Automated Online Solid Phase Extraction Ultra High Performance

Jun 9, 2010 - abuse), through an automated online solid phase extrac- tion ultra high performance liquid chromatography method coupled with tandem mas...
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Anal. Chem. 2010, 82, 5636–5645

Automated Online Solid Phase Extraction Ultra High Performance Liquid Chromatography Method Coupled with Tandem Mass Spectrometry for Determination of Forty-Two Therapeutic Drugs and Drugs of Abuse in Human Urine Ugo Chiuminatto,† Fabio Gosetti,*,‡ Paolo Dossetto,† Eleonora Mazzucco,‡ Davide Zampieri,‡ Elisa Robotti,‡ Maria Carla Gennaro,‡ and Emilio Marengo‡ AB Sciex, via Tiepolo 18, 20052 Monza, Italy, and University of Piemonte Orientale, DISAV Dipartimento di Scienze dell’Ambiente e della Vita, viale Michel 11, 15121 Alessandria, Italy The study deals with a fast (analysis times of around 11 min) simultaneous identification and quantification in human urine of 42 drugs (21 therapeutic and 21 of abuse), through an automated online solid phase extraction ultra high performance liquid chromatography method coupled with tandem mass spectrometry (SPE UHPLCMS/MS). In the method validation, particular attention was devoted to the matrix effect, through matrix-matched calibration in blank urine, suitably diluted. For all the abuse drugs investigated, the limit of quantitation (LOQ) values are lower than the legal threshold concentration levels, making the method suitable for routine control. The whole procedure was applied in the analysis of urine of patients positive to the I level screening test. Consumption and abuse of drugs is continuously and progressively increasing all over the world and represent a serious social problem. Consumption is widely diffused also among adolescent and young people. These substances, that cause changes in perception and behavior and may produce hallucinogenic effects, give apparently positive effects such as euphoria, relaxation, and enhanced empathy. However, they also cause moodiness, intolerance, and dangerous consequences for health: psychological and physical dependence can lead to death. Moreover, drug misuse can cause social problems such as violence, motor vehicle accidents, homicides, and suicides. The widespread increase in the use of both illicit and therapeutic drugs is clearly evidenced by the more recent screening methods of analysis and control of river and sewage waters.1 A typical case is represented by benzodiazepines, therapeutically prescribed as a depressant to produce sedation and induce sleep but frequently abused. Large doses and/or daily use often cause physical and psychological dependences, like illicit drugs. * Corresponding author. Fax: +39 0131 360365. E-mail: fabio.gosetti@ mfn.unipmn.it. † AB Sciex. ‡ University of Piemonte Orientale. (1) Postigo, C.; Lopez de Alda, M. J.; Barcelo`, D. Anal. Chem. 2008, 80, 3123– 3134.

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The European Union has elaborated the EU Drugs Strategy 2005-2012 and EU action plan (2009-2012), which include a chapter concerning international collaborations addressing the need of balanced approaches among the states to face the problem. At present anyway, each European State applies its own legislation sanctions.2,3 As an example in Italy, the use of drug is ruled out by the DPR 309/1990 that deals with the licit trade, treatment, and prevention, as well as with prohibition and punishment of illicit activities.4 The number of the drugs consumed is steadily increasing, and drug lists must be continuously updated. When dealing with drug analysis in biological fluids (urine, blood, hair, and oral fluid), it must be underlined that most of the drugs are characterized by a short permanence time, that also depends on the dose and the kind of assumption, kind of matrix, and individual metabolism. In the human body, drugs are generally metabolized to watersoluble compounds and eliminated through urine. Anyway, their presence can be later detected as derivative species in plasma, urine, sweat, and hair. For example, heroin is transformed into morphine and 6-monoacetylmorphine (6-MAM); cocaine is transformed into benzoylecgonine (BE) and ecgonine methyl ester (EME),5 while ∆9-tetrahydrocannabinol (Cannabis THC) is metabolized to 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH) and to 11-hydroxy-∆9-tetrahydrocannabinol (THC-OH). Cannabis THC can be detected in plasma only up to 5 h from consumption and 10 h in urine, while the metabolite THCCOOH reaches in plasma persistence times up to 20-57 h in occasional users and 3-13 days in regular users.6 The analysis of drugs in biological samples is a very important routine task in clinical-medical and forensic fields. Generally, drugs of abuse in biological samples are screened in a I level test (2) Council of European Union. EU drugs action plan (2009-2012); Brussels, 2008. (3) EU drugs action plan (2009-12); Council of the European Union 2008. (4) Decree of the President of the Italian Republic n. 309/1990. Testo unico delle leggi in materia di disciplina degli stupefacenti e sostanze psicotrope, prevenzione, cura e riabilitazione dei relativi stati di tossicodipendenza; Rome, 1990. (5) Berg, T.; Lundanes, E.; Christophersen, A. S.; Strand, D. H. J. Chromatogr., B 2009, 877, 421–432. (6) Verstraete, A. Ther. Drug Monit. 2004, 26, 200–205. 10.1021/ac100607v  2010 American Chemical Society Published on Web 06/09/2010

