyrine clearance.

The effect of gabapentin on antipyrine clearance was assessed in 12 healthy male volunteers, using a known enzyme inducer, phenytoin, as control. Subjects received gabapentin 400 mg or phenytoin 100 mg three times daily for 2 weeks. Antipyrine tests were performed before, during, and after treatment with gabapentin or phenytoin. In contrast to phenytoin, chronic administration of gabapentin did not affect antipyrine clearance. Gabapentin appears to have little potential for drug interactions.

Comparison by external quality assessment of performance of analytical systems for measurement of therapeutic drugs in serum.

The precision and accuracy of analytical methods currently in use for therapeutic drug monitoring were evaluated from proficiency test data provided by laboratories participating in the international Healthcontrol external quality assessment scheme for a range of eight antiepileptic drugs, theophylline, caffeine, and digoxin. Different analytical systems were assessed after grouping according to the reagent source and the analyzer used. The majority of analytical methods produced comparable levels of performance with coefficient of variation of < 10% and accuracy within +/-7% of the spike value. Emit reagents were implemented successfully on diverse analyzers but data from the Cobas Mira were generally in the technique group with significantly lower precision. Bias problems were evident for a number of FPIA assays for specific drugs. For example, caffeine interference was present in theophylline measurements by Sigma FPIA reagents whereas use of nonhuman matrix caused a negative bias in Abbott FPIA measurements of carbamazepine. Measurements in the group with highest positive bias were produced by Roche FPIA reagents for phenytoin, phenobarbitone, and carbamazepine. Chromatographic and turbidimetric techniques performed satisfactorily. The variable performance of the different reagent/analyzer combinations demonstrates the value of the narrower technique classification in the assessment of assay performance.

External quality assessment of Syva Emit and Abbott TDx II assays for methotrexate in serum.

The Syva Emit and Abbott TDx II kits for determination of methotrexate in serum were compared using data from 14 samples distributed by the United Kingdom National External Quality Assessment Scheme to a mean of 38 European laboratories. For methotrexate concentrations above 0.2 mumol/L, there was no significant difference in the bias or coefficient of variation of measurements between the two techniques. A significantly greater number of measurements by Syva Emit (6.9%) were rejected as outliers > 3 SD from the sample mean compared with Abbott TDx (1.3%). The lower sensitivity of the Syva Emit assay was evident in data reported for samples containing methotrexate concentrations below 0.2 mumol/L.

Time course of appearance of cotinine in human beard hair after a single dose of nicotine.

The time course of appearance of cotinine in saliva and in daily collections of beard clippings was monitored by gas chromatography with mass spectrometry in six volunteers who were nonsmokers following a single 30-min buccal administration of nicotine as a chewing gum. Salivary cotinine concentrations reached a plateau after 1.5 h and were nondetectable (< 0.3 ng/ml) 24 h after drug administration. Cotinine was detected in extracts of sodium hydroxide digests of beard hair in all subjects on the third day after drug administration. Mean concentrations in beard peaked on day 5, and cotinine was not detected (< 0.03 ng/mg) after day 7. The data indicate the main route for cotinine incorporation into beard is during hair growth. Transfer into beard from sweat is of little importance. A role for transfer via sebum is equivocal.

Phenobarbital in newborns with neonatal seizures. A study of plasma levels after intravenous administration.

Phenobarbital plasma levels were studied in a group of 25 newborn infants. Phenobarbital was administered i.v. in all cases throughout the study period. The mean loading dose was 19.4 mg/kg, ranging from 16.4 to 20.5, and the mean maintenance dose was 4.0 mg/kg/day, varying from 2.6 to 5.0. We obtained mean plasma levels of 22.9 micrograms/ml, 24 h after administering the loading dose. Mean plasma levels at 4, 7, 14 and 21 days were in the therapeutic range (15-40 micrograms/ml), with only a few cases falling outside of it. There was no difference in plasma phenobarbital levels between term and pre-term infants. Side effects were not seen in infants without a severe neurological impairment prior to drug administration.

Intra- and interlaboratory sources of imprecision in drug measurements by different techniques.

