A diagnostic test for cocaine and benzoylecgonine in urine and oral fluid using portable mass spectrometry†

Mahado Ismail *a, Mark Baumert b, Derek Stevenson a, John Watts a, Roger Webb a, Catia Costa a, Fiona Robinson c and Melanie Bailey a aUniversity of Surrey, Guildford, Surrey GU2 7XH, UK. E-mail: [email protected] bAdvion Biosciences Ltd., Harlow Enterprise Hub, Edinburgh Way, Harlow, Essex CM20 2NQ, UK cSurrey and Borders Partnership NHS Foundation Trust, Guildford Road, Chertsey, Surrey KT16 0PZ, UK

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First published on 21st November 2016

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Introduction

Surface mass spectrometry is used in a wide array of disciplines to obtain chemical information from the surface of a sample. Since 2004, there has been an increase of techniques that can be used to liberate molecules from a surface under ambient pressure, followed by mass spectrometry detection. This has resulted in a step-change in sample throughput, due to the fact that samples no longer need to be analysed under vacuum and require minimal sample preparation. Surface mass spectrometry techniques include desorption electrospray ionisation (DESI),1-4 atmospheric pressure matrix assisted laser desorption ionisation mass spectrometry (AP-MALDI),5-7 plasma assisted desorption ionisation (PADI),8,9 direct analysis in real time (DART),10 MeV secondary ion mass spectrometry (MeV-SIMS)11,12 and desorption atmospheric pressure chemical ionisation (DAPCI).13,14 These techniques have shown strength in many areas, but the absence of a chromatography step leaves the methods vulnerable to ion suppression effects.

In parallel to the development of ambient ionisation methods, a range of mass spectrometers have been miniaturised and tested for portable mass spectrometry.15-21 Various combinations of these have been used in conjunction with methods such as DESI and paper spray to support portable analysis of drugs.15,18,22 However, the limitation of miniaturised or portable mass spectrometers is that they are less powerful than lab based instruments in terms of resolving power and sensitivity.23 This is problematic when encountering complex mixtures of samples. This combined with the issue of ion suppression effects, limits the selectivity, sensitivity and quantitative power of the portable mass spectrometry approaches to surface analysis.

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In this work, we present a method that attempts to overcome the limitations of surface analysis by introducing a chromatography step. In the method, a solvent is flushed across the sample surface under pressure to extract analytes and the resulting solution is passed through a chromatography column before analysis using a portable mass spectrometer. The result is a low cost, sensitive and selective method of surface analysis.

Recent developments in a related technique, liquid extraction surface analysis (LESA)24-30 have demonstrated the capability of using a liquid microjunction to extract analytes from a sample surface. The extracted analytes are collected in a pipette tip and are sprayed into a mass spectrometer using a nano-electrospray source. However, LESA has limited portability as it requires a separate instrument to facilitate the chromatography step.31

Recent work by Oliveira et al.32 has shown that therapeutic drugs in dried blood spots can be extracted from a surface, passed through a chromatography column and analysed using high resolution mass spectrometry. In this work, we test for the first time the feasibility of using a similar method to Oliveira et al. of surface extraction and chromatography, but using a portable mass spectrometer and in-source fragmentation for portable diagnostics. Our method can be broadly applied to the detection and quantification of compounds on flat surfaces without sample preparation. We show how the method could be applied as a portable and rapid diagnostic test for cocaine and its primary metabolite (benzoylecgonine) in urine and oral fluid, with potential applicability to roadside or workplace drug testing.

Experimental methods

Reagents and materials

Solvents (LC-MS grade) used for analysis were all purchased from Fisher Scientific (Loughborough, UK). Formic acid (99%) used for the mobile phase was purchased from Fisher Scientific (Loughborough, UK). The certified reference materials (CRM) for cocaine and benzoylecgonine (at 1 mg ml−1 in solution) and cocaine-d3 (100 μg ml−1 in solution used as internal standard) were purchased from Sigma Aldrich (Dorset, UK). All drugs and deuterated internal standards were stored at −20 °C. Whatman grade 1-chromatography paper was purchased from VWR (Leicestershire, UK) and used as a sample substrate. Sterilin™ polystyrene containers used for collection of urine was purchased from Scientific Laboratory Supplies (Nottingham, UK).

Sample collection

Negative control oral fluid samples were collected from healthy (drug-free) volunteers (n = 10, 5 male and 5 female) in the laboratory at the University of Surrey using a syringe. The oral fluid samples, 1 ml per volunteer, were pooled and used to prepare blank and spiked solutions of cocaine and benzoylecgonine. In addition, urine samples were collected in carcinogen containers (Scientific Laboratory Supplies, Nottingham, United Kingdom) from healthy (drug-free) volunteers (n = 3 males). The urine samples were pooled. An aliquot of pooled urine was reserved as negative control, and the remainder was spiked with cocaine and benzoylecgonine.

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Urine and oral fluid samples were also collected from two individuals seeking treatment for drug dependency at a drug and alcohol service, to show the applicability of the method to real samples. Urine samples were collected in Sterilin™ polystyrene containers and corresponding oral fluid samples were collected using Quantisal™ collection devices (Alere Toxicology, United Kingdom). Samples were stored at 4 °C and analysed 4 weeks after sample collection. A favourable ethical opinion was granted from the National Research Ethics Service (NRES) for the collection and analysis of these samples (IRAS ID: 142223 and study ID: 17487). Informed consent was obtained from all subjects prior to sample collection.

Sample preparation

Instrumentation

Results and discussion

Analytical performance – urine samples

Analytical performance – oral fluid samples

Extracted ion chromatograms (XIC) of blank and spiked (200 ng ml−1 cocaine and benzoylecgonine) pooled oral fluid are shown in Fig. 4A and B, respectively. The respective retention times for cocaine and benzoylecgonine were 4.12 min and 4.01 min. XICs for the fragment ions corresponding to cocaine (m/z 182.1) and benzoylecgonine (m/z 168.1) are also shown in Fig. 4 (with the same retention times as the respective molecular ions). The chromatograms of blank (drug-free) oral fluid (see Fig. 4A) showed no interferences from endogenous analytes for both analytes.

Cocaine and benzoylecgonine in samples from patients

Conclusions

The method presented here offers a low cost, flexible and portable set up for analysis of analytes on flat surfaces. Whilst we have demonstrated the proof of concept for mobile drug testing of oral fluid and urine, there is a wide range of potential applications for which this methodology could be used. We have developed a new way to screen and quantify cocaine and related metabolite (benzoylecgonine) in biological fluids, using a combination of surface extraction, liquid chromatography and portable mass spectrometry. We have demonstrated the proof of concept for testing for cocaine in urine and oral fluid from patients. We have shown relevant levels of sensitivity (<30 ng ml−1) in these matrices, good linearity (R2 0.998) and relative standard deviations below 23% for replicate measurements. We therefore conclude that this configuration could be a candidate for roadside drug testing investigations in the future.

Acknowledgements

The authors would also like to thank Helen Adams and the service users of Surrey and Borders Partnership NHS Foundation Trust for their help with sample collection. The National Institute of Health Research (NIHR) is thanked for funding the Clinical Research Network Portfolio (ID: 17487). In addition, the authors thank Julien Demarche and Vladimir Palitsin from the Ion Beam Centre for their help with the instrument set up and Inga Zudovaite for assisting in the analyses of samples. The authors also thank Clive Aldcroft from Advion Biosciences for his support with the instrument and Hazim F EL-Sharif for his support in data handling. We would also like to thank the EPSRC Impact Acceleration Account for funding this work.

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