Table 1. Chemical Class, CAS Number, Molecular Weight (*+HCl; **+2HCl), pKa, Cutoff Concentration Levels for Screening and Confirmation Tests for Each Drug Compounda

drug compound alprazolam amphetamine benzoylecgonine (BE) bromazepam brotizolam buprenorphine carbamazepine chlordiazepoxide clobazam clotiazepam cocaine codeine delorazepam dihydrocodeine (DHC) diazepam dimethyltryptamine (DMTA) 2-ethyllidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) ecgonine methyl ester (EME) estazolam flunitrazepam flurazepam heroin ketamine lorazepam lormetazepam lysergic acid diethylamide (LSD) 3,4-methylenedioxy-N-ethylamphetamine (MDEA) 6-monoacetylmorphine (6-MAM) 3,4-methylenedioxymethamphetamine (MDMA) medazepam methadone methamphetamine midazolam morphine nitrazepam nordazepam pinazepam prazepam temazepam ∆9-tetrahydrocannabinol (THC) 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THC-COOH) 11-hydroxy-∆9-tetrahydrocannabinol (THC-OH) a

chemical class benzodiazepine amphetamine alkaloid benzodiazepine benzodiazepine opiate benzodiazepine benzodiazepine benzodiazepine benzodiazepine alkaloid opiate benzodiazepine opiate benzodiazepine opiate alkaloid benzodiazepine benzodiazepine benzodiazepine opiate benzodiazepine benzodiazepine amphetamine opiate amphetamine benzodiazepine opiate amphetamine benzodiazepine opiate benzodiazepine benzodiazepine benzodiazepine benzodiazepine benzodiazepine cannabinoid cannabinoid cannabinoid

cutoff cutoff concentration concentration of screening of confirmation test (ng L-1) test (ng L-1)

CAS Number

molecular weight

pKa

28981-97-7 2706-50-5 519-09-5 1812-30-2 57801-81-7 53152-21-9 298-46-4 438-41-5 22316-47-8 33671-46-4 50-36-2 76-57-3 2894-67-9 125-28-0 439-14-5 61-50-7 30223-73-5

308.76 171.67* 289.22 315.15 392.71 504.10* 236.27 336.22* 300.74 318.84 303.35 299.36 305.16 301.38 284.74 188.27 277.41

2.4 10.1 >8 2.9; 11.0 N.A. N.A N.A. 4.6 6.7 N.A. >8 8.2 N.A. 8.8 3.3 N.A. N.A.

200 500 300 200 200 5 200 200 200 200 300 300 200 300 200 N.A. 100

200 200 150 200 200 5 200 200 200 200 50 50 200 50 200 N.A. 100

7143-09-1 29975-16-4 1622-62-4 1172-18-5 561-27-3 1867-66-9 846-49-1 848-75-9 50-37-3 82801-81-8 2784-73-8 69610-10-2 2898-12-6 76-99-3 537-46-2 59467-70-8 57-27-2 146-22-5 1088-11-5 52463-83-9 2955-38-6 846-50-4 1972-08-3 64280-14-4 36557-05-8

199.25 294.74 313.28 460.80** 369.41 274.19* 321.16 335.20 323.43 207.27 327.37 193.25 270.81 345.45* 149.23 325.78 285.34 281.32 270.37 308.23 324.18 300.74 314.45 344.45 330.46

>8 N.A 1.8 1.9; 8.2 7.6 7.5 1.3; 11.5 N.A. 7.5 9.3 N.A. N.A. 6.2 10.1 N.A. 6.1 8.2 3.2; 10.8 3.5; 12.0 5.4 2.7 1.6 10.6 N.A. N.A.

300 200 200 200 300 300 200 200 N.A. 500 300 500 200 300 500 200 300 200 200 200 200 200 50 50 50

50 200 200 200 50 100 200 200 N.A. 200 10 200 200 100 200 200 200 200 200 200 200 200 5 15 5

“N.A.” for not available.

performed through immunochemical techniques (enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorimetric polarization immunoassay (FPIA)).7-15 Positive samples are further confirmed with a II level test characterized by higher (7) Allen, K. R.; Azad, R.; Field, H. P.; Blake, D. K. Ann. Clin. Biochem. 2005, 42, 277–284. (8) Moore, C.; Lewis, D.; Cone, E. J. J. Anal. Toxicol. 2003, 27, 169–172. (9) Coulter, C.; Tuyay, J.; Taruc, M.; Moore, C. Forensic Sci. Int. 2010, 196, 70–73. (10) Pujol, M. L.; Cirimele, V.; Tritsch, P. J.; Villain, M.; Kintz, P. Forensic Sci. Int. 2007, 170, 189–192. (11) Han, E.; Miller, E.; Lee, J.; Park, Y.; Lim, M.; Chung, H.; Wylie, F. M.; Oliver, J. S. J. Anal. Toxicol. 2006, 30, 380–385. (12) Moore, K. A.; Werner, C.; Zanelli, R. M.; Levine, B.; Smith, M. L. Forensic Sci. Int. 1999, 106, 93–102. (13) Moore, C.; Ross, W.; Coulter, C.; Adams, L.; Rana, S.; Vincent, M.; Soares, J. J. Anal. Toxicol. 2006, 30, 413–418. (14) Miller, E. I.; Torrance, H. J.; Oliver, J. S. J. Anal. Toxicol. 2006, 30, 115– 119. (15) Moeller, K. E.; Lee, K. C.; Kissack, J. C. Mayo Clin. Proc. 2008, 83, 66– 76.

sensitivities to minimize the number of false positive responses. The cutoff concentrations for the screening test of drugs in biological fluids (in Europe) are reported in Table 1.6,16 The screening cutoff values in urine samples are not the same for all the countries; for example, concerning opiates, the threshold concentration is 300 ng mL-1 in Europe and Australia and 2000 ng mL-1 in the U.S.17 Rapid, accurate, precise, and possibly cheap multicomponent methods are, therefore, required to overcome the long analysis times of both screening and confirming tests.18 The techniques more largely employed in the determination of drugs of abuse (16) Quality Commission of Italian Toxicology Forensic Group. Guidelines of the laboratories of abuse drug analysis for forensic purposes; 2008. http:// tossicologia-forense.unina2.it/it/linee_guida_per_i_laboratori_di_analisi.htm (accessed June 2010). (17) Scientific Section Division for Operations and Analysis. Rapid on-site screening of drugs of abuse of United Nations International Drug Control Programme; Vienna, 2001. (18) http://www.toxlab.co.uk/dasguide.htm (accessed June 2010).

