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Occupational Drug Screening using High Resolution Accurate Mass LCMS Simon Hudson, Bob Gray, Jeremy Cook. HFL Sport Science, LGC Health Sciences, Fordham, UK Overview Historically, workplace drug testing has been based on immunoassay technology operating on large clinical analysers. The advantages to this have been low cost, high throughput and adequate performance in terms of drug coverage and sensitivity. The main disadvantages have been the availability of tests and, more recently, the ability to keep up with the emergence of new ‘legal’ highs. A recent phenomenon is that of syn- thetic cannabinoids, otherwise known as ‘Spice’. Reagents are available for clinical analyser based screening but the coverage of the 70 or more potential compounds is very limited. The current availability of robust and sensitive high resolution accurate mass (HRAM) LCMS instrumentation has presented an opportunity to deliver a truly fit for purpose workplace test that can cover a wide range of analytes and that is easy to add new analytes to. The application of this technology to deliver an ‘Enhanced’ workplace test is described. Methods A batch of samples for analysis for ‘designer’ drugs was submitted to LGC Health Sciences for ‘workplace drug testing’. The routine workplace analysis using Olympus AU2700 clinical analysers with standard test kits was performed on each sample. The same samples were also submitted for the ‘Enhanced Workplace Test’ as follows. Beta glucuronidase hydrolysed urine samples were prepared for analysis using a multi eluate sold phase extraction methodology to prepare both an acid/neutral and a basic fraction. The extracts were then combined, with a portion of the acid/ neutral fraction being retained in case of any requirement for anabolic steroid testing. The combined extracts were analysed using full scan HRAM LCMS on a Thermo LTQ Orbitrap operating at a resolution of 30,000 FWHM in positive ion electrospray mode. The chromatography was performed on a ballistic acetic acid/acetonitrile gradient using a Water Atlantis T3 column. Starting conditions were 100% aqueous ending with 98% organic in a 5 minute analysis. The resulting data file was processed using Thermo ExactFinder software against a database of over 1600 analytes using accurate mass, retention time, isotopic profiling and in source generated qualifier fragment ions as determinands. The same protocol was also employed on a study of drug use in the night time economy of central London. Temporary urinals (pissoirs) are placed in strategic positions in city centres to provide toilet facilities for late night visitors, typically on a Saturday night. These are emptied on a Sunday morning and at this point a sample is retained for analysis Results Workplace Tests A comparison of results from the ‘Conventional’ and ‘Enhanced’ workplace test for this batch of samples shows a large increase in findings in the LCMS analysis. Samples found positive for cocaine on the conventional test were also positive for cocaine (as benzoylecgonine) on the enhanced screen. Other metabolites (hydroxy cocaine, ecgonine methyl ester, ecgonine ethyl ester) were also identified by LCMS. Four samples were positive for pentedrone using the enhanced test. Pentedrone alone, as in Sample13 did not trigger a positive in the conventional test. Likewise, 4-methylmethcathinone, fluoroephedrine, benzylpiperazine, trifluoro- methylphenylpiperazine and methylhexanamine did not generate a positive result on the clinical analyser. 4-methylmethcathinone was detected in the conventional workplace test for sample 12. A positive response was seen on the amphetamine immunoassay. The GCMS follow up to the immunoassay detected the presence of 4-methylmethcathinone. This however was anomalous with the fact that a 4-methylmethcatrhinone standard at high concentration does not give a response on the amphetamine test. In addition, none of the other 4-methylmethcathinone samples were positive on the clinical analyser. The cause of the initial positive on the amphetamine test is therefore unknown and the finding of 4-methylmethcathinone in sample 12 is by pure chance. City Centre Samples The analysis of the samples from the city centre urinals identified a large number of ‘normal’ prescription or over the counter drugs. In addition, most samples contained large amounts—in the 100’s ng to low µg/ml range— of ketamine, benzoylecgonine, amphetamine and MDMA. Nearly 150 drugs were detected, either as parent compound or metabolite. Other findings of ‘legal high’ type compounds to date include:- cathinone variants including:cathinone, methcathinone, 4-methylmethcathinone, 4-methylethcathinone and beta ethyl methcathinone (pentedrone) piperazines including 1,4 trifluoromethylphenylpiperazine, benzylpiperazine and methylphenylpiperazine other analytes including 4-fluorephedrine 6-APB, methoxetamine and methiopropamine. Other related analytes have been seen, including various cutting agents such as levamisole and benzocaine. Conclusions Current available information demonstrates the rapid growth in the availability and use of substances collectively known as ‘legal highs’. This in turn presents a challenge to the delivery of effective meaningfull workplace drug tests. The comparison of traditional immunoassay/clinical analyser based workplace drug testing with a broad coverage LCMS approach demonstrates clearly