PAPER

TOXICOLOGY

Sarah Kerrigan,1,2 Ph.D.; Ashley Mott,1 M.S.; Breanna Jatzlau,1 M.S.; Francisco Ortiz,1 M.S.;Laura Perrella,1 M.S.; Sarah Martin,1 M.S.; and Kelsie Bryand,1 M.S.

Designer Psychostimulants in Urine by LiquidChromatography–Tandem MassSpectrometry*,†

ABSTRACT: Designer psychostimulants are known by recreational drug users to produce a complex array of adrenergic and hallucinogeniceffects. Many of these drugs are not targeted during routine toxicology testing and as a consequence, they are rarely reported. The purpose ofthis study was to develop a procedure for the detection of 15 psychostimulants in urine using liquid chromatography–tandem mass spectrometry(LC-MS/MS), specifically 2,5-dimethoxy-4-bromophenethylamine (2C-B), 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-methyl-phenethylamine (2C-D), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), 2,5-dimethoxyphenethylamine (2C-H), 2,5-dimethoxy-4-iodophenethylamine(2C-I), 2,5-dimethoxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4-propylthiophen-ethylamine (2C-T-7), 2,5-dimethoxy-4-bromoamphetamine (DOB), 2,5-dimethoxy-4-chloroamphetamine (DOC), 2,5-dimethoxy-4-ethylamphetamine(DOET), 2,5-dimethoxy-4-iodoamphetamine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), and 4-methylthioamphetamine (4-MTA). Analyticalrecoveries using solid-phase extraction were 64–92% and the limit of detection was 0.5 ng/mL for all drugs except 2C-B (1 ng/mL). The assay wasevaluated in terms of analytical recovery, precision, accuracy, linearity, matrix effect, and interferences. The technique allows for the simultaneousdetection of 15 psychostimulants at sub-ng/mL concentrations.

KEYWORDS: forensic science, toxicology, urine, designer drugs, amphetamines, liquid chromatography–tandem mass spectrometry

A recent review suggests that more than 100 new psychotro-pic substances or designer drugs have been introduced of late(1). Following regulation of conventional counterparts, synthesisof these new derivatives is often a direct attempt to circumventregulation and criminalization (2). The increasing prevalence ofdesigner drugs stems largely from control measures, which serveto schedule or regulate drugs that have the potential for abuse ordeleterious effects. Designer drugs are often perceived by drugusers to be advantageous from a legal standpoint and may beviewed as having more desirable pharmacologic effects. Smallalterations in structure may produce drugs with similar subjectiveeffects, but circumvent existing drug legislation. The innovationof suppliers to create and effectively “market” designer drugs torecreational users via the Internet significantly outpaces the abil-ity of government to regulate, legislate, and enforce thoseactions.One of the largest and most important classes of designer drugs

are the phenethylamine derivatives. Early examples of these are

3,4-methylenedioxyamphetamine (MDA) and 3,4-methylenedi-oxymethamphetamine (MDMA), which emerged as popular rec-reational drugs in the 1960s and 1980s, respectively. Today,many of the phenethylamine-based designer drugs have becomepopularized by young recreational drug users, and as a conse-quence, are of growing concern from a public safety perspective.Many of the newer drugs were initially developed to harness

the psychedelic and introspective potential of certain phenethyl-amines. Alexander Shulgin synthesized numerous compoundsbearing structural or pharmacological similarity to many of thetraditional psychedelics. This report focuses on three classes ofphenethylamine-type drugs that can produce a combination ofhallucinogenic and stimulant effects. The 2C series are dimeth-oxyphenethylethanamines, containing two methoxy groups at the2 and 5 positions of the benzene ring and two carbons (“2C”)between the amine and benzene ring (3). Some within the 2Cseries also contain a halogen on the benzene ring at the 4 posi-tion; the 2C-T series are dimethoxyphenethylethanamines thattypically contain an alkylated thio- group on the 4 position ofthe ring; the DO-series are dimethoxyphenylpropanamines, con-taining a methyl on the aminoethyl chain and a halogen or alkylgroup on the 4 position of the benzene ring (3).The 2C, 2C-T, and DO-series of designer drugs are not well

characterized in humans. There are some notable differencesbetween traditional hallucinogenic amphetamines and thesenewer alternatives. Despite the structural similarity of many ofthese synthetic derivatives (Fig. 1), minor alterations can havesignificant impact in terms of potency and onset of action(Table 1). The delayed onset of action and increased potency of

1Department of Forensic Science, College of Criminal Justice, Sam Hous-ton State University, Box 2525, 1003 Bowers Blvd., Huntsville, TX 77341.

