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Home , Methylhexanamine, Pentedrone

Occupational Drug Screening using High Resolution Accurate Mass LCMS

Simon Hudson, Bob Gray, Jeremy Cook. HFL Sport Science, LGC Health Sciences, Fordham, UK

Overview Methods Conclusions

Current available information demonstrates the rapid growth in the availability Historically, workplace drug testing has been based on immunoassay technology A batch of samples for analysis for ‘designer’ drugs was submitted to LGC Health Four samples were positive for using the enhanced test. Pentedrone and use of substances collectively known as ‘legal highs’. This in turn presents a operating on large clinical analysers. The advantages to this have been low cost, Sciences for ‘workplace drug testing’. The routine workplace analysis using alone, as in Sample13 did not trigger a positive in the conventional test. Likewise, challenge to the delivery of effective meaningfull workplace drug tests. high throughput and adequate performance in terms of drug coverage and Olympus AU2700 clinical analysers with standard test kits was performed on each 4-methylmethcathinone, fluoroephedrine, , trifluoro- The comparison of traditional immunoassay/clinical analyser based workplace sensitivity. The main disadvantages have been the availability of tests and, more sample. methylphenylpiperazine and did not generate a positive result drug testing with a broad coverage LCMS approach demonstrates clearly that the recently, the ability to keep up with the emergence of new ‘legal’ highs. The same samples were also submitted for the ‘Enhanced Workplace Test’ as on the clinical analyser. traditional approach is severely limited in terms of drug coverage. The use of high 60 follows. 4-methylmethcathinone was detected in the conventional workplace test for Beta glucuronidase hydrolysed urine samples were prepared for analysis using a resolution accurate mass LCMS delivers several performance advantages:- 50 A recent phenomenon is that of syn- sample 12. A positive response was seen on the immunoassay. multi eluate sold phase extraction methodology to prepare both an acid/neutral 40 thetic cannabinoids, otherwise known The GCMS follow up to the immunoassay detected the presence of as ‘Spice’. Reagents are available for and a basic fraction. The extracts were then combined, with a portion of the acid/  Broad Analyte Coverage—demonstrated by the analysis of city centre pooled 30 New Psychoactive 4-methylmethcathinone. This however was anomalous with the fact that a Substances Reported in urine samples. Europe - source clinical analyser based screening but neutral fraction being retained in case of any requirement for anabolic steroid 20 EMCDDA 4-methylmethcatrhinone standard at high concentration does not give a response the coverage of the 70 or more testing.  Rapid Response—it is very easy to add new analytes to data processing 10 on the amphetamine test. In addition, none of the other 4-methylmethcathinone potential compounds is very limited. The combined extracts were analysed using full scan HRAM LCMS on a Thermo databases to extend drug coverage. No modification to instrument methodology 0 samples were positive on the clinical analyser. LTQ Orbitrap operating at a resolution of 30,000 is required. 2008 2009 2010 2011 The cause of the initial positive on the amphetamine test is therefore unknown and FWHM in positive ion electrospray mode. The  High Selectivity—first pass identification of drug compound is usually possible. the finding of 4-methylmethcathinone in sample 12 is by pure chance. chromatography was performed on a ballistic  Reduced False Positives—due to high selectivity of primary HRAM LCMS The current availability of robust and sensitive high resolution accurate mass acetic acid/acetonitrile gradient using a Water screen. Reduction in wasted confirmation work. (HRAM) LCMS instrumentation has presented an opportunity to deliver a truly fit City Centre Samples for purpose workplace test that can cover a wide range of analytes and that is Atlantis T3 column. Starting conditions were  Reduced False Negatives—increased coverage hence less chance of missing 100% aqueous ending with 98% organic in a 5 The analysis of the samples from the city centre urinals identified a large number ‘positive’ samples easy to add new analytes to. The application of this technology to deliver an of ‘normal’ prescription or over the counter drugs. In addition, most samples ‘Enhanced’ workplace test is described. minute analysis.  High Sensitivity—method capability into the low ppb and high ppt ranges. contained large amounts—in the 100’s ng to low µg/ml range— of , The resulting data file was processed using Thermo ExactFinder software against a benzoylecgonine, amphetamine and MDMA. Nearly 150 drugs were detected, Possible drawbacks to the use of this approach include the cost and the need for database of over 1600 analytes using accurate either as parent compound or metabolite. high throughput. mass, retention time, isotopic profiling and in