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are liquid chromatography with electrochemical (HPLC-ED)19,20 and diode array detectors (HPLC-DAD),21,22 capillary electrochromatography-time-of-flight mass spectrometry (CEC-TOF),23 and liquid-liquid extraction online coupled with atomic absorption spectrometry (AAS).24 More recent methods are based on gas or liquid chromatography, hyphenated with mass spectrometry detection,25,26 GC/MS with sample pretreatment or derivatization,27-33 HPLC-MS/MS1,34-42 and ultra high performance liquid chromatography (UHPLC)-MS methods.5,25,26,43,44 The matrixes analyzed to identify and quantify drugs of abuse can be obtained through invasive techniques, such as serum and plasma, and less invasive ones, such as urine, hair, oral fluid, and sweat.25,45 Since urine samples might be subjected to adulteration, through substitution, dilution, or addition of “masking agents”, a method clinically accepted to check for adulterations or dilutions consists of the determination of typical urinary characteristics such as creatinine content. A sample pretreatment of urine is strictly necessary to eliminate matrix interfering species (endogenous substances like proteins and nonvolatile species), to preconcentrate the analytes of interest, and to reduce, when HPLC-MS techniques are used, possible signal suppression/enhancement (effects) due to the matrix (19) Schleyer, E.; Lohmann, R.; Rolf, C.; Gralow, A.; Kaufmann, C. C.; Unterhalt, M.; Hiddemann, W. J. Chromatogr., B 1993, 614, 275–283. (20) Kramer, E.; Kovar, K.-A. J. Chromatogr., B 1999, 731, 167–177. (21) Mercolini, L.; Mandrioli, R.; Conti, M.; Leopardi, C.; Gerra, G.; Raggi, M. A. J. Chromatogr., B 2007, 847, 95–102. (22) Ferna´ndez, P.; Morales, L.; Va´zquez, C.; Lago, M.; Bermejo, A. M. J. Appl. Toxicol. 2008, 28, 998–1003. (23) Blas, M.; McCord, B. R. Electrophoresis 2008, 29, 2182–2192. (24) Montero, R.; Gallego, M.; Valcarcel, M. Anal. Chim. Acta 1991, 252, 83– 88. (25) Maquille, A.; Guillarme, D.; Rudaz, S.; Veuthey, J. - L. Chromatographia 2009, 70, 1373–1380. (26) Eichhorst, J. C.; Etter, M. L.; Rousseaux, N.; Lehotay, D. C. Clin. Biochem. 2009, 42, 1531–1542. (27) Kudo, K.; Ishida, T.; Hikiji, W.; Hayashida, M.; Uekusa, K.; Usumoto, Y.; Tsuji, A.; Ikeda, N. Forensic Toxicol. 2009, 27, 21–31. (28) Gentili, S.; Torresi, A.; Marsili, R.; Chiarotti, M.; Macchia, T. J. Chromatogr., B 2002, 780, 183–192. (29) Kim, J. Y.; Shin, S. H.; In, M. K. Forensic Sci. Int. 2010, 194, 108–114. (30) Guthery, B.; Bassindale, A.; Pillinger, C. T.; Morgan, G. H. Rapid Commun. Mass Spectrom. 2009, 23, 340–348. (31) Valente-Campos, S.; Yonamine, M.; de Moraes Moreau, R. L.; Alves Silva, O. Forensic Sci. Int. 2006, 159, 218–222. (32) Barroso, M.; Dias, M.; Vieira, D. N.; Queiroz, J. A.; Lo´pez-Rivadulla, M. Rapid Commun. Mass Spectrom. 2008, 22, 3320–3326. (33) Chung, L. W.; Liu, G. J.; Li, Z. G.; Chang, Y. Z.; Lee, M. R. J. Chromatogr., B 2008, 874, 115–118. (34) Bouzas, N. F.; Dresen, S.; Munz, B.; Weinmann, W. Anal. Bioanal. Chem. 2009, 395, 2499–2507. (35) Wu, T.-Y.; Fuh, M.-R. Rapid Commun. Mass Spectrom. 2005, 19, 775–780. (36) Liu, A.-C.; Lin, T.-Y.; Su, L.-W.; Fuh, M.-R. Talanta 2008, 75, 198–204. (37) Link, B.; Haschke, M.; Wenk, M.; Krahenbuhl, S. Rapid Commun. Mass Spectrom. 2007, 21, 1531–1540. (38) Tabernero, M. J.; Felli, M. L.; Bermejo, A. M.; Chiarotti, M. Anal. Bioanal. Chem. 2009, 395, 2547–2557. (39) Gray, T. R.; Shakleya, D. M.; Huestis, M. A. Anal. Bioanal. Chem. 2009, 393, 1977–1990. (40) Ishida, T.; Kudo, K.; Hayashida, M.; Ikeda, N. J. Chromatogr., B 2009, 877, 2652–2657. (41) Marchi, I.; Schappler, J.; Veuthey, J. - L.; Rudaz, S. J. Chromatogr., B 2009, 877, 2275–2283. (42) Yao, J.; Hou, L.; Zhou, J.; Zhang, Z. Chromatographia 2009, 70, 83–88. (43) Stephanson, N.; Josefsson, M.; Kronstrand, R.; Beck, O. J. Chromatogr., B 2008, 871, 101–108. (44) Bijlsma, L.; Sancho, J. V.; Pitarch, E.; Iba´nez, M.; Herna´ndez, F. J. Chromatogr., A 2009, 1216, 3078–3089. (45) Wood, M.; Laloup, M.; Samyn, N.; Ramirez Fernandez, M. M.; de Bruijn, E. A.; Maes, R. A. A.; De Boeck, G. J. Chromatogr., A 2006, 1130, 3–15.

