Elsevier

Journal of Chromatography B

Volume 878, Issue 30, 15 November 2010, Pages 3095-3105
Journal of Chromatography B

Determination of 17 illicit drugs in oral fluid using isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry with three atmospheric pressure ionizations

https://doi.org/10.1016/j.jchromb.2010.09.014Get rights and content

Abstract

The collection of oral fluid for drug testing is easy and non-invasive. This study developed a drug testing method using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC–MS/MS) in selected-reaction monitoring (SRM) mode. We tested the method on the analysis of four opiates and their metabolites, five amphetamines, flunitrazepam and its two metabolites, and cocaine and its four metabolites in oral fluid. 100-μL samples of oral fluid were diluted with twice the amount of water then spiked with isotope-labeled internal standards. After the samples had undergone high-speed centrifugation for 20 min, we analyzed the supernatant. The recovery of the sample preparation ranged from 81 to 108%. We compared the performance of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). The ion suppression of most analytes on ESI (28–78%) was lower than that of APCI and APPI. A post-column flow split (5:1) did not reduce the matrix effect on ESI. Direct APPI performed better than dopant-assisted APPI using toluene. ESI, APCI and APPI limits of quantitation mostly ranged from 0.11 to 1.9 ng/mL, 0.02 to 2.2 ng/mL and 0.02 to 2.1 ng/mL, respectively, but were much higher on amphetamine and ecgonine methyl ester (about 2.7–4.7 ng/mL, 8.7–14 ng/mL, and 10–19 ng/mL, respectively). Most of the bias percentages (accuracy) and relative standard deviations (precision) on spiked samples were below 15%. This method greatly simplifies the process of sample preparation and shortens the chromatographic time to only 7.5 min per run and is able to detect analytes at sub-ppb levels.

Introduction

Drug abuse comes with serious health problems, increased criminal activity, and the spread of some diseases. According to the 2008 World Drug Report, almost 5% of the world's populations have abused at least one drug in the past twelve months and 0.6% of the world's adults are severely drug addicted [1].

Morphine, heroin (diacetyl morphine) and codeine are narcotics. Amphetamine and its derivatives, including methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethamphetamine (MDEA), and 3,4-methylenedioxyamphetamine (MDA), act to stimulate the central nervous system (CNS), increasing alertness and decreasing fatigue and appetite. Flunitrazepam, a hypnotic for short-term treatment of chronic insomnia, can debilitate persons and is often used as a date rape drug or for robbery. Cocaine is a CNS stimulant and topical anaesthetic able to produce a euphoric state similar to that induced by amphetamines.

Oral fluid has emerged for drug testing for the last twenty years [2], [3]. It is a much simpler matrix than traditional specimens like urine and plasma because it is composed of 99% water, 0.3% proteins, and 0.3% mucin [4]. Its collection is non-invasive, easily performed, and can be done under surveillance or in clinics and workplaces [4], minimizing the chances of sample substitution or adulteration. Parent drugs in oral fluid are usually found in concentrations highly correlated with those in plasma [5], [6], [7] and they are much less susceptible to dilution by fluid intake [8], [9], [10], [11], [12], [13].

Certain chemicals are better detected than others in suited in the oral fluid from users of illicit drugs. Because heroin has a short half-life in the blood (2–7 min), a better biomarker of heroin use might be 6-acetylmorphine in the oral fluid [14], [15]. Codeine is the primary chemical found in plasma and oral fluids of codeine users [16], [17]. Amphetamine and methamphetamine are found at higher concentrations in the oral fluid than in the plasma if they are taken orally [18], [19]. MDMA and its metabolite MDA are the major chemicals found in the oral fluid from MDMA users [20], [21]. The flunitrazepam metabolite, 7-aminoflunitrazepam, is the primary analyte detected in the oral fluid in flunitrazepam users [22]. Besides cocaine, its major metabolite benzoylecgonine is also the main detectable chemical in the oral fluid in cocaine users [10], [15], [23].

Illicit drugs can be detected in oral fluid using gas chromatography–mass spectrometry (GC–MS) or GC–tandem MS (GC–MS/MS) with the use of solid-phase extraction (SPE) [3], [24], solid-phase microextraction [25], small volume liquid extraction [26], or a single-step extraction and derivatization [27]. The limits of quantitation (LOQs) for these methods range between 5 and 25 ng/mL. Another method, liquid chromatography (LC) coupled with MS/MS, does not require chemical derivatization. This method is often more sensitive than GC–MS, and has LOQs ranging from 0.5 to 2 ng/mL [28], [29], [30], [31]. It is especially useful when the amount of oral fluid is limited as is often found in samples from amphetamine users who often have “dry mouth” [32], [33].