2Sam Houston State University Regional Crime Laboratory, The Wood-lands, TX 77381.

*Supported by Award No. 2008-DN-BX-K126 awarded by the NationalInstitute of Justice, Office of Justice Programs, U.S. Department of Justice.

†The opinions, findings, and conclusions or recommendations expressed inthis publication are those of the author(s) and do not necessarily reflect thoseof the Department of Justice.

Received 26 June 2012; and in revised form 14 Nov. 2012; accepted 23Nov. 2012.

© 2013 American Academy of Forensic Sciences 175

J Forensic Sci, January 2014, Vol. 59, No. 1doi: 10.1111/1556-4029.12306

Available online at: onlinelibrary.wiley.com

some drugs within this class relative to MDMA has been noted.This may increase the potential for adverse reactions, toxicity(4), or fatal overdose (5). These drugs are sometimes

misrepresented, marketed, and sold as other substances, whichmay increase the risks associated with their use and result inunintended consequences.

TABLE 1––Dose and duration of action of 2C, 2C-T, and DO-series drugs*.

Drug Chemical Name Street Names Dosage (mg) Duration (h)

2C-B 2,5-dimethoxy-4-bromophenethylamine Nexus, 2’s; Toonies; Bromo; TC; Spectrum; Venus; Bees; Erox 12–24 4–82C-C 2,5-dimethoxy-4-chlorophenethylamine – 20–40 4–82C-D 2,5-dimethoxy-4-methylphenethylamine LE–25 20–60 4–62C-E 2,5-dimethoxy-4-ethylphenethylamine Europa 10–25 8–122C-H 2,5-dimethoxyphenethylamine – Unknown Unknown2C-I 2,5-dimethoxy-4-iodophenethylamine i 14–22 6–102C-T-2 2,5-dimethoxy-4-ethylthiophenethylamine T2 12–25 6–82C-T-4 2,5-dimethoxy-4-isopropylthiophenethylamine T4 8–20 12–182C-T-7 2,5-dimethoxy-4-propylthiophenethylamine T7; Blue Mystic; Beautiful; Tripstacy; Tweety-Bird Mescaline; Belladona 10–30 8–154-MTA 4-methylthioamphetamine Flatliner; Golden Eagle Unknown UnknownDOB 2,5-dimethoxy-4-bromoamphetamine Bob; Dr. Bob; Brolamfetamine 1–3 18–30DOC 2,5-dimethoxy-4-chloroamphetamine – 1.5–3 12–24DOET 2,5-dimethoxy-4-ethylamphetamine – 2–6 14–20DOI 2,5-dimethoxy-4-iodoamphetamine – 1.5–3 16–30DOM 2,5-dimethoxy-4-methylamphetamine STP (Serenity, Tranquility, and Peace) 3–10 14–20

*Dosage and duration of action are reported from testimonial and nonscientific publications (3,42).

FIG. 1––Structures of the 15 target analytes and internal standard.

176 JOURNAL OF FORENSIC SCIENCES

Many of the psychedelic phenethylamines derive their effectsfrom their action as 5-HT2 agonists. The profound hallucino-genic effect associated with several of these drugs is likely dueto their strong affinity toward serotonin receptor sites (6,7). Thedifferences in their potency and duration of effects come fromminor structural differences. The metabolic fate of several of thedrugs under investigation has been investigated and reported inanimal models. 2C-B is perhaps the most widely studied in ratsand mice (8–12). The majority of the drug undergoes a combina-tion of O-demethylation, N-acetylation, deamination with oxida-tion to the corresponding acid, or reduction to the alcohol.Transformative pathways for 2C-I (13) and 2C-E (14) are notdissimilar, and in general, the O-demethylation of methoxygroups, beta-oxidation of the alkyl side chain, and oxidativedeamination can be likened to mescaline. In rat studies, the2C-T series may undergo combinations of N-acetylation,O-demethylation, sulfoxidation, S-dealkylation and S-methyla-tion, and hydroxylation and demethylation (15–17). DOI hasbeen shown to undergo O-demethylation, as well as sulfate andglucuronide conjugation (18). In general, the DO-series has beenshown to primarily be metabolized via O-demethylation as wellas hydroxylation (18–21). Metabolism is believed to be cata-lyzed primarily via CYP2D6 isoenzymes (22).Although animal pharmacokinetic models are useful, pharma-