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effect.34 Liquid-liquid extraction (LLE),23-25 off-line solid phase extraction (SPE),30,44 and online SPE34-36 are the most used techniques. The online SPE strategy seems to be the best choice to improve method sensitivity, to shorten pretreatment and analysis times, and to increase the number of the samples analyzed in the same time. This study presents the development of an online SPE UHPLCMS/MS method for the determination in human urine of 42 analytes, belonging to different chemical classes. Table 1 reports the compounds studied, their chemical classes, the CAS number, and some physicochemical properties. To our knowledge, no online SPE UHPLC-MS/MS method for the simultaneous identification and quantitation of such a high number of both therapeutic drugs and drugs of abuse is present in literature. The online extraction procedure employed here is based on the use of a cationic extraction column coupled with a LC-MS hybrid mass spectrometer through a two-way switching valve, which allows the loading and the injection steps. EXPERIMENTAL SECTION Reagents. The drugs studied were reported in Table 1. All analytes, methanol, acetonitrile, formic acid, ammonium acetate, ultrapure water, β-glucuronidase (type L-II from limpets), sodium acetate, and glacial acetic acid were purchased from Sigma-Aldrich (Milwaukee, WI). All reagents were characterized by LC-MS grade. Stock solutions for all the drugs were prepared at 10 µg mL-1 in methanol as the solvent, with the exception of heroin, prepared in acetonitrile/ultrapure water 50/50 (v/v) due to its poor stability in methanol solution. The real urine samples were kindly provided by Medicina Legale of Modena General Hospital, and the blank urine samples were obtained from the laboratory analysts. Instrumentation. The chromatographic analyses were performed using a Dionex (Sunnyvale, CA) Ultimate 3000 UHPLC system constituted of an Ultimate 3000 Degasser, an Ultimate 3000 Pump, an Ultimate 3000 RS autosampler, and an Ultimate 3000 RS column compartment. The system was interfaced with a 3200 QTrap LC-MS/MS system (Applied Biosystems, Foster City, CA) by a Turbo V interface equipped with an electrospray ionization probe. Data were processed by Analyst 1.5 software (Toronto, Canada). An IEC CL31R multispeed centrifuge (Thermo Electron Corp., Milford, MA) was employed in the sample pretreatment. UHPLC-MS/MS Conditions. The stationary phase was a Zorbax Eclipse XDB-C18 column (4.6 mm × 50.0 mm, 1.8 µm) acquired from Agilent (Milan, Italy). The mobile phase was a mixture of 0.5% HCOOH in ultrapure water (A) and 0.5% HCOOH in acetonitrile (B) eluting under the gradient conditions reported in Table 2a. The flow rate was 900 µL min-1, and the injection volume was 50 µL. The temperatures of the sampler and the column oven were set at 5 and 55 °C, respectively. The turbo ion spray (TIS) ionization was obtained using the Turbo V interface working in positive polarity ion mode (PI). The instrumental parameters were set as follows: curtain gas (N2) at 30 psig, nebulizer gas GS1 and GS2 at 45 and 60 psig, respectively, desolvation temperature (TEM) at 650 °C, collision activated dissociation gas (CAD) at 6 units of the arbitrary scale of the instrument, and ionspray voltage (IS) at 4300 V. The 3200QTrap was used in scheduled multiple reaction monitoring

Table 2. (a) UHPLC Conditionsa; (b) On-Line SPE Conditions a UHPLC time (min)

A (%)

B (%)

flow rate (µL min-1)

0.0 1.3 3.1 5.5 5.6 11.0

90 90 0 0 90 90

10 10 100 100 10 10

900 900 900 900 900 900

b SPE -1

time (min)

flow rate (µL min )

valve position

0.0 1.5 2.5 5.0 6.0 11.0

3000 3000 300 300 3000 3000

load inject inject inject load load

a A (%) ) percentage of ultrapure water (0.5%) in HCOOH and B (%) ) percentage of acetonitrile (0.5%) in HCOOH.

(sMRM) considering the transitions of each species at a prefixed retention time. Unit mass resolution was established and maintained in each mass-resolving quadrupole by keeping a full width at half-maximum (fwhm) of about 0.7 u. Online SPE Conditions. An online SPE system was applied to pretreat the urine samples, using a SPE Strata X-CW (2 mm × 20 mm, 25 µm) from Phenomenex (Bologna, Italy). The loading solution was ammonium acetate, 2.0 mM, prepared in a mixture

of ultrapure water and methanol 85/15 (v/v), and the elution agent was the mobile phase (composition as at time ) 0 min) used in the chromatographic separation. The system setup for online SPE consists of three steps (Figure 1). In the first step (loading), 50 µL of urine samples are loaded onto the cartridge using the UHPLC autosampler. The trap cartridge is fitted into the loading position of Valco 6 port switching valve. The Dionex Ultimate 3000 RS Dual pump (left pump) is used to load the sample at high flow (3000 µL min-1) onto the trapping cartridge. The sample matrix is flushed to waste, while the analytes are retained on the SPE column, and simultaneously, the analytical LC column is equilibrated with the chromatographic pump (right pump). In the second step (injection), after 1.5 min, the valve is switched to injection position, that couples the SPE column with the chromatographic column, in which the analytes are transferred. The Dionex right pump is used to provide the gradient elution. In the third final step (separation), while the analytes are separated in the analytical column, after 6.0 min, the switching valve is switched back to the loading position to re-equilibrate the online SPE cartridge with the loading phase, prior to the analysis of the next sample. The detailed online SPE conditions are reported in Table 2b. Urine Sample Preparation. The biological samples of urine were treated by enzymatic hydrolysis in order to remove the glucuronide bonds and to ensure solubility and the quantitative extraction of the drug compounds. The β-glucuronidase enzyme was dissolved in 4.0 mL of acetate buffer prepared by dissolving 73.8 mg of sodium acetate in 100.0 mL of ultrapure water, previously brought to pH 4.5 with glacial acetic acid.