Matrix effects can influence the efficiency of ionization and measurement of analytes and confound LC–MS(/MS) results [32], [34], [35], [36]. To reduce their effects, oral fluid can be pretreated with SPE because it usually provides lower ion suppression on electrospray ionization (ESI) than either liquid–liquid extraction (LLE) or protein precipitation [31], [33], [37], [38]. However, SPE concentrates some matrix components as well as the analytes and it may not lower ion suppression any better than a simple dilution of samples with water [37]. In addition to a better sample preparation, use of different ionization interface may also reduce matrix effects. For example, two studies performed by Dams et al. found that samples subjected to atmospheric pressure chemical ionization (APCI) were less susceptible to matrix effects than those undergoing ESI when measuring the amount of illicit drugs in biofluids [37], [39].

Atmospheric pressure photoionization (APPI) is a relatively new means of ionization. This method uses an ultraviolet lamp to emit photons and initiates the ionization by direct interaction with analytes or indirect ionization via dopant [40], [41]. To date, no published paper has assessed the use of APPI to analyze illicit drugs in oral fluid. In addition, there has been a recent increase in the use of ultra-high performance liquid chromatography (UHPLC) in bio-analysis because it provides better resolution and higher throughput than high-performance liquid chromatography (HPLC) [42], [43]. Because UHPLC columns can be packed with smaller particles (less than 2.0 μm), the resulting increase in flow rates can shorten the chromatographic time and sharpen the peaks, providing better sensitivity and peak capacities without sacrificing separation efficiencies.

The aim of this study was to develop and assess analytical methods involving the use of UHPLC–MS/MS combined with ESI, APCI and APPI to determine four opiates and metabolites (heroin, morphine, 6-actylmorphine, codeine), five amphetamines (amphetamine, methamphetamine, MDMA, MDA, MDEA), flunitrazepam and its two metabolites (7-aminoflunitrazepam and N-desmethylflunitrazepam), cocaine and its four metabolites (norcocaine, benzoylecgonine, ecgonine methyl ester, and cocaethylene) in oral fluid. The sample was pretreated with steps of dilution and centrifugation before instrumental analysis; the matrix effect and detection sensitivity on the three different ionization sources were evaluated. Use of stable isotope-labeled internal standards for quantitation proved to be precise and accurate. This method greatly simplifies sample preparation and requires only 7.5-min chromatography including re-equilibration. The improved sensitivity greatly enhances the detection window for the detection of illicit drugs in oral fluid and the high throughput makes it possible to process a large number of samples in short amount of time.

Section snippets

Chemicals and reagents

We obtained heroin, 6-acetylmorphine, cocaine, norcocaine: hydrochloride, ecgonine methyl ester, and cocaethylene in acetonitrile at 1.0 mg/mL, 7-aminoflunitrazepam in acetonitrile at 100 μg/mL, morphine, codeine, (+/−)-amphetamine, (+/−)-methamphetamine, (+/−)-MDMA, (+/−)-MDA, (+/−)-MDEA, flunitrazepam, N-desmethylflunitrazepam, and benzoylecgonine in methanol at 1.0 mg/mL from Cerilliant (Austin, TX, USA). Their molecular structures are shown in Fig. 1. We also purchased the isotope-labeled

Chromatography

Acetonitrile as the organic mobile phase provided better separation and sharper analyte peaks than methanol on the high strength silica (HSS) T3 column. Simões et al. also chose acetonitrile as the organic mobile phase using a Waters Atlantis T3 column, which has the same packing material but in a larger particle size than the HSS T3, to separate 24 illicit drugs and medicines in oral fluid [46]. Similarly, two other studies have also used acetonitrile to analyze illicit drugs in oral fluid

Conclusions

This study developed and validated a method using UHPLC-MS/MS to detect 17 illicit drugs in only 100-μL oral fluid and was sensitive enough to provide LODs reaching sub-ppb levels at three atmospheric pressure ionization sources. The throughput was significantly increased by simplifying the pretreatment process and the chromatographic run was shortened to 7.5 min. This method makes it possible to handle a large number of oral-fluid samples containing trace amounts of illicit drugs and makes it

Acknowledgements

This study was supported by the National Bureau of Controlled Drugs, Department of Health, Taiwan (project number DOH97-NNB-1014). The UHPLC–MS/MS was provided by the Center for Environment and Occupational Health Research, College of Public Health, National Taiwan University.

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