cology in humans has yet to be fully explored. Metabolite drugstandards are not commercially available, posing additional chal-lenges for toxicological detection in the laboratory. The harmassociated with these drugs is not well understood, although it isclear that they are not without risk (23). These drugs may beencountered illicitly in a variety of forms, including tablets,capsules, powders (2C and 2C-T series), and LSD-like blottersor liquids for DO-series drugs. Despite the frequency with whichthey are encountered in controlled substance exhibits, they areinfrequently encountered in biological samples submitted fortoxicological testing in the United States. Although recreationaldrug users report a complex array of stimulant and hallucino-genic effects, a relatively small number of fatal and nonfatalreports have been published in the literature (5,24–27).The DO-series produce more powerful hallucinogenic effects

compared with either the 2C or 2C-T series. The methyl groupadjacent to the amine is reportedly responsible for the increasedpotency and duration of action of this group (28). The DO-seriesof drugs appear to show affinity for 5-HT2 receptors and act asagonists and antagonists at different receptor subtypes. The phar-macological activity of the 2C and 2C-T series is attributed to asimilar mode of action, although it is less well characterized.Partial agonism of a-adrenergic receptors has been described for2C-B (28). The hallucinogenic properties of these drugs is gener-ally attributed to the two carbon spacer that separates the amineform the phenyl ring, methoxy groups at positions 2 and 5, anda hydrophobic substituent in the 4-position (alkyl, thio,alkylthio).Toxicology laboratories frequently rely upon immunoassay

screening methodology to identify presumptive positive sam-ples. A recent systematic evaluation of commercial immunoas-says directed toward amphetamine-like psychostimulantsshowed virtually no cross-reactivity toward the 2C, 2C-T orDO-series (29). As a consequence, laboratories that rely uponimmunoassay rather than broad-spectrum chromatographicscreening techniques may fail to detect these powerful psyche-delic substances.Forensic toxicology laboratories in the U.S. do not typically

target these recreational drugs during routine testing procedures.

Published studies, largely from Europe, have described the useof liquid chromatography methods for the detection of some, butnot all of the drugs described here (4,24,25,30–37). Hyphenatedcapillary electrophoretic procedures have also been described(38–40), but these techniques enjoy less widespread use in toxi-cology laboratories. We previously reported a gas chromatogra-phy/mass spectrometry (GC/MS) procedure for the analysis often of these designer drugs in urine samples (29). In this study,we report a liquid chromatography–tandem mass spectrometry(LC-MS/MS) procedure with improved detection limits for 15psychedelic amphetamines: 2,5-dimethoxy-4-bromophenethyl-amine (2C-B), 2,5-dimethoxy-4-chlorophenethylamine (2C-C),2,5-dimethoxy-4-methylphenethylamine (2C-D), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), 2,5-dimethoxyphenethylamine(2C-H), 2,5-dimethoxy-4-iodophenethylamine (2C-I), 2,5-dimeth-oxy-4-ethylthiophenethylamine (2C-T-2), 2,5-dimethoxy-4-iso-propylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4-bromoamphetamine(DOB), 2,5-dimethoxy-4-chloroamphetamine (DOC), 2,5-dimeth-oxy-4-ethylamphetamine (DOET), 2,5-dimethoxy-4-iodoamphet-amine (DOI), 2,5-dimethoxy-4-methylamphetamine (DOM), and4-methylthioamphetamine (4-MTA). The structures of the targetanalytes are shown in Fig. 1. Recent legislation in the UnitedStates now means that 12 of the 15 drugs under investigationare listed in Schedule I of the Federal Controlled Substances Act(CSA) due to their high potential for abuse and absence of eithermedical use or accepted safety. DOM, DOET, 2C-B, DOB, and2C-T-7 have been federally scheduled drugs for some time.However, in July 2012, the Food and Drug AdministrationSafety and Innovation Act (S.3187), which includes the Syn-thetic Drug Abuse Prevention Act of 2012 was signed into law.This new legislation updated Schedule I drugs listed in the Con-trolled Substances Act (21 U.S.C. 812(c)) to include seven addi-tional psychedelic amphetamines that are the subject of thisstudy (2C-C, 2C-D, 2C-E, 2C-H, 2C-I, 2C-T-2, and 2C-T-4)(41). DOI, DOC, and 4-MTA remain unscheduled but may beregulated by the Federal Analogue Act, which states that anydrug substantially similar to a scheduled drug may be treated asthough it were scheduled, if intended for human consumption.