We compared the intra- and interlaboratory precision of seven techniques used to measure eight antiepileptic drugs, digoxin, and theophylline by using data from the international Healthcontrol external quality-assessment scheme. Scheme participants were supplied blind with 6 or 12 sets of duplicate lyophilized serum samples. Each set contained different drug concentrations, and duplicates were analyzed separately, 1 to 6 months apart. The intra- and interlaboratory components of assay variance were isolated and compared by Bartlett's test for homogeneity of variance. Fluorescence polarization immunoassay (Abbott) showed the best overall intra- and interlaboratory performance for a range of analytes. The largest intralaboratory errors were produced by techniques using the Syva EMIT assays. Our analysis of the data shows that for most analyte/technique combinations, intralaboratory sources of variation were more important than interlaboratory sources. Gains in assay precision will therefore result from attention to internal laboratory procedures.

Performance of techniques for measurement of therapeutic drugs in serum. A comparison based on external quality assessment data.

Ten assay techniques were compared using measurements of a range of 15 drugs spiked in freeze-dried samples of serum reported to the Heathcontrol External Quality Assessment Scheme between November 1988 and January 1991. Three measures of performance were studied: frequency of outliers greater than 3 standard deviations from the sample mean, the coefficient of variation (CV) of sample measurements, and the difference of the sample mean from the spike value. The most consistently precise technique was polarisation fluoroimmunoassay (PFIA). It was in the group of techniques producing significantly fewer outliers and lower CVs than other techniques for all its target analytes. However, a specific interaction with the animal serum used as sample matrix resulted in significant negative bias in PFIA measurements of carbamazepine. Other immunoassay techniques and high-performance liquid chromatography also performed well for a range of analytes, in most cases giving less than 6% of outliers with CVs of less than 13% and less than 5% bias. The least satisfactory techniques were nephelometry and gas-liquid chromatography with derivatisation, which for several analytes gave significantly more outliers and higher CV values than other techniques. In samples containing carbamazepine-10, 11-epoxide, immunoassay measurements of carbamazepine showed cross-reactivity with the epoxide metabolite of between 7 and 15%.

Cognitive functions of epileptic patients on monotherapy with phenobarbitone and healthy controls.

Quantitative measurements have indicated that heredity, cerebral damage, psycho-social aspects, ictal and inter-ictal phenomena and antiepileptic drugs may interfere in the cognitive dysfunction of epileptic patients. In the present study objective methods included immediate and late recall and recognition of pictures, Stroop test and auditory selection. Twenty patients with symptomatic localized epilepsy aged 17-52 years (27 +/- 10, mean +/- sd) were compared to age and socially matched healthy controls. Patients were on therapeutic serum concentrations (25 +/- 12 mu/ml) of phenobarbitone and had active epilepsy with 1.94 generalized tonic-clonic, 0.85 simple partial and 6.28 complex partial seizures monthly (means). Patients performed worse than controls in all 6 tests (p less than 0.05 to p less than 0.001), indicating a generalized cognitive deficit related to seizures and/or barbiturate therapy. We suggest further studies should be carried out in populations with uniform monotherapeutic regimens and epileptic syndromes in order to isolate factors related to the cognitive dysfunction of epileptic patients.

Determination of vigabatrin in plasma by reversed-phase high-performance liquid chromatography.

A method is described for the determination of vigabatrin in 50 microliters of plasma by isocratic high-performance liquid chromatography using fluorescence detection. The procedure involves protein precipitation with methanol followed by precolumn derivatisation with o-phthaldialdehyde reagent. Separation of the derivatised vigabatrin was achieved on a Microsorb C18 column using a mobile phase of 10 mM orthophosphoric acid:acetonitrile:methanol (6:3:1) at a flow rate of 2.0 ml/min. Assay time is 15 min and chromatograms show no interference from commonly coadministered anticonvulsant drugs. The total analytical error within the range of 0.85-85 micrograms/ml was found to be 7.6% with the within-replicates error of 2.76%. The minimum detection limit was 0.08 micrograms/ml and the minimum quantitation limit was 0.54 micrograms/ml.

Comparison of human, bovine, and newborn calf serum in the preparation of external quality assurance samples for therapeutic drugs.