Figure 1. Valco valve position (A ) loading, B ) injection) and instrumental configuration of online SPE. Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Table 3. sMRM Transitions (Q1 and Q3 Masses) and Mass Spectrometry Parametersa drug compound

Q1 mass (m/z)

Q3 mass (m/z)

time (min)

DP (volts)

EP (volts)

CEP (volts)

CE (volts)

CXP (volts)

*alprazolam alprazolam *amphetamine amphetamine *BE BE *bromazepam bromazepam *brotizolam brotizolam *buprenorphine buprenorphine *carbamazepine carbamazepine *chlordiazepoxide chlordiazepoxide *clobazam clobazam *clotiazepam clotiazepam *cocaine cocaine *codeine codeine *delorazepam delorazepam *DHC DHC *diazepam diazepam *DMTA DMTA *EDDP EDDP *EME EME *estazolam estazolam *flunitrazepam flunitrazepam *flurazepam flurazepam *heroin heroin *ketamine ketamine *lorazepam lorazepam *lormetazepam lormetazepam *LSD LSD *MDEA MDEA *6-MAM 6-MAM *MDMA MDMA *medazepam medazepam *methadone methadone *methamphetamine methamphetamine *midazolam midazolam *morphine morphne *nitrazepam nitrazepam *nordazepam nordazepam *pinazepam pinazepam *prazepam prazepam *temazepam temazepam *THC THC *THC-COOH THC-COOH *THC-OH THC-OH

309.14 309.14 136.10 136.10 290.14 290.14 316.03 318.03 393.00 393.00 468.26 468.26 237.18 237.18 300.12 300.12 301.15 301.15 319.07 319.07 304.16 304.16 300.14 300.14 305.05 305.05 302.14 302.14 285.14 285.14 189.13 189.13 278.20 278.20 200.13 200.13 295.14 295.14 314.12 314.12 388.23 388.23 370.12 370.12 238.13 238.13 321.08 323.08 335.06 337.06 324.20 324.20 208.15 208.15 328.11 328.11 194.13 194.13 271.18 271.18 310.21 310.21 150.17 150.17 326.08 326.08 286.13 286.13 282.12 282.12 271.14 271.14 309.10 309.10 325.17 325.17 301.10 301.10 315.22 315.22 345.16 345.16 331.20 331.20

205.10 281.10 91.10 119.20 168.00 105.10 182.10 182.10 314.10 210.00 396.10 414.10 194.20 193.10 227.10 283.10 259.00 224.20 291.00 154.00 182.10 77.00 115.00 152.20 140.00 242.10 199.00 128.10 193.10 154.00 58.10 144.10 234.10 249.10 82.10 83.00 205.10 267.20 268.10 239.10 315.10 317.10 165.10 58.10 125.00 89.10 275.00 277.10 289.00 291.00 223.10 208.00 163.00 135.00 152.20 165.00 163.10 105.00 207.20 91.10 265.00 105.10 91.10 119.20 291.10 249.00 152.00 115.20 236.00 180.20 139.90 165.10 241.10 163.10 271.10 140.00 255.10 257.10 193.30 123.00 193.00 299.20 313.20 193.10

3.7 3.7 2.9 2.9 3.1 3.1 3.4 3.4 3.8 3.8 3.4 3.4 3.6 3.6 3.2 3.2 3.8 3.8 3.8 3.8 3.2 3.2 2.7 2.7 3.8 3.8 2.7 2.7 3.9 3.9 2.9 2.9 3.4 3.4 2.2 2.2 3.6 3.6 3.8 3.8 3.3 3.3 3.1 3.1 3.0 3.0 3.7 3.7 3.8 3.8 3.2 3.2 3.0 3.0 2.9 2.9 2.9 2.9 3.3 3.3 3.5 3.5 2.9 2.9 3.3 3.3 2.4 2.4 3.6 3.6 3.7 3.7 3.7 3.7 4.2 4.2 3.8 3.8 4.8 4.8 4.3 4.3 4.3 4.3

51.00 51.00 21.00 21.00 31.00 31.00 46.00 46.00 46.00 46.00 76.00 76.00 31.00 31.00 31.00 31.00 36.00 36.00 51.00 51.00 36.00 36.00 51.00 51.00 56.00 56.00 46.00 46.00 51.00 51.00 16.00 16.00 56.00 56.00 36.00 36.00 46.00 46.00 51.00 51.00 36.00 36.00 61.00 61.00 26.00 26.00 36.00 36.00 31.00 31.00 41.00 41.00 21.00 21.00 56.00 56.00 21.00 21.00 51.00 51.00 31.00 31.00 21.00 21.00 56.00 56.00 61.00 61.00 46.00 46.00 41.00 41.00 51.00 51.00 36.00 36.00 26.00 26.00 41.00 41.00 36.00 36.00 26.00 26.00

8.00 8.00 2.50 2.50 8.00 8.00 8.50 8.50 8.00 8.00 9.00 9.00 8.00 8.00 4.50 4.50 8.00 8.00 9.50 9.50 8.00 8.00 8.50 8.50 8.00 8.00 9.50 9.50 8.00 8.00 8.00 8.00 8.50 8.50 8.00 8.00 6.50 6.50 8.50 8.50 8.00 8.00 9.50 9.50 9.00 9.00 6.00 6.00 6.50 6.50 8.50 8.50 8.00 8.00 9.00 9.00 8.00 8.00 8.00 8.00 6.50 6.50 4.50 4.50 8.00 8.00 9.00 9.00 8.00 8.00 9.00 9.00 8.50 8.50 7.50 7.50 8.50 8.50 5.00 5.00 5.00 5.00 5.00 5.00

17.97 17.97 12.26 12.26 17.34 17.34 18.20 18.26 20.74 20.74 23.22 23.22 15.60 15.60 17.67 17.67 17.71 17.71 18.30 18.30 17.81 17.81 17.67 17.67 17.84 17.84 17.74 17.74 16.00 16.00 14.01 14.01 16.95 16.95 14.37 14.37 17.51 17.51 18.14 18.14 18.00 18.00 19.98 19.98 15.63 15.63 18.37 18.43 18.83 18.89 18.47 18.47 14.64 14.64 18.60 18.60 14.18 14.18 14.00 14.00 18.01 18.01 12.73 12.73 18.53 18.53 17.21 17.21 17.08 17.08 16.72 16.72 17.97 17.97 22.00 22.00 17.71 17.77 18.17 18.17 19.16 19.16 18.70 18.70