Materials and Methods

Reagents

Mescaline-d9 (internal standard) was obtained from Cerriliant(Round Rock, TX) in a methanolic solution at 0.1 mg/mL.The hydrochloride salts 2C-B, 4-MTA, DOET, DOM, 2C-T-2,2C-T-4, 2C-T-7, 2C-H, 2C-I, and DOB were obtained from Lip-omed (Cambridge, MA) in 1 mg/mL solutions. The hydrochlo-ride salt of DOI was obtained as a powder from Sigma-Aldrich(St. Louis, MO). The Drug Enforcement Agency (DEA) SpecialTesting and Research Laboratory (Dulles, VA) provided solidreference standards for 2C-C, 2C-D, 2C-E, and DOC. Methanol-ic stock solutions for all target analytes were routinely preparedat 1 mg/mL (free base) and stored at �10°C. Working standardscontaining all target analytes (0.01, 0.001, and 0.0001 mg/mL)were used for the preparation of the calibrators and controls.For the interference study, methanolic standards for

amphetamine, methamphetamine, MDA, MDMA, 3,4-methylen-edioxyethylamphetamine (MDEA), ephedrine, pseudoephedrine,phentermine, phenylpropanolamine, alprazolam, amitriptyline,cocaine, codeine, dextromethorphan, diazepam, hydrocodone,ketamine, meperidine, methadone, nordiazepam, oxycodone,

KERRIGAN ET AL. . PSYCHOSTIMULANTS IN URINE BY LC/MS/MS 177

phencyclidine (PCP), tramadol, and zolpidem were obtained fromCerilliant (Round Rock, TX). Standards of phenethylamine,putrescine, tryptamine, tyramine were obtained from Sigma-Aldrich and N-methyl-l-(3,4-methylenedioxyphenyl)-2-buta-namine (MBDB) was obtained from Lipomed.Acetic acid, acetonitrile, ethyl acetate, hexane, isopropanol,

methanol, and methylene chloride were obtained from Mallink-rodt-Baker (Hazelwood, MO). Concentrated ammonium hydrox-ide and sodium fluoride were obtained from Fisher Scientific(Pittsburgh, PA). Ammonium acetate and monobasic sodiumphosphate were purchased from Sigma-Aldrich. Dibasic sodiumphosphate heptahydrate was obtained from VWR International(West Chester, PA). Concentrated hydrochloric acid was obtainedfrom EMD Chemical (Gibbstown, NJ). A Millipore Milli-Q (Bill-erica, MA) was used to purify all deionized water (DIW) used inthis study. All reagents were of ACS or HPLC grade. Acidicmethanol consisted of 1% (v/v) concentrated hydrochloric acid inmethanol. Pooled drug-free urine preserved with 1% (w/v)sodium fluoride was obtained from human volunteers.

Sample Preparation and SPE Extraction

Drugs were extracted from urine by solid-phase extraction(SPE) using PolyChrom ClinII mixed-mode polymeric columnsfrom SPEware (Baldwin Park, CA). All extractions were per-formed using 2 mL of urine and silanized glassware. Followingthe addition of mescaline–d9 internal standard to achieve a finalconcentration of 100 ng/mL, 2 mL of pH 6.0 phosphate bufferwas added, samples were vortex mixed, and transferred to SPEcolumns. Samples were drawn through the column under gravityor sufficient vacuum to maintain continuous flow. Columns werethen washed with 1 mL of deionized water, 1 mL of 1M aceticacid, and then dried at full vacuum for 5 min. Columns werewashed with hexane (1 mL), ethyl acetate (1 mL), and methanol(1 mL) in a successive fashion. Target analytes were eluted with1 mL methylene chloride/isopropanol (95:5, v/v) containing 2%concentrated ammonium hydroxide. Acidic methanol (30 lL)was added to each sample prior to evaporation at 50°C using aTurboVap� II (Caliper Life Sciences, Hopkinton, MA). Thisextraction protocol was optimized previously and described else-where (29). Extracts were reconstituted in 50 lL of mobilephase A (described below) and transferred to autosampler vialsfor analysis.

LC/MS/MS Conditions

Separation was achieved using a Shimadzu high-performanceliquid chromatography (HPLC) system (Columbia, MD) with aPhenomenex Luna 5 lm C18 Colum (100 9 2.0 mm) (Tor-rance, CA) equipped with a C18 guard column (4.0 9 2.0 mm).An API 3200 tandem mass spectrometer from AB Sciex (FosterCity, CA) and Analyst 1.4.2 software from Applied Biosystemswere used for detection.Mobile phase A consisted of 50 mM ammonium acetate in

DIW/methanol (95:5). Mobile phase B consisted of 50 mMammonium acetate in a mixture of acetonitrile/DIW (90:10). Aflow rate of 0.4 mL/min was used in accordance with the fol-lowing gradient profile: 20% mobile phase B for 0–1 min,increased to 65% by 4 min, held at 65% until 4.5 min, and thendecreased to 20% by 6 min. Positive electrospray ionization andmultiple reaction monitoring (MRM) were used throughout.Acquisition parameters and optimized conditions are summarizedin Table 2.

