Drugs of abuse in oral fluid collected by two different sample kits – Stability testing and validation using ultra performance tandem mass spectrometry analysis
Introduction
Oral fluid (OF) has gained focus as an alternative matrix for monitoring drugs of abuse in workplace testing, clinical toxicology, criminal justice, and driving under the influence of drugs programs (DUID) [1], [2]. OF is suitable for detection of drugs that have been taken recently and is easily available for collection. Sample adulteration is more difficult compared to urine, as it is unproblematic to observe the collection process [3]. Both drugs and their metabolites can be detected in OF. The volume of the collected samples is often less than 1 mL, therefore multicomponent methods with low detection limits are needed [2], [4]. Developments within analytical technology using liquid chromatography with tandem mass spectrometry (LC–MS/MS) have made it possible to simultaneously quantify several drugs at low concentrations in OF [5], [6], [7], [8], [9]. Ultra performance liquid chromatography (UPLC) has been introduced as a replacement for high performance liquid chromatography (HPLC) with potential of faster analysis, less solvent consumption, and improved resolution [10], [11], [12], [13], [14], and is considered as a promising technique for analysis of small volumes of OF [15], [16], [17].
We have previously used a method for detection of 32 drugs in OF with HPLC coupled with tandem mass spectrometry (MS/MS) [18]. The run time was 14 min and several components co-eluates. Switching to UPLC could give shorter analysis time and similar or better separation of the drugs. Badawi et al. has developed an UPLC-method for screening and quantification of 29 drugs in OF [15]. The run time was 20 min and satisfactory separations were achieved. We wanted to develop a UPLC-method for screening of drugs in OF with a shorter run time to facilitate analysis of larger volumes of samples. Two other UPLC–MS/MS methods with run times of approximately 8 min have been published [17], [19]. These do however have smaller repertoire lacking e.g., benzodiazepines [19] and THC [17]. Sample preparation only by dilution as presented in these methods might in addition give long term instrumental problems as commercial sampling kits contain preservatives, surfactants and in some cases dyes, which might build up instrumental back-ground noise if not removed.
The suitability of 9 different commercial sampling kits for collecting samples for drug analysis have been evaluated by Langel et al. [20]. In this paper we have used two of these sampling kits, Intercept® and StatSure Saliva Sampler™, in the development of the analysis method. We also wanted to study the influence of different short term storage conditions and stability during storage past the 28 days tested in the paper by Langel et al. and in addition include more substances.
Section snippets
Chemicals, reagents and materials
The reference compounds were obtained from several companies. 3,4-Methylenedioxyamphetamine (MDA) and 3,4-ethylenedioxyethylamphetamine (MDEA) were obtained from Alltech (Lexington, KY, USA); 3,4-methylenedioxymethamphetamine (MDMA), lysergic acid diethylamide (LSD), Δ9-tetrahydrocannabinol (THC), 3,4-methylenedioxymethamphetamine-d5, 7-aminoflunitrazepam-d7, amphetamine-d11, benzoylecgonine-d8, buprenorphine-d4, clonazepam-d4, cocaine-d3, methadone-d3, methamphetamine-d11 and nordiazepam-d5
Identification and quantitation
The retention times are shown with chromatograms in Fig. 1 and the MRM transitions are shown in Table 2. Several of the components co-eluates, but the separations were satisfactory because of the variation in m/z for the compounds. The 32 components and their internal standards were separated in less than 7 min.
Calibration curves
The average values of R2 are shown in Table 3. All of the components except for 6-MAM, codeine, benzoylecgonine, zopiclone, cocaine, 7-AN, meprobamat, LSD, zolpidem, carisoprodol and
Conclusions
The developed screening method is intended for determination of drugs of abuse in OF. 32 compounds were analysed in 7 min (cycle time 9 min), a significant reduction from our previous method. The stability testing showed that 6-MAM, cocaine, and zopiclone were unstable the first week of the testing. The results were in accordance with earlier studies, and there was no significant difference in the long term stability in the two sampling kits we tested. In the testing for stability at short term,
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