Quantification of Drugs of Abuse in Municipal Wastewater Via SPE and Direct Injection Liquid Chromatography Mass Spectrometry
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Anal Bioanal Chem (2010) 398:2701–2712 DOI 10.1007/s00216-010-4191-9 ORIGINAL PAPER Quantification of drugs of abuse in municipal wastewater via SPE and direct injection liquid chromatography mass spectrometry Kevin J. Bisceglia & A. Lynn Roberts & Michele M. Schantz & Katrice A. Lippa Received: 2 June 2010 /Revised: 30 August 2010 /Accepted: 2 September 2010 /Published online: 24 September 2010 # US Government 2010 Abstract We present an isotopic-dilution direct injection 50 ng/L. We also present a modified version of this method reversed-phase liquid chromatography–tandem mass spec- that incorporates solid-phase extraction to further enhance trometry method for the simultaneous determination of 23 sensitivity. The method includes a confirmatory LC separa- drugs of abuse, drug metabolites, and human-use markers in tion (selected by evaluating 13 unique chromatographic municipal wastewater. The method places particular emphasis phases) that has been evaluated using National Institute of on cocaine; it includes 11 of its metabolites to facilitate Standards and Technology Standard Reference Material 1511 assessment of routes of administration and to enhance the Multi-Drugs of Abuse in Freeze-Dried Urine. Seven analytes accuracy of estimates of cocaine consumption. Four opioids (ecgonine methyl ester, ecgonine ethyl ester, anhydroecgo- (6-acetylmorphine, morphine, hydrocodone, and oxycodone) nine methyl ester, m-hydroxybenzoylecgonine, p-hydroxy- are also included, along with five phenylamine drugs benzoyl-ecgonine, ecgonine, and anhydroecgonine) were (amphetamine, methamphetamine, 3,4-methylenedioxy- detected for the first time in a wastewater sample. methamphetamine, methylbenzodioxolyl-butanamine, and 3,4-methylenedioxy-N-ethylamphetamine) and two human- Keywords Wastewater . Illicit drugs . Urinary metabolites . use markers (cotinine and creatinine). The method is LC/MS/MS . Direct injection . SPE sufficiently sensitive to directly quantify (without preconcen- tration) 18 analytes in wastewater at concentrations less than Introduction Electronic supplementary material The online version of this article (doi:10.1007/s00216-010-4191-9) contains supplementary material, The analysis of drugs of abuse in wastewater (increasingly which is available to authorized users. called sewer epidemiology) has emerged as an appealing means for routinely monitoring drug consumption in K. J. Bisceglia : A. L. Roberts Department of Geography and Environmental Engineering, municipalities. The approach is unobtrusive and has the Johns Hopkins University, potential to provide data at much better temporal and spatial 3400 North Charles Street, resolution than is afforded by traditional interview-based Baltimore, MD 21218, USA survey sampling. Since first being proposed [1], “drug ” – K. J. Bisceglia : M. M. Schantz : K. A. Lippa (*) tests have been conducted on a number of regions [2 15]. Analytical Chemistry Division, Such studies, which rely almost exclusively on reversed- National Institute of Standards and Technology, phase liquid chromatography–tandem mass spectrometry 100 Bureau Drive, Stop 8392, (RPLC/MS/MS), have successfully measured the parent Gaithersburg, MD 20899-8392, USA e-mail: [email protected] and/or metabolites of many drugs of abuse, including cocaine, heroin, prescription pain killers, amphetamine Present Address: and methamphetamine, ecstasy (3,4-methylenedioxy-meth- K. J. Bisceglia amphetamine or MDMA), and marijuana [11]. Chemistry Department, Bard High School Early College, 30-20 Thomson Avenue, Cocaine is particularly well suited to sewer epidemiology. Queens, NY 11101, USA It is excreted almost exclusively (85% to 90%) in urine [16– 2702 K.J. Bisceglia et al. 18], predominantly as the parent drug and three metabolites: has been the technique of choice [29, 30], although it is benzoylecgonine, ecgonine methyl ester, and ecgonine [19]. insufficiently sensitive to detect most cocaine metabolites in Its human metabolites are all chemically unique in that they environmental samples. RPLC/MS/MS has been employed have no licit sources. Equally important, the urinary profile with some success [28, 31, 32], although the poor retention of cocaine metabolites is dependent on the route of of these analytes makes them susceptible to matrix effects administration (ROA); metabolites can include pyrolysis and interferences from salts [33]. Recently, Gheorghe et al. products such as anhydroecgonine and anhydroecgonine [10] and Giroud et al. [34] used hydrophilic interaction methyl ester if smoked as crack cocaine [20, 21], or ethylated chromatography (HILIC) to successfully quantify the alkyl derivatives such as cocaethylene and ecgonine