Reconstituted extracts were injected (30 lL) onto the LC/MS/MS using a Shimadzu Sil-20A HT autosampler equipped withtwo LC-20AT pumps. Analytical recovery was evaluated todetermine the extraction efficiency of the SPE method. Addition-ally, the effect of “salting out” prior to evaporation was investi-gated to determine whether sample losses could occur due to thevolatility of some target drugs in the base (uncharged) form.Acidic methanol was used for this purpose. Analytical recoverywas determined by fortifying drug-free urine with target analytesat 100 ng/mL (n = 5).The limit of quantitation (LOQ) and limit of detection (LOD)

were determined empirically by analyzing successively lower con-centrations. Drug-free urine was fortified with target analyte todetermine the lowest concentration that met the following criteria.For LOD, relative retention time within 2% of the expected value;signal-to-noise ratio of at least 3:1; ion ratios within 25% ofexpected value. For LOQ, relative retention time within 2% of theexpected value; signal-to-noise ratio of at least 10:1; ion ratioswithin 25% of expected value; and calculated concentrations ofindependently fortified controls within 20% of the expectedvalues. Intra-assay precision and accuracy were evaluated at 50and 250 ng/mL by replicate analysis (n = 4) of quantitative con-trols. Interassay precision was evaluated at 100 ng/mL (n = 4).Due to the anticipated low concentration of target analytesexpected in actual casework, accuracy was further evaluated atmuch lower concentrations (between 0.5 and 5 ng/mL).

TABLE 2––LC-MS/MS acquisition parameters and optimized conditions. TheMRM transitions used for quantitation are shown in bold.

Drug m/z TransitionAbundance

(%) DP EP CEP CE CXP

mescaline-d9 221 ? 204 100 26 10 16 25 4221 ? 178 29 26 10 16 25 4

2C-H 182 ? 165 100 26 4 16 13 4182 ? 150 133 26 4 16 27 4

4-MTA 182 ? 165 224 26 3 16 13 4182 ? 117 100 26 3 16 27 4

2C-D 196 ? 179 100 21 6.5 15 13 4196 ? 164 50 21 6.5 15 25 4

2C-C 216 ? 199 100 26 4.5 15 15 4216 ? 184 53 26 4.5 15 29 4

2C-B 262 ? 245 100 26 3 14 15 6262 ? 230 49 26 3 14 27 4

DOM 210 ? 193 100 21 4 16 13 4210 ? 178 38 21 4 16 27 4

DOC 230 ? 213 100 26 7.5 16 13 4230 ? 185 71 26 7.5 16 21 4

DOB 276 ? 259 100 27 10 18 17 5276 ? 231 47 27 10 18 25 5

2C-T-2 242 ? 225 100 26 4 18 15 4242 ? 134 12 26 4 18 37 4

2C-I 308 ? 291 100 26 4.5 20 17 6308 ? 276 50 26 4.5 20 25 6

2C-E 210 ? 193 100 26 6 16 15 4210 ? 178 41 26 6 16 31 4

DOI 322 ? 305 100 36 8 18 17 6322 ? 105 26 36 8 18 57 4

DOET 224 ? 207 100 26 3.5 18 15 4224 ? 179 37 26 3.5 18 25 4

2C-T-4 256 ? 239 100 26 3.5 14 15 4256 ? 197 77 26 3.5 14 27 4

2C-T-7 256 ? 239 603 26 3.5 14 15 4256 ? 197 100 26 3.5 14 27 4

DP, declustering potential; EP, entrance potential; CEP, cell entrancepotential; CE, collision energy; CXP, cell exit potential; MRM, multiplereaction monitoring.