The accuracy and precision of drug measurements made by different analytical techniques of a range of eight antiepileptic drugs, four tricyclic antidepressants, theophylline, and digoxin in three different matrices, human, bovine, and newborn calf serum, were compared using data reported to the Heathcontrol External Quality Assurance Scheme over 18 months. Neither of the animal serum samples was universally satisfactory compared with human serum. The newborn calf serum sample produced results that were closer to the spiked drug concentrations than those in bovine serum for most of the analytes covered by the survey. Both animal samples were unsuitable for digoxin, for which the use of scarce human material is recommended.

External quality assurance of tricyclic antidepressant measurements in serum: eight years of progress?

Comparison of the precision and accuracy of measurements made between December 1979 and January 1988 of tricyclic antidepressant drug concentrations in freeze-dried external quality-assurance samples of human serum showed only occasional significant differences between the different chromatographic techniques used for drug assay. Larger differences between data collected in different years correlated with changes in the source of the sample matrix and the personnel responsible for sample preparation. Measurements of amitriptyline and imipramine were more precise than those of nortriptyline and desipramine; the mean coefficients of variation of measurements were 20.2, 19.6, 26.6 and 25.3%, respectively. The data indicate the need for further interlaboratory studies directed at reducing the high level of variability in tricyclic measurements.

Comparison of nonisotopic and radioimmunoassay techniques for digoxin: a study based on external quality assurance measurements.

A comparison of the precision and accuracy of three nonisotopic and of radioimmunoassay (RIA) techniques for digoxin was made using data from 141 samples of spiked human serum assayed between 1984 and 1987 by members of the Heathcontrol external quality assurance scheme. There were significant differences between techniques in the number of observations rejected as outliers greater than 3 SD from the sample mean, the percentages of rejected measurements being Abbott TDX 1.4, RIA 3.3, Ames TDA 4.2, and Syva EMIT 6.3. Ames TDA and Syva EMIT were shown to have a significantly lower precision than the other techniques in measurements of digoxin concentrations less than 1 nmol/L. A significant 4% negative bias was observed for Ames TDA measurements of digoxin concentrations between 1 and 2.6 nmol/L. A concentration-related bias was demonstrated for Abbott TDX measurements that varied from 14% overestimates at concentrations less than 1 nmol/L to 6.7% underestimates of digoxin at concentrations greater than 2.6 nmol/L.

Evaluation of assay techniques for the measurement of antiepileptic drugs in serum: a study based on external quality assurance measurements.

The accuracy and precision of eight techniques used to measure a range of eight antiepileptic drugs in human serum were compared using data from 80 samples from the Heathcontrol External Quality Assurance scheme. The fluorescence polarization immunoassay was significantly more precise than other techniques for several analytes, producing the lowest number of measurements rejected as outliers and measurements with the lowest coefficient of variation. Other techniques had a significantly lower precision. Nephelometry (Neph) produced most outliers for phenytoin and carbamazepine and had the highest variability for phenytoin. Gas-liquid chromatography (GLC) with derivatization was most variable for phenobarbitone and primidone. Measurements by GLC with or without derivatization contained greater than 10% of outliers and were least precise for carbamazepine. Differences between the majority of techniques were in many cases, however, not significant. The accuracy of techniques was assessed from differences between sample means and spiked drug concentrations. Neph was notable in producing overestimates at lower concentrations. Immunoassay methods had a 16-21% cross-reactivity with carbamazepine 10,11-epoxide when measuring carbamazepine. All techniques reported underestimates for valproic acid that were thought to result from the hygroscopic nature of the salt used for spiking.

Evaluation of chromatographic and kit immunoassay techniques for the measurement of theophylline in serum: a study based on external quality assurance measurements.