that the traditional approach is severely limited in terms of drug coverage. The use of high resolution accurate mass LCMS delivers several performance advantages:- Broad Analyte Coverage—demonstrated by the analysis of city centre pooled urine samples. Rapid Response—it is very easy to add new analytes to data processing databases to extend drug coverage. No modification to instrument methodology is required. High Selectivity—first pass identification of drug compound is usually possible. Reduced False Positives—due to high selectivity of primary HRAM LCMS screen. Reduction in wasted confirmation work. Reduced False Negatives—increased coverage hence less chance of missing ‘positive’ samples High Sensitivity—method capability into the low ppb and high ppt ranges. Possible drawbacks to the use of this approach include the cost and the need for high throughput. References The use of high resolution accurate mass spectrometry for rapid throughput, multi -drug screening, S. Hudson, S. Maynard and S. Timbers, Proceedings of the 18th In- ternational Conference of Racing Analysts and Veterinarians, New Zealand 2010 Analysis of anonymous pooled urine from portable urinals in central London con- firms the significant use of novel psychoactive substances, Archer JR, Dargan PI, Hudson S, Wood DM., QJM. 2013 Feb;106(2):147-52. doi: 10.1093/qjmed/hcs219. Epub 2012 Nov 22 Taking the Pissoir – a novel and reliable way of knowing what drugs are being used in nightclubs, Archer, J. R. H,, Dargan, P. I., Hudson, S., Davies, S., Puchnarewicz, M., Kicman, A. T., Ramsey, J., Measham, F., Wood, M., Johnston, A., Wood, D. M. Journal of Substance Use. Posted online on May 6, 2013. (doi:10.3109/14659891.2012.740139) Introduction Standard urine-based occupational drug screening is a two-stage process, involv- ing initial immunoassay screening to identify possible positive samples followed by more detailed and targeted gas or liquid chromatography coupled with mass spectrometry to confirm and quantify the materials present. This combination provides a cost-effective means of conducting large scale testing, but means that materials which do not trigger the initial immunoassay will not be detected. Current urine-based workplace drug testing schemes cover a relatively limited range of illicit substances which can be broken down into the following groups :- Opiates Amphetamines Benzodiazepines Cannabis Cocaine Other targeted analytes depending on availability of kits There is limited coverage for many of the designer drugs and so called ‘legal highs’ that are now widely abused, and due to the nature of the testing, conventional workplace drug testing will always struggle to keep up with the latest tastes. Recent data from Europe (EMCDDA) shows the scale of the problem and how the popularity/availability of different drug classes changes year on year. 0 10 20 30 40 50 60 2008 2009 2010 2011 New Psychoactive Substances Reported in Europe - source EMCDDA Sample No. Conventional Work- place Test Enhanced Workplace Test Sample 1 4 methylmethcathinone/fluorephedrine Sample 2 negative Sample 3 Cocaine pentedrone/benzoylecgonine Sample 4 Cocaine pentedrone/benzoylecgonine Sample 5 4 methylmethcathinone/fluorephedrine/ trifluoromethylphenylpiperazine/benzylpiperazine Sample 6 Cannabis 4-methylmethcathinone Sample 7 methylhexanamine Sample 8 negative Sample 9 negative Sample 10 4-methylmethcathinone Sample 11 Cocaine Pentedrone/trifluoromethylphenylpiperazine/benzoylecgonine Sample 12 4-methylmethcathinone 4 methylmethcathinone/4-methyl ethcathinone/fluorephedrine/ trifluoromethylphenylpiperazine/benzylpiperazine Sample 13 pentedrone 1,4 methoxyphenylpiperazine chlordiazepoxide gabapentin morphine propranolol 4 methylethcathinone chloroquine guaifenesin Nandrolone pyrimethamine 4-ethylmethcathinone chlorpheniramine HMMA naproxen quetiapine 4-fluoroephedrine Chlorthalidone hordenine nefopam quinine 4-methylmethcathinone cimetidine hydrochlorthiazide nevirapine ranitidine 5/6-APB citalopram Ibuprofen nicotine risperidone ambroxol clenbuterol isometheptene niflumic acid salbutamol amisulpiride clobazam ketamine nordazepam sertaline amitriptylline clomipramine Ketoprofen nortriptyline sildenafil amphetamine clozapine lamotrigine noscapine sotalol ampyrone codeine lansoprazole Olanzapine Stanozolol arecoline cotinine Lidocaine omeprazole sulpiride atenolol cyclobenzaprine MDA opipramol tamoxifen benzocaine DEET MDMA oripavine temazepam benzoylecgonine desloratidine Meclofenamic acid orphenadrine tetracycline benzydamine dextromethorphan mefenamic acid oxazepam tetramisole bisoprolol dextrorphan methadone oxprenolol theobromine bromhexine Diazepam methamphetamine oxycodone theophylline buprenorphine diclofenac methcathinone oxytetracycline tramadol buprenorphine-nor dihydrocodeine methiopropamine papaverine Trazodone caffeine dihydromorphine methoxetamine paracetamol trenbolone camphor diphenhydramine methylhexaneamine paroxetine trifluoromethylphenylpiperazine capsaicin dipyridamole methylphenidate pentedrone trimethoprim carbamazepine doxycycline metoclopramid pheniramine vardenafil Carboxy THC enalapril metoprolol Phenylpropanolamine venlafaxine cathine ephedrine/pseudoephedrine metronidazole Pholcodine xylometazolone cathinone ethylphenidate midazolam pipradrol Yohimbine celecoxib Etoricoxib minoxidil pregabalin zolpidem cetirizine fluoxetine mitragynine prilocaine chlorcylizine furosemide modafinil Propoxyphene