178 JOURNAL OF FORENSIC SCIENCES

Linearity was evaluated using regression analysis. The limit oflinearity was evaluated by adding successively higher calibratorsto a calibration curve between 1 and 2000 ng/mL (1, 5, 10, 25,50, 100, 200, 300, 500, 1000, 2000 ng/mL). Linearity was main-tained if the R2 value was at least 0.99, if the gradient changedby 10% or less and the back-calculated concentration of the cali-brator was within 20% of the expected value.Matrix effects were evaluated qualitatively and quantitatively

using two well-established approaches: postcolumn infusion andpostextraction addition. During method development and duringthe preliminary stages of validation, ion suppression or enhance-ment was evaluated using a T-connector to allow postcolumninfusion of drugs. Target drugs were infused postcolumn, whiledrug-free urine extracts from different individuals (n = 20) wereinjected on the LC-MS/MS. This approach allowed potentialmatrix interferences to be identified qualitatively over the entirechromatographic run. Matrix effects were also assessed quantita-tively using postextraction addition. Target analytes were forti-fied into drug-free urine extracts from different volunteers(n = 20). The transition ion abundance for analytes fortified intomatrix (A) was compared quantitatively with target analytes forti-fied into nonmatrix, specifically mobile phase (B). The percentmatrix effect (ME) was calculated numerically using% ME =(A–B)/B.Potential interferences from other amphetamine-like drugs,

endogenous bases, and common basic drugs were also investi-gated. Interferences were evaluated qualitatively and quantita-tively using both negative and positive controls. Drug-free andpositive controls containing 0 and 100 ng/mL of each target ana-lyte, respectively were fortified with potential interferences at aconcentration of 1000 ng/mL. The common amphetamine-likedrugs included in the study were amphetamine, methamphetamine,MDA, MDMA, MDEA, MBDB, ephedrine, pseudoephedrine, andphentermine. Endogenous bases included phenethylamine,putrescine, tryptamine, and tyramine. Finally, the following com-

monly encountered drugs were also investigated: alprazolam, ami-triptyline, cocaine, codeine, dextromethorphan, diazepam,hydrocodone, ketamine, meperidine, methadone, nordiazepam,oxycodone, phencyclidine, tramadol, and zolpidem.

Casework Samples

The presence of 4-MTA, 2C, 2C-T and DO-series drugs wasdetermined in a population of adjudicated casework samples thatwere due for destruction. Urine samples (n = 2021) obtainedfrom individuals apprehended for suspicion of being under theinfluence of a controlled substance (California Health and SafetyCode Section 11550) were provided by the California Depart-ment of Justice Toxicology Laboratory, Bureau of ForensicServices (Sacramento, CA). The study was subject to Institu-tional Review Board (IRB) approval by the Protection of HumanSubjects Committee of Sam Houston State University.

Results and Discussion

Recovery, Precision, and Accuracy

Overall, the addition of acidic methanol to extracts prior toevaporation significantly improved the extraction efficiency oftarget analytes (Fig. 2). The abundance of drugs evaporated inthe base form (uncharged) was significantly lower (19–75%),relative to those treated with acidic methanol prior to evapora-tion (Table 3). t-tests (p = 0.05) were used to show that theseresults were significant. Not surprisingly, the evaporative lossesalso introduced significant imprecision, with CVs in the range12–31% compared with 2–9% when acidic methanol was used.Consequently, all extracts were “salted out” using acidic metha-nol prior to evaporation for the remainder of the study. The sus-ceptibility to evaporative losses (particularly the marked decreasewith 4-MTA) is not unexpected given the similarity of the target

FIG. 2––Effect of “salting out” target drugs with acidic methanol prior to evaporation. Data show the mean peak area (n = 10) and 1 SD (error bars).

KERRIGAN ET AL. . PSYCHOSTIMULANTS IN URINE BY LC/MS/MS 179

analytes to other amphetamines that are known to be volatile.Evaporative losses might have been minimized using a lowertemperature (<50°C), but as this method was being developed toscreen a large number of samples in a high-throughput setting,

the addition of acidic methanol was preferable to extendingassay time. The mean extraction efficiency (n = 5) from urineranged from 64 to 93% for all target analytes (Table 4). Meanaccuracies for controls evaluated at 50 and 250 ng/mL were 96–120% with corresponding intra-assay CVs between 0.5 and 5.6%(Table 5). Interassay CVs at 100 ng/mL ranged from 2.1 to20.8%. Quantitative performance at much lower concentrationswas also evaluated as part of LOQ (see below).