53.00 35.00 21.00 11.00 23.00 41.00 41.00 41.00 29.00 55.00 49.00 37.00 27.00 41.00 31.00 17.00 23.00 41.00 25.00 39.00 23.00 75.00 93.00 77.00 41.00 41.00 43.00 81.00 43.00 35.00 23.00 23.00 39.00 29.00 35.00 35.00 51.00 31.00 29.00 43.00 29.00 23.00 57.00 50.00 39.00 71.00 29.00 29.00 25.00 25.00 33.00 39.00 17.00 29.00 93.00 63.00 17.00 33.00 35.00 41.00 19.00 41.00 23.00 15.00 37.00 47.00 73.00 87.00 33.00 47.00 37.00 37.00 41.00 65.00 29.00 49.00 29.00 29.00 27.00 45.00 33.00 23.00 17.00 35.00

4.00 4.60 4.00 4.00 4.00 4.00 4.00 4.00 4.60 4.00 5.00 5.00 4.00 4.00 4.00 4.60 4.00 4.00 6.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 2.00 2.00 4.00 4.60 4.00 4.00 4.60 4.60 4.00 2.00 4.00 4.00 4.40 4.40 4.60 4.60 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.60 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.00 4.60 4.60 4.00

a DP (declustering potential), EP (entrance potential), CEP (collision cell entrance potential), CE (collision energy), and CXP (collision cell exit potential). For each species, the most sensitive transition, marked as *, was used for quantitation (quantifier) and the second one was used for confirmation (qualifier).

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Table 4. Regression Coefficient (R2), Linearity Range, LOD, LOQ, Intra- and Interday RSD (%) on Concentration, j (%), and Matrix Effect (ME) for the 42 Analytes Considered Average Recovery Yields R drug compound

R2

alprazolam amphetamine BE bromazepam brotizolam buprenorphine carbamazepine chlordiazepoxide clobazam clotiazepam cocaine codeine delorazepam DHC diazepam DMTA EDDP EME estazolam flunitrazepam flurazepam heroin ketamine lorazepam lormetazepam LSD MDEA 6-MAM MDMA medazepam methadone methamphetamine midazolam morphine nitrazepam nordazepam pinazepam prazepam temazepam THC THC-COOH THC-OH

0.9998 0.9981 0.9990 0.9833 0.9995 0.9964 0.9947 0.9994 0.9985 0.9995 0.9966 0.9976 0.9946 0.9986 0.9991 0.9969 0.9928 0.9992 0.9971 0.9995 0.9950 0.9971 0.9881 0.9966 0.9995 0.9984 0.9877 0.9965 0.9945 0.9945 0.9981 0.9990 0.9975 0.9987 0.9883 0.9940 0.9802 0.9995 0.9982 0.9981 0.9991 0.9955

linearity range (µg L-1) LOD (µg L-1) LOQ (µg L-1) intraday RSD (%) interday RSD (%) 2.42-100.00 0.73-100.00 5.00-100.00 5.00-100.00 2.64-100.00 4.80-100.00 1.70-100.00 3.12-100.00 5.00-100.00 4.08-100.00 2.93-100.00 2.50-100.00 4.01-100.00 1.80-100.00 2.14-100.00 3.58-100.00 0.66-100.00 4.45-100.00 3.81-100.00 2.50-100.00 2.38-100.00 4.70-100.00 3.93-100.00 4.72-100.00 5.00-100.00 1.09-100.00 0.82-100.00 5.00-100.00 2.71-100.00 2.37-100.00 1.11-100.00 1.83-100.00 1.42-100.00 5.00-100.00 5.00-100.00 5.00-100.00 5.00-100.00 1.67-100.00 2.09-100.00 2.50-100.00 2.50-100.00 1.80-100.00

0.73 0.22 1.52 1.52 0.80 1.45 0.52 0.95 1.52 1.24 0.89 0.76 1.22 0.55 0.65 1.08 0.20 1.35 1.15 0.76 0.72 1.42 1.19 1.43 1.52 0.33 0.25 1.52 0.82 0.72 0.34 0.55 0.43 1.52 1.52 1.52 1.52 0.51 0.63 0.76 0.76 0.55

A volume of 10.0 µL of the enzymatic solution was added to 500.0 µL of acetate buffer and 500.0 µL of urine sample. The whole solution was kept in an incubator at 65 °C for 2 h, and then, 200.0 µL of the hydrolyzed solution was diluted with 800.0 µL of SPE loading solution and subjected to centrifugation for 30 s at 10 000 rpm. Finally, the supernatant was analyzed by the online SPE UHPLC-MS/MS method. RESULTS AND DISCUSSION Mass Spectrometry Characterization of the Analytes. The analytes were previously subjected to a MS/MS characterization study with TIS (PI mode) source, to identify the fragmentation patterns formed under increasing collisional energy. The experiments were carried out for direct infusion (flow rate of 10 µL min-1) of 300.00 µg L-1 solutions of each analyte, with the SPE loading solution as the solvent. All the species presented many transitions: for each species, the most intense transition was used for the quantitative analysis and was referred to as the “quantifier” transition, while the second one (the “qualifier” transition) was employed in the identification step, as a confirmation. The “quantifier” and “qualifier” transitions and instrumental potential values for each compound are reported in Table 3.