source generated qualifier fragment ions as determinands. Other findings of ‘legal high’ type compounds to date include:- Introduction The same protocol was also employed on a study of drug use in the night time References economy of central London. Temporary urinals (pissoirs) are placed in strategic  variants including:cathinone, , 4-methylmethcathinone, The use of high resolution accurate mass spectrometry for rapid throughput, multi Standard urine-based occupational drug screening is a two-stage process, involv- positions in city centres to provide toilet facilities for late night visitors, typically on 4-methylethcathinone and beta ethyl methcathinone (pentedrone) -drug screening, S. Hudson, S. Maynard and S. Timbers, Proceedings of the 18th In- a Saturday night. These are emptied on a Sunday morning and at this point a ing initial immunoassay screening to identify possible positive samples followed by  including 1,4 trifluoromethylphenylpiperazine, benzylpiperazine and ternational Conference of Racing Analysts and Veterinarians, New Zealand 2010 more detailed and targeted gas or liquid chromatography coupled with mass sample is retained for analysis methylphenylpiperazine Analysis of anonymous pooled urine from portable urinals in central London con- firms the significant use of novel psychoactive substances, Archer JR, Dargan PI, spectrometry to confirm and quantify the materials present. This combination  other analytes including 4-fluorephedrine 6-APB, methoxetamine and Hudson S, Wood DM., QJM. 2013 Feb;106(2):147-52. doi: 10.1093/qjmed/hcs219. Epub provides a cost-effective means of conducting large scale testing, but means that . Results 2012 Nov 22 materials which do not trigger the initial immunoassay will not be detected. 1,4 methoxyphenylpiperazine chlordiazepoxide gabapentin morphine propranolol Taking the Pissoir – a novel and reliable way of knowing what drugs are being Current urine-based workplace drug testing schemes cover a relatively limited Workplace Tests 4 methylethcathinone chloroquine guaifenesin Nandrolone pyrimethamine used in nightclubs, Archer, J. R. H,, Dargan, P. I., Hudson, S., Davies, S., range of illicit substances which can be broken down into the following groups :- A comparison of results from the ‘Conventional’ and ‘Enhanced’ workplace test for 4-ethylmethcathinone chlorpheniramine HMMA naproxen quetiapine 4-fluoroephedrine Chlorthalidone quinine Puchnarewicz, M., Kicman, A. T., Ramsey, J., Measham, F., Wood, M., Johnston, A.,  Opiates this batch of samples shows a large increase in findings in the LCMS analysis. 4-methylmethcathinone cimetidine hydrochlorthiazide nevirapine ranitidine Wood, D. M. Journal of Substance Use. Posted online on May 6, 2013. 5/6-APB citalopram Ibuprofen risperidone  Samples found positive for on the conventional test were also positive for (doi:10.3109/14659891.2012.740139) cocaine (as benzoylecgonine) on the enhanced screen. Other metabolites ambroxol niflumic acid  Benzodiazepines amisulpiride clobazam ketamine nordazepam sertaline  Cannabis (hydroxy cocaine, ecgonine methyl ester, ecgonine ethyl ester) were also identified amitriptylline clomipramine Ketoprofen nortriptyline sildenafil amphetamine clozapine lamotrigine noscapine sotalol  Cocaine by LCMS. ampyrone codeine lansoprazole Olanzapine Stanozolol  Other targeted analytes depending on availability of kits Lidocaine omeprazole sulpiride atenolol cyclobenzaprine MDA opipramol tamoxifen Conventional Work- Sample No. Enhanced Workplace Test benzocaine DEET MDMA oripavine temazepam There is limited coverage for place Test benzoylecgonine desloratidine Meclofenamic acid orphenadrine tetracycline dextromethorphan mefenamic acid oxazepam tetramisole Sample 1 4 methylmethcathinone/fluorephedrine many of the designer drugs and bisoprolol dextrorphan methadone oxprenolol Sample 2 negative bromhexine Diazepam oxycodone so called ‘legal highs’ that are now Sample 3 Cocaine pentedrone/benzoylecgonine buprenorphine diclofenac methcathinone oxytetracycline tramadol widely abused, and due to the Sample 4 Cocaine pentedrone/benzoylecgonine buprenorphine-nor dihydrocodeine methiopropamine papaverine Trazodone dihydromorphine methoxetamine paracetamol trenbolone 4 methylmethcathinone/fluorephedrine/ nature of the testing, conventional Sample 5 camphor diphenhydramine methylhexaneamine paroxetine trifluoromethylphenylpiperazine trifluoromethylphenylpiperazine/benzylpiperazine capsaicin dipyridamole pentedrone trimethoprim workplace drug testing will always Sample 6 Cannabis 4-methylmethcathinone carbamazepine doxycycline metoclopramid pheniramine vardenafil struggle to keep up with the latest Sample 7 methylhexanamine Carboxy THC enalapril metoprolol / metronidazole Pholcodine xylometazolone tastes. Sample 8 negative cathinone midazolam

Sample 9 negative celecoxib Etoricoxib minoxidil pregabalin zolpidem Sample 10 4-methylmethcathinone cetirizine fluoxetine mitragynine prilocaine chlorcylizine furosemide Propoxyphene Sample 11 Cocaine Pentedrone/trifluoromethylphenylpiperazine/benzoylecgonine Recent data from Europe (EMCDDA) shows the scale of the problem and how the 4 methylmethcathinone/4-methyl /fluorephedrine/ Sample 12 4-methylmethcathinone Other related analytes have been seen, including various cutting agents such as popularity/availability of different drug classes changes year on year. trifluoromethylphenylpiperazine/benzylpiperazine Sample 13 pentedrone levamisole and benzocaine.