Detection and Quantitation

With the exception of 2C-B, the LOD was 0.5 ng/mL for allanalytes. An evaluation of ion ratio stability over a wide rangeof concentrations (0.5–50 ng/mL) indicated highly favorableresults (�25%), and the reproducibility of ion ratios (%CV) was<10%. 2C-B was the only exception, with a LOD of 1 ng/mL.For all target analytes except 2C-B, the criteria for the LOQwere also met at 0.5 ng/mL (1 ng/mL for 2C-B). However, theLOQ was arbitrarily set at 1 ng/mL for all target analytes.Signal-to-noise ratios and accuracy for all target drugs at theLOQ are summarized in Table 6. It should be noted that% accu-racy for all 15 drugs was 80–120% between 0.5 and 5 ng/mL.MRM transitions for a representative group of target analytesfrom the 2C, 2C-T, DO, and 4-MTA are shown in Fig. 3. Thelimit of linearity was reached at 500 ng/mL, and calibrationcurves were routinely run in the 0–300 ng/mL range. In thisrange, R2 values of at least 0.99 were achieved for all drugs.

Matrix Effects

Matrix effects were evaluated using a combination of post-column infusion and postextraction addition techniques. A pre-liminary evaluation of matrix effect during the developmentstage revealed significant loss of signal intensity at approxi-mately 0.5 min, causing potential suppression of the mesca-line-d9 internal standard. This suppression early on in thechromatogram is commonplace in LC-MS/MS procedures. Thiswas addressed early on by modifying the gradient elution pro-gram (from 25% mobile phase B initially, to 20%). Thisslight shift in retention time was sufficient to prevent suppres-sion of mescaline-d9. This highlights the value of postcolumninfusion during method development, as this approach allowsthe influence of the matrix on ionization to be evaluated over

TABLE 3––Effect of “salting out” on evaporative losses.

Drug

Salt Form Base Form

Mean RelativePeak Area

% CV(n = 10)

Mean RelativePeak Area % CV (n = 10)

2C-B 100 6 64 182C-C 100 2 49 222C-D 100 5 35 242C-E 100 6 39 242C-H 100 5 27 242C-I 100 2 69 162C-T-2 100 2 71 152C-T-4 100 9 73 162C-T-7 100 3 75 124-MTA 100 2 19 31DOB 100 2 54 23DOC 100 2 42 29DOET 100 2 34 23DOI 100 2 68 16DOM 100 2 31 22

TABLE 4––Extraction efficiency of target analytes from urine samples.

DrugRecovery

Mean � SD (n = 5)

2C-B 87 � 4%2C-C 89 � 4%2C-D 86 � 4%2C-E 80 � 4%2C-H 80 � 5%2C-I 87 � 5%2C-T-2 64 � 4%2C-T-4 75 � 5%2C-T-7 66 � 3%4-MTA 75 � 3%DOB 92 � 6%DOC 93 � 3%DOET 90 � 2%DOI 86 � 1%DOM 84 � 4%

TABLE 5––Precision and accuracy of target analytes from urine samples.

Drug

Intra-Assay50 ng/mL (n = 4)

Intra-Assay250 ng/mL (n = 4)

Inter Assay100 ng/mL (n = 4)

Mean � SD (ng/mL) Accuracy (%) CV (%) Mean � SD (ng/mL) Accuracy (%) CV (%) Mean � SD (ng/mL) Accuracy (%) CV (%)

2C-B 55 � 0.9 111 1.6 250 � 4.9 100 2.0 107 � 2 107 2.12C-C 55 � 2.3 109 4.2 245 � 4.3 98 1.7 101 � 6 101 6.12C-D 56 � 2.6 111 4.7 244 � 8.3 98 3.4 106 � 20 106 18.72C-E 58 � 3.3 116 5.6 252 � 5.7 101 2.3 109 � 12 109 11.12C-H 53 � 2.8 106 5.3 250 � 3.3 100 1.3 103 � 9 103 8.52C-I 54 � 1.5 109 2.7 249 � 7.7 99 3.1 102 � 6 102 5.62C-T-2 56 � 0.9 113 1.6 251 � 6.4 101 2.6 94 � 11 94 11.72C-T-4 56 � 1.5 112 2.7 243 � 10.6 97 4.4 97 � 13 97 13.42C-T-7 56 � 3.1 111 5.6 249 � 12.0 100 4.8 105 � 22 105 20.84-MTA 56 � 1.1 112 2.0 252 � 8.3 101 3.3 86 � 14 86 16.4DOB 57 � 1.7 115 3.0 253 � 3.4 101 1.3 94 � 10 94 10.9DOC 58 � 0.6 116 1.1 251 � 8.6 100 3.4 103 � 5 103 4.9DOET 60 � 2.2 120 3.6 242 � 4.8 97 2.0 114 � 9 114 8.3DOI 54 � 0.3 108 0.5 246 � 6.2 98 2.5 95 � 9 95 9.9DOM 60 � 2.0 120 3.3 241 � 13.8 96 5.7 113 � 10 113 8.9

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the entire chromatographic run. Following method develop-ment and optimization, a more rigorous statistical approach tomatrix effect was taken during the final validation of theassay using postextraction addition. The mean percent matrixeffect for all drugs was �1 to �18%, with CVs in the rangeof 5.5–9.0% (n = 20).