2.42 0.73 5.00 5.00 2.64 4.80 1.70 3.12 5.00 4.08 2.93 2.50 4.01 1.80 2.14 3.58 0.66 4.45 3.81 2.50 2.38 4.70 3.93 4.72 5.00 1.09 0.82 5.00 2.71 2.37 1.11 1.83 1.42 5.00 5.00 5.00 5.00 1.67 2.09 2.50 2.50 1.80

0.1 0.1 3.5 5.3 1.8 6.1 0.8 1.0 1.0 0.3 1.2 0.1 0.5 0.6 0.5 3.0 0.2 2.3 1.3 0.1 2.1 6.6 2.8 1.9 2.8 0.9 0.1 0.1 0.5 2.0 0.4 0.1 0.3 2.1 0.1 0.8 0.9 0.4 0.1 1.1 0.1 2.3

0.3 0.5 4.5 9.3 2.8 8.1 2.8 4.0 6.0 3.3 2.2 0.9 3.5 1.6 5.5 4.0 1.2 5.3 3.3 4.0 3.1 9.6 5.8 3.9 3.8 1.9 2.1 2.0 2.5 3.0 2.4 0.4 1.3 3.1 4.1 7.8 1.9 1.4 2.0 3.1 5.0 3.3

¯ (%) R

ME (%)

100.5 (±0.4) 94.4 (±1.0) 97.7 (±5.1) 102.0 (±9.5) 98.6 (±3.2) 102.0 (±8.8) 99.2 (±3.2) 101.4 (±4.0) 97.8 (±6.8) 99.1 (±4.1) 99.0 (±2.8) 104.7 (±1.7) 103.9 (±3.7) 102.5 (±2.3) 99.8 (±6.0) 98.5 (±4.8) 97.6 (±1.7) 100.5 (±5.6) 105.3 (±4.2) 98.8 (±4.0) 98.8 (±3.6) 98.2 (±9.7) 95.7 (±6.0) 100.0 (±4.2) 98.1 (±4.8) 96.6 (±1.9) 96.0 (±2.5) 97.2 (±2.9) 94.1 (±2.8) 98.0 (±3.9) 98.7 (±2.6) 101.0 (±0.9) 100.6 (±2.3) 96.2 (±4.1) 93.8 (±4.6) 101.8 (±7.9) 101.7 (±2.4) 100.8 (±2.0) 100.4 (±2.4) 99.6 (±4.1) 101.9 (±5.8) 99.2 (±4.0)

-20 -6 -30 +3 -5 -1 -10 -13 -20 -5 -50 +10 +4 +2 0 -10 -10 -49 +2 0 -20 -20 -10 0 +10 -1 -10 -20 -6 -8 -19 -15 +5 -33 +3 +4 -10 +7 +10 0 +14 0

Development and Optimization of the Analytical Method. It is important to underline that the analytes considered belong to different chemical classes and are characterized by different functionalities and different acidic properties, that largely affect the choice of the extraction and of the chromatographic conditions. Different types of SPE sorbents and elution phases were tested, and the results were compared in terms of chromatographic resolution and peak symmetry. While C18 sorbent characterized by hydrophobic interactions did not allow the retention of the more polar drugs, C18 sorbent bound with polar groups permitted good sorption and chromatographic separation, with the only exception of EME. To also include EME, a cationic/ hydrophobic SPE column (Strata X-CW) was used. While benzodiazepines undergo hydrophobic interactions, the cationic/ hydrophobic interactions retain more efficaciously basic polar compounds (like EME, morphine, cocaine, etc.). Great attention must be addressed to the SPE loading phase composition: a good compromise between SPE column sorption and chromatographic elution was reached with ammonium acetate. At pH ) 7.0, THC (pKa ) 10.6) and its metabolites are present in their undissociated forms, which better interact with Strata X-CW sorbent, Analytical Chemistry, Vol. 82, No. 13, July 1, 2010

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Figure 2. Online SPE UHPLC-MS/MS chromatogram of a mixture of the analytes at 25.00 µg L-1, each prepared in a blank urine sample, diluted 1/10 (v/v). The chromatographic conditions are reported in the Experimental Section.

leading to an increase in the recovery values. In addition, for a good and fast separation, sharp changing of the pH value from 7.0 (SPE loading phase) to 2.5 (chromatographic mobile phase) favors the separation in terms of both resolution and analysis time since it increases the elution of BE and morphine from the SPE column and decreases their retention time, also avoiding peak tailing. Moreover, the use of acetonitrile in the mobile phase allows a greater symmetry of the chromatographic peaks. In order to evaluate the maximum injection volume, some experiments carried out by increasing the injection volume (5, 10, 25, 50, 75, and 100 µL) showed that 50 µL represents the maximum tolerable volume for the online SPE UHPLC-MS system. For higher volumes, the more polar compounds do not show linearity of response with concentration, very likely due to the overload of SPE cartridge. Validation of the Analytical Method. To develop the method and to evaluate the matrix effect, 10 blank urine samples coming from different analysts of our laboratory were diluted 1/10 (v/v) with the loading solution used in SPE and spiked with the mixture of all the analytes at five concentration levels (namely, 5.00, 10.00, 25.00, 50.00, and 100.00 µg L-1). To test possible differences among the blank samples, calibration plots were built for each analyte and the slopes and intercepts of the plots were compared through a t-test at a 99% confidence level. The relative standard deviation (RSD, %) was on the order of 3% on the slopes and around 20% on the intercepts, to indicate no 5642

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statistically significant difference among the 10 blank samples. The results were likely due to the good cleanup obtained with the washing process of the SPE column, performed during the loading step with a volume of 150-fold of that of the column. The limit of detection (LOD) was calculated as the concentration of the analyte that gives a signal (peak area) equal to the average background (Sblank) plus three times the standard deviation sblank of the blank (LOD ) Sblank + 3sblank), while the limit of quantitation (LOQ) is given as LOQ ) Sblank + 10sblank.46 For each drug, a calibration plot, reporting the peak area of the “quantifier” transition (y) versus the concentration of standard solutions prepared spiking a blank urine sample (x), was built. A linear regression fit was used for all analytes, with a weighting factor of 1/x. Five concentration levels in the range between LOQ and 100.00 µg L-1 were considered. For all the analytes, a good linearity was observed between peak area and concentration. Linearity ranges, R2, LODs, and LOQs for all the analytes are reported in Table 4. Figure 2 shows a typical chromatogram recorded for the mixture of the analytes at 25.00 µg L-1, each prepared in a blank urine sample. Intra- and interday precision on retention time and on concentration was determined by analyzing blank urine samples previously diluted 1/10 (v/v) and spiked with the mixture of all the analytes (30.00 µg L-1 each) five times every day for a week. (46) Miller, J. N.; Miller, J. C. Statistics and Chemometrics for Analytical Chemistry, 4th ed.; Prentice Hall: Upper Saddle River, NJ, 2000.