ethyl ester if ester metabolites of cocaine (ecgonine methyl ester and co-ingested with alcohol [21, 22]. These minor metabolites ethyl ester, and anhydroecgonine methyl ester) in body can provide valuable information on lifestyle behaviors fluids and tissue [34] and in wastewater [10]. Gheorghe et associated with cocaine use; their inclusion in wastewater al. [10] also reported the successful separation of ecgonine monitoring programs could provide data useful to public using HILIC (though this metabolite was not detected in health and law enforcement communities. At present, wastewater samples and solid-phase extraction (SPE) although cocaine has been the most extensively investigated recoveries were not reported). The ability of HILIC/MS/ illicit drug in municipal wastewater, such analyses have been MS to successfully quantify complex mixtures of illicit limited to the parent drug, two of its three major metabolites drug metabolites remains, however, untested. (benzoylecgonine and ecgonine methyl ester), and three Most current methods for the analysis of drugs of abuse minor metabolites, only one of which (cocaethylene) can in wastewater incorporate a sample pre-concentration step. provide ROA information [11, 14, 15]. Off-line SPE is the most commonly used approach [11], Expanding cocaine monitoring programs to include although online SPE [12] has also been employed with additional metabolites has another benefit in that, by notable success. These techniques can be costly and time capturing a greater fraction of the total cocaine load, it consuming, and sometimes require specialized instrumen- might be possible to reduce uncertainties in wastewater- tation. In addition, they can concentrate interferences along derived measurements of cocaine consumption. Current with target analytes. While such approaches have histori- procedures, which entail a number of assumptions [6, 14, cally been necessary for the analysis of illicit drugs and 23–26], estimate cocaine usage from a single metabolite pharmaceuticals in environmental samples, the additional (usually benzoylecgonine), multiplying measured concen- cost and labor involved have limited the spatial and trations by a correction factor that incorporates the molar temporal resolution at which analyses could be performed. mass ratio (parent/metabolite) as well as the fraction Fortunately, recent improvements in MS/MS sensitivity excreted as the metabolite in question [6, 14, 23–26]. may enable the analysis of at least some drugs of abuse via Using a total measured cocaine load rather than a single direct injection. This is especially likely for the major metabolite would reduce errors that might be associated cocaine metabolites, which are routinely measured in the with individual variations in the extent of metabolism, or 100 to 1,000 ng/L range [11]. In a notable recent with the degradation of key metabolites within the sewage advancement, Chiaia et al. [9] used large volume injection system [27]. Ecgonine, in particular, can represent as much and RPLC/MS/MS to quantify 22 chemicals (with an as 23% of total cocaine in human urine [19]; as the final emphasis on illicit drugs) in municipal wastewater. Unfor- hydrolysis product of cocaine, it is likely to represent an tunately, their analyte suite included only three cocaine even greater fraction of the total cocaine load in wastewater. metabolites along with the parent drug. To our knowledge, there are no published LC/MS/MS Sewer epidemiology holds great promise as a tool for methods for the analysis of ecgonine in municipal sewage, monitoring the population-based consumption of drugs of although an initial reversed-phase LC/MS/MS methodology abuse. At present, however, this approach is still in its infancy. by Peck et al. that would capture a broad array of cocaine A need remains for robust and reliable methods that can metabolites (including ecgonine) has been previously estimate drug use with minimal analytical effort and reduced reported [28]. measurement uncertainty. In this paper and with our recent The small molecular size, high polarity, zwitterionic hydrolysis method for the simplified analysis of total cocaine character, and structural similarity of most cocaine metab- residues [35], we provide improved methods for determining olites make them challenging analytes in matrices as cocaine residues in municipal wastewater using LC/MS/MS. complex as municipal wastewater. The low molecular mass The RPLC method described herein maximizes the informa- metabolites, such as ecgonine and anhydroecgonine, are tion that is obtained from each measurement by simulta- seldom investigated, even indrugscreeningtestsor neously analyzing cocaine and 11 of its metabolites, forensic studies. When such metabolites are analyzed, gas including those with the potential to provide ROA informa- chromatography–mass