Interferences

No interferences were present for any of the common amphet-amines, endogenous bases, or common drugs investigated. Noneof the 29 interferences evaluated produced either a qualitative orquantitative interference for the negative control (0 ng/mL) or

FIG. 3––Multiple reaction monitoring (MRM) transitions of representative drugs at the limit of detection (LOD): 2C-B, 4-MTA, DOI, and 2C-T-7.

KERRIGAN ET AL. . PSYCHOSTIMULANTS IN URINE BY LC/MS/MS 181

positive control (100 ng/mL) when evaluated independently. Allquantitative values for the positive control were within 20% ofexpected, and no false positives were obtained for the negativecontrols. However, when the positive control (100 ng/mL) wasevaluated with a mixture containing all of the interfering sub-stances (1000 ng/mL), suppression of 2C-T-2 (4.12 min) andDOB (4.10 min) was evident. Although this might be attributedto meperidine (4.05 min), the substance that eluted with closestproximity, no interference was present when samples were eval-uated with meperidine alone. The excess quantity of so manydrugs in the interference mix may overload the source anddecrease the efficiency of ionization, but the presence of such alarge number of drugs in an actual sample appears most unli-kely. However, it does highlight a limitation of ultra sensitiveLC-MS/MS techniques, whereby ionization is somewhat capacitylimited.

Casework Samples

Only two of the 2021 urine samples tested contained any ofthe target analytes. Both samples in question contained DOI atconcentrations of 1 and 2 ng/mL in urine. A search of the litera-ture revealed no other reports of DOI in human subjects to date.The samples tested were adjudicated urine specimens that weredue for destruction and subsequently, several years old. Speci-mens were routinely stored at room temperature followingrelease of the report and adjudication of the case. Although sam-ples were stored refrigerated prior to testing in this study, verylittle is known of the stability of these drugs. This is a signifi-cant limitation. The two samples tested contained DOI at verylow concentrations, consistent with the high potency of theDO-series (Table 1). Recreational doses of DOI are very low(1.5–3 mg). Frequently encountered on LSD-like blotters, DOIis renowned for its profound hallucinogenic effects and is some-times referred to as “synthetic acid”. Although the limitationsregarding storage and stability should be considered, it is clearthat many of these substances will pose an analytical challengeto many laboratories that perform routine toxicology testing.

Conclusions

A technique capable of detecting very low concentrations of15 designer psychostimulants was developed using SPE and

LC-MS/MS. The 2C, 2C-T, and DO-series of designer drugs areused recreationally for their hallucinogenic and stimulant effects.Detection in toxicological samples is challenging due to poorcross-reactivity using common immunoassay techniques, lowrecreational doses for several of the drugs, limited pharmacologi-cal data in humans, absence of commercial metabolite referencematerials, and availability of confirmatory techniques. Using thistargeted procedure, concentrations as low as 0.5 ng/mL weredetected in urine. In a retrospective analysis of adjudicated case-work, the presence of DOI in two human subjects was confirmedat concentrations of 1 and 2 ng/mL, respectively. This representsthe first report of DOI in human subjects to date and highlightsthe analytical challenges associated with the detection of thesedrugs during routine toxicological testing.

Acknowledgments

We gratefully acknowledge the DEA Special Testing andResearch Laboratory in Virginia and Darrell L. Davis, formerLaboratory Director of the DEA/South Central Laboratory inDallas, TX for providing reference materials for substances thatwere not commercially available. We also acknowledge KenjiOta, Timothy Appel, and Dan Coleman of the California Depart-ment of Justice, Bureau of Forensic Services Toxicology Labora-tory (Sacramento, CA) for providing urine specimens. This workwas supported by Award No. 2008-DN-BX-K126 (NationalInstitute of Justice, Office of Justice Programs, U.S. Depart-ment of Justice). he opinions, findings, and conclusions orrecommendations expressed in this publication are those of theauthor(s) and do not necessarily reflect those of the Departmentof Justice.

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Additional information and reprint requests:Sarah Kerrigan, Ph.D.Forensic Science ProgramSam Houston State UniversityBox 2525, 1003 Bowers Blvd.Huntsville, TX 77341E-mail: sarah.kerrigan@shsu.edu

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