Lolita Tsanaclis

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

Summary: A method is described for the determination of vigabatrin in 50 μl 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 μg/ml was found to be 7.6% with the within-replicates error of 2.76%. The minimum detection limit was 0.08 μg/ml and the minimum quantitation limit was 0.54 μg/ml.

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

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%.

Workplace drug testing, different matrices different objectives

Drug testing is used by employers to detect drug use by employees or job candidates. It can identify recent use of alcohol, prescription drugs, and illicit drugs as a screening tool for potential health and safety and performance issues. Urine is the most commonly used sample for illicit drugs. It detects the use of a drug within the last few days and as such is evidence of recent use; but a positive test does not necessarily mean that the individual was impaired at the time of the test. Abstention from use for three days will often produce a negative test result. Analysis of hair provides a much longer window of detection, typically 1 to 3 months. Hence the likelihood of a falsely negative test using hair is very much less than with a urine test. Conversely, a negative hair test is a substantially stronger indicator of a non-drug user than a negative urine test. Oral fluid (saliva) is also easy to collect. Drugs remain in oral fluid for a similar time as in blood. The method is a good way of detecting current use and is more likely to reflect current impairment. It offers promise as a test in post-accident, for cause, and on-duty situations. Studies have shown that within the same industrial settings, hair testing can detect twice as many drug users as urine testing.

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.

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 >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.

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.

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 >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.

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.

Commentary on current changes of the SoHT 2016 consensus on alcohol markers in hair and further background information.

The consensus on alcohol markers in hair was revised for the fourth time by an expert group of the Society of Hair Testing based on current state of research. This revision was adopted by the members of the Society during the business meeting in Brisbane on August 29th 2016. For both markers, ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs), two cut-off values for discrimination between teetotalers or occasional low amount consumption and moderate alcohol drinking (low cut-off), and between non-excessive (abstinence up to moderate alcohol intake) and chronic excessive drinking (high cut-off value) were critically examined. For the current revision, the cut-off values for EtG (7pg/mg and 30pg/mg, respectively) remained unchanged despite different findings or discussions published in the meantime. This was mainly due to the lack of broader data collections from new studies with great numbers of volunteers following thorough study concepts. In contrast, an essential change of the consensus was accepted for the FAEEs, where the concentration of ethyl palmitate (E16:0) can be used autonomously for interpretation instead of the concentration sum (ΣFAEE) of the four esters ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate, as previously applied. After evaluation of the data from seven laboratories, the E16:0 cut-off for abstinence assessment was defined at 0.12ng/mg for the 0-3cm segment and at 0.15ng/mg for the 0-6cm segment. The cut-off for chronic excessive drinking was fixed at 0.35ng/mg for the 0-3cm segment and at 0.45ng/mg for the 0-6cm segment. The use of E16:0 with these cut-offs in place of ΣFAEE for alcohol intake assessment produces only a minor loss in discrimination power, leads to no essential difference in the interpretation concerning chronic excessive alcohol consumption and is suitable to confirm EtG results in abstinence assessment if ethanol containing hair sprays or lotions are excluded.