The accuracy and precision of assay techniques used to measure theophylline concentrations in human serum were compared using data from 96 samples from the Heathcontrol external quality assurance scheme. Abbott TDX had the highest precision, with a mean coefficient of variation (CV) of measurements of 6.4%, and Ames Fluorostat was most accurate, with a mean bias of +0.1%. Differences between the better techniques, however, were not significant. The Beckman ICS assay gave the lowest precision (CV 9.3%) and, in addition, produced the highest proportion (7.5%) of rejected observations greater than 3 standard deviations from the sample mean. The least accurate method was radioimmunoassay, with a -9.9% bias. Measurements from two samples spiked with paraxanthine demonstrated that 76% of laboratories using high-pressure liquid chromatography (HPLC) were unable to distinguish between paraxanthine and theophylline. The +4% bias observed at low concentrations and the high variability (CV 8.3%) of measurements made by HPLC were thus attributed to interference by paraxanthine present in the sample matrix. Discriminant analysis of a range of HPLC column and mobile phase parameters indicated that separation of theophylline and paraxanthine was achieved by the use of lower flow rates with mobile phases of solvent mixtures with lower proton donator selectivity.

[Loading-doses of carbamazepine and diphenylhydantoin: use in high-risk patients]. / Doses-carga de carbamazepina e difenil-hidantoína: utilização em pacientes de alto risco.

Loading-doses of phenytoin (1000 mg) and carbamazepine (600 mg) were given orally respectively to 10 and 6 patients with uncontrolled epileptic seizures secondary to acute neurological disorders or alcohol withdrawal. In the phenytoin group age varied between 12-73 years and serum concentrations at 2, 4, 6, 8, 12 and 18 hours after drug administration were 7.6, 8.8, 8.7, 8.7, 7.2 and 6.5 micrograms/ml (means). A quantitative method did not detect important side-effects. In the carbamazepine group age varied between 25-56 years and serum concentrations at the same times were 3.9, 5.3, 6.5, 7.5, 7.4 and 8.2 micrograms/ml. Side-effects were discrete. Further medication was not necessary in the 24 hours after drug administration. Although both regimens controlled the clinical situation without relevant side-effects serum concentrations were sub-therapeutic in the case of phenytoin. We suggest the ideal phenytoin oral loading-dose is 1500 mg. The carbamazepine load produced therapeutic concentrations. The stability of serum concentrations for the period of the study shows that those regimens are useful in the subacute control of epileptic seizures in the maintenance treatment of status epilepticus and in alcohol withdrawal.

Pharmacokinetics of antipyrine in epileptic patients.

The pharmacokinetics of antipyrine were examined after oral and intravenous administration to 20 epileptic subjects receiving antiepileptic drug therapy. Bioavailability was essentially complete (mean bioavailability 101.2% +/- 14.4 (s.d.] indicating that even in enzyme induced subjects, antipyrine behaves as a restrictively eliminated compound with negligible presystemic elimination in the gut or liver. Of the generally used measures of enzyme induction (oral clearance, oral half-life and intravenous half-life) oral clearance was the most closely related to the intravenous clearance of antipyrine (r = 0.919, P less than 0.001). Oral antipyrine administration is an alternative to intravenous administration in epileptic subjects who are enzyme-induced.

Calcium homeostasis in mentally handicapped epileptic patients.

Three groups of subjects have been studied to evaluate the role of long term hospitalization and chronic anti-epileptic therapy on calcium metabolism. The first group consisted of 32 epileptic patients, randomly selected from a population of inpatients in a hospital for the mentally handicapped, receiving various combinations of anti-epileptic drugs for at least 3 years. The second group was made up of 32 non-epileptic residents of the same hospital, individually matched for age and sex with epileptic patients and who had not received any anti-epileptic drugs in the last 3 years. The third group of 22 normal subjects was randomly selected from the staff of the University Hospital of Wales, matched for age and sex against the epileptic group, who had not received any anti-epileptic medication within the last 3 years. None of the subjects received any drugs (except anti-epileptic drugs) known to have effect on calcium metabolism. Significant differences in the serum levels of the total calcium, ionised calcium, total alkaline phosphatase and its liver iso-enzyme were seen. Serum total alkaline phosphatase and its liver iso-enzyme were significantly elevated in the epileptic group, showing the effect of anti-epileptic drugs. On the other hand serum total calcium was significantly lower in both residential groups compared to the normal population, epileptics being lower than non-epileptics showing the combined effect of hospitalization and anti-epileptic drugs. No significant difference was detected among the groups in the serum concentration of the bone alkaline phosphatase iso-enzyme.