Kaarina Langel

A validated method for the detection and quantitation of 50 drugs of abuse and medicinal drugs in oral fluid by gas chromatography-mass spectrometry.

Oral fluid is of increasing interest as an alternative sample matrix in drugs of abuse analysis. Compared to the conventional sample matrix blood, sample volumes of oral fluid are smaller and the concentrations of some drugs can be much lower. This imposes some restrictions on the analysis method, which has to be able to detect and quantify multiple analytes from a small sample volume at low concentrations. A sensitive multi-component method for quantitative determination of 50 drug compounds from oral fluid samples collected with the StatSure SalivaSampler™ device was developed. The compounds analysed include cannabis, cocaine, amphetamines, opioids, benzodiazepines and other psychoactive medicines. Both liquid-liquid-extraction (LLE) and solid-phase-extraction (SPE) are employed in the sample pre-treatment and the samples are analysed using gas chromatography-mass spectrometry (GC-MS) with the mass selective detector (MSD) operating in either electron ionization (EI) or negative-ion chemical ionization (NICI) mode. The method was fully validated, and the validated parameters included linearity, selectivity, accuracy, precision, and extraction efficiency. Stability of the collected samples during storage at -18°C was also studied, and even after over a years storage all analyte concentrations were more than 60% of the original concentrations. The described method is suitable for routine analysis of oral fluid samples and it has been applied to analysis of more than 4000 oral fluid samples collected anonymously from volunteer road users in Finland during 2007-2009 as a part of the EU project DRUID (Driving under the Influence of Drugs, Alcohol and Medicines). At the moment the developed method is the most comprehensive validated analysis method for oral fluid samples.

Comparison of drug concentrations between whole blood and oral fluid

The relationship of drug concentrations between oral fluid and whole blood was evaluated by studying the linear correlation of concentrations and calculating the oral fluid to blood concentration ratios (OF/B) for different substances. Paired oral fluid and whole blood samples were collected from volunteers and persons suspected of drug use in four European countries. Oral fluid samples were collected with the Saliva∙Sampler™ device. All samples were analyzed for drugs of abuse and psychoactive medicines with validated gas and liquid chromatography-mass spectrometric methods. The median OF/B ratios were, for amphetamines 19-22, for opioids 1.8-11, for cocaine and metabolites 1.7-17, for tetrahydrocannabinol (THC) 14, for benzodiazepines 0.035-0.33, and for other psychoactive medicines 0.24-3.7. Most of the these results were close to theoretical values based on the physicochemical properties of the drugs and to values presented earlier, but there was a lot of inter-individual variation in the OF/B ratios. For all substances, except for lorazepam (R(2)  = 0.031) and THC (R(2)  = 0.030), a correlation between the oral fluid and whole blood concentrations was observed. Due to large variation seen here, drug findings in oral fluid should not be used to estimate the corresponding concentrations in whole blood (or vice versa). However, detection of drugs in oral fluid is a sign of recent drug use and oral fluid can be used for qualitative detection of several drugs, e.g. in epidemiological prevalence studies. By optimizing the sampling and the analytical cut-offs, the potential of oral fluid as a confirmation matrix could be enhanced.

Alcohol and drugs in seriously injured drivers in six European countries

The objective of this study was to determine the presence of alcohol and drugs in drivers severely injured in traffic crashes in six European countries. Data were collected from 2492 seriously injured drivers of cars and vans in Belgium, Denmark, Finland, Italy, Lithuania, and the Netherlands, between 2007 and 2010. Toxicological analysis was performed with chromatographic techniques on whole blood for 23 substances. The percentage of drivers positive for at least one psychoactive substance ranged between 28% (Lithuania) and 53% (Belgium). Alcohol (≥0.1 g/L) was the most common finding with the highest percentage in Belgium (42.5%). Among the alcohol-positive drivers, 90.5% had a blood alcohol count (BAC) ≥0.5 g/L and 65.7% had a BAC ≥1.3 g/L. Benzodiazepines (0.0-10.2%) and medicinal opioids (0.5-7.8%) were the most prevailing medicinal drugs, but half of the concentrations were lower than therapeutic. Cannabis (0.5-7.6%) was the most prevailing illicit drug. Alcohol was found in combination with drugs in 2.3-13.2% of the drivers. Drug combinations were found in 0.5-4.3% of the drivers. This study confirms the high prevalence of psychoactive substances in injured drivers, but we observed large differences between the participating countries. Alcohol was the most common finding, followed by cannabis and benzodiazepines. Notable are the many drivers having a BAC ≥ 1.3 g/L. The majority of the substances were found in combination with another psychoactive substance, mostly alcohol. The high prevalence of high BACs and combinations (compared to roadside surveys) suggest that those drivers are most at risk and that preventive actions should target them preferentially.