Figure 3. xy-plot of Cobs vs Cref for nitrazepam (the lowest recovery), estazolam (the greatest recovery), and EME (in the range of recovery). The analyses were replicated three times for each concentration level.

The results showed that intraday precision ranged from 0.01% to 0.43% and interday precision ranged from 0.01% to 3.54%. The intraday and interday RSDs (%) on concentration ranged from 0.1% to 6.6% and 0.3% to 9.6%, respectively (Table 4). To check the stability of the quantitative response, at random intervals along the analyses, standard QC quality control solutions at concentrations of 30.00 and 90.00 µg L-1 were injected: all the results obtained for QC solutions lay within the ±3σ control limits of the calibration plots. In order to evaluate the recovery R and investigate a possible recovery dependence on concentration, two sets of multicomponent solutions of each analyte at three different levels of concentration (50.00, 75.00, and 100.00 µg L-1) (of each analyte) were analyzed, repeating each analysis three times.47 The first set of solutions was analyzed by injecting volumes of 20 µL without the use of the SPE column. (The injection volume was established both to have chromatographic peaks with a satisfactory sensitivity and to not overload the chromatographic column.) The second set of solutions was analyzed by injecting volumes of 20 µL into the online SPE UHPLC-MS/MS system. The results of the first set define the expected concentrations (Cref), and the results of the second one gives the concentrations observed (Cobs). The ratio Cobs/Cref gives the recovery R. A t-test showed that for all the analytes the R values obtained for the different concentration levels were not significantly different, at a 95% confidence level. In these conditions and in the explored concentration range, recovery does not depend on the concentration level of the analytes. Cobs as a function of Cref was graphically reported for some drugs in Figure ¯ (%) values for all the analytes, 3. The average percentage recovery R together with the respective deviations, are reported in Table 4: they are all greater than 93.8%. The possible elimination of matrix effect was then evaluated, since it is a very important aspect in the LC/MS analysis of biological fluids, that may affect the results of both qualitative and quantitative analyses. First, experiments were performed to evaluate the optimum dilution factor (2, 5, 10, and 20 times) suitable to gain, for all the analytes, the best compromise between method sensitivity and (47) Maroto, A.; Boque´, R.; Riu, J.; Rius, F. X. Anal. Chim. Acta 2001, 446, 133–145.

Figure 4. xy-plot of Aint vs Aext for benzoylecgonine (signal suppression), lorazepam (no matrix effect), and temazepam (signal enhancement). The plot reports the average area of three analyses.

relevance of the matrix effect. A dilution factor 1/10 (v/v) satisfies these requirements.48 To quantify the matrix effect, the use of the standard addition method was preferred here to the expensive use of 42 labeled internal standards.49 For each analyte, samples with concentrations laying in the linearity range (namely, 5.00, 10.00, 25.00, 50.00, and 100.00 µg L-1) were prepared both in SPE loading solution and in blank urine previously diluted 1/10 (v/v) in SPE loading solution. This dilution of urine was necessary to avoid overload of the cartridge, as also recommended by producers. The analyses were repeated three times. For each analyte, the average matrix effect was estimated, considering, for each concentration level, the ratio Aint/Aex, where Aint is the average peak area of the analyte in the analysis of the blank urine and Aex is the average peak area of the analyte in SPE loading solution. The results evidenced that the matrix effect depends on the analyte (Table 4). While no instrumental matrix effect was evidenced for diazepam, flunitrazepam, lorazepam, THC, and THC-OH, it ranges from a signal suppression of 50% (for cocaine) to a signal enhancement of 14% (for THC-COOH). Around 60% of the compounds showed a signal suppression, more typical of the ESI process, while 29% of the drugs were affected by a signal enhancement. For example, Figure 4 shows the different behaviors (suppression, enhancement, and no matrix effect) of three analytes. However, it was not possible to generalize and attribute the matrix effect to specific chemical structures. Benzodiazepines evidenced a matrix effect ranging from a signal suppression of 20% to a signal enhancement of 20%.48 The analytes mostly affected by the matrix effect were morphine and cocaine with its two metabolites (EME and BE), likely due to difficulties to optimize the chromatographic conditions, due to the polar characteristics of the analytes. In order to compensate for the LC/MS matrix effect, the matrix-matched calibration plots in blank urine sample were built. A comparison was performed among the slopes already obtained for the blank diluted urine and the slopes of the plots of the standard addition method (2-, 3-, and 4-fold of the native concen(48) Applied Biosystem Application Note (2007) n. 114AP66-01. http:// www3.appliedbiosystems.com/cms/groups/psm_marketing/documents/ generaldocuments/cms_046758.pdf (accessed June 2010). (49) Gosetti, F.; Mazzucco, E.; Zampieri, D.; Gennaro, M. C. J. Chromatogr., A 2010, 1217, 3929-3937.

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Figure 5. Online SPE UHPLC-MS/MS chromatogram of urine sample 4 diluted 1/10 (v/v) in SPE loading solution. The chromatographic conditions are reported in the Experimental Section.

Table 5. Quantification Data of the Analytes in Urine Samples of Different Patientsa drug compound BE bromazepam buprenorphine chlordiazepoxide clobazam clotiazepam cocaine codeine delorazepam DHC diazepam DMTA EDDP EME estazolam flurazepam flunitrazepam heroin lorazepam lormetazepam 6-MAM methamphetamine midazolam morphine nitrazepam nordazepam temazepam THC THC-COOH THC-OH a

sample 1

sample 2

sample 3