Estimation of Equivalent Cutoff Thresholds in Blood and Oral Fluid for Drug Prevalence Studies

Oral fluid is an easily available specimen for studying drug use in a cohort or population. The prevalence of drugs in samples of oral fluid is the same as the prevalence in blood if using equivalent cutoff concentrations. The cutoffs in oral fluid may be higher or lower than that in blood in accordance with the median oral fluid-to-blood (OF/B) concentration ratio, but it is also influenced by the skewness of the distribution of OF/B ratios. The aim of this study was to determine formulae for the estimation of equivalent cutoff concentrations in oral fluid and blood for 12 commonly used illegal and medicinal psychoactive drugs when oral fluid was collected with Statsure Saliva·Sampler™. Paired samples from 4,080 persons were collected and analyzed with chromatographic methods and mass spectroscopic detection. Regression formulae for the concentrations corresponding to selected percentiles in oral fluid versus the same concentration percentiles in blood were determined. The accuracy when multiplying the cutoff thresholds in blood with the average and median OF/B ratios to estimate equivalent cutoffs in oral fluid was also investigated. Prevalence regression gave the most accurate results. The regression formulae can be used to estimate equivalent cutoff concentrations in oral fluid and blood.

Detection of illicit drugs in oral fluid from drivers as biomarker for drugs in blood.

AIM To assess whether analysis of oral fluid can be used to identify individual drivers with drug concentrations in blood above 25ng/mL for amphetamine and methamphetamine, 10ng/mL for cocaine and 1.0ng/mL for tetrahydrocannabinol (THC), which are the cut-off concentrations used in the European DRUID Project, by calculating the diagnostic accuracies when using the analytical cut-off concentrations in oral fluid as well as for the optimal cut-off concentrations. METHODS Paired samples of whole blood and oral fluid collected with the Statsure SalivaSampler were obtained from 4080 drivers in four European countries and analysed for amphetamine, methamphetamine, cocaine and THC using GC-MS or LC-MS. The vast majority (89%) were random drivers not suspected of drug-impaired driving. Receiver-Operating Characteristic analysis was used to evaluate the analytical results. RESULTS The prevalence of drug findings above the cut-off concentrations in blood was 1.3% for amphetamine, 1.0% for methamphetamine, 0.6% for cocaine and 1.3% for THC. The cut-off concentrations in oral fluid that gave the highest diagnostic accuracy were for amphetamine 130ng/mL (accuracy 99.8%), methamphetamine 280ng/mL (accuracy 99.9%), cocaine 570ng/mL (accuracy 99.6%), and THC 38ng/mL (accuracy 98.3%). The proportion of false positives were 0.2%, 0.1%, 0.1% and 0.9%; and the proportion of false negatives were 0.1%, 0.0%, 0.3% and 0.8%, respectively, when using those cut-offs. The positive predictive values were 87.9%, 92.9%, 84.6% and 35.7% for amphetamine, methamphetamine, cocaine and THC, respectively. CONCLUSIONS Analysis of concentrations of illicit drugs in oral fluid could not be used to accurately identify drivers with drugs concentrations above the selected cut-offs in blood in a cohort of drivers with low prevalence of drugs.

Detection of 4 benzodiazepines in oral fluid as biomarker for presence in blood.

Background: Analysis of samples of oral fluid (mixed saliva) is increasingly being used to detect recent drug use. The aim of this investigation was to assess the suitability of testing oral fluid as a biomarker for the presence of 4 benzodiazepines in blood and its possible application in clinical settings and in research on drug use. Methods: Paired samples of oral fluid and blood from 4080 individuals in 4 European countries were collected and analyzed for benzodiazepines using gas or liquid chromatography with mass spectroscopic detection. Results: Concentration data for the 4 most commonly detected benzodiazepines were studied: alprazolam, clonazepam, diazepam, and nordiazepam. Large variations in oral fluid to blood concentration ratios were observed for the studied benzodiazepines. The interquartile ranges for the oral fluid to blood concentrations ratios corresponded to 88%–197% of the median values. Selecting cutoff concentrations in oral fluid that gave the best accuracy in identifying individuals with benzodiazepine concentrations in blood above chosen thresholds produced accuracies of 74%–85% and the fraction of false negatives was 9%–23%. Conclusions: The concentration of the 4 studied benzodiazepines in oral fluid can neither be used to accurately estimate the concentrations in blood nor to correctly identify patients with blood drug concentrations below or above recommended therapeutic levels. When using analytical methods with limits of quantitation corresponding to concentrations less than 0.5 ng/mL in undiluted oral fluid, it may be used to confirm a recent intake of benzodiazepines. However, it is likely that some false negatives may occur.