A Novel Study of Screening and Confirmation of Modafinil, Adrafinil and Their Metabolite Modafinilic Acid Under EI-GC-MS and ESI-LC-MS-MS Ionization S

Research Article

A novel study of screening and confirmation of modafinil, adrafinil and their metabolite modafinilic acid under EI-GC-MS and ESI-LC-MS-MS ionization S. Dubey, S. Ahi, I. M. Reddy, T. Kaur, A. Beotra, S. Jain

ABSTRACT

OObjective:bjective: Adrafinil and modafinil have received wide publicity and have become controversial in the sporting world when several athletes were discovered allegedly using these drugs as doping agents. By acknowledging the facts, the World Anti-Doping Agency (WADA) banned these drugs in sports since 2004. The present study explores the possibility of differentiating adrafinil and modafinil and their major metabolites under electron impact National Dope Testing Laboratory, ionization in gas chromatograph–mass spectrometer (GC-MSD) and electrospray ionization Ministry of Youth Affairs and Sports, in liquid chromatograph–mass spectrometer (LC-MS/MS) by studying the fragmentation J. N. Stadium, New Delhi - 100 03, pattern of these drugs. India MMaterialsaterials aandnd MMethods:ethods: Adrafinil, modafinil and their major metabolite, modafinilic acid were analyzed on EI-GC-MSD and ESI-LC-MS/MS using various individual parameters RReceived:eceived: 06.05.2009 on both the instruments. The analytical technique and equipment used in the analysis RRevised:evised: 30.06.2009 were an Agilent 6890N GC with 5973 mass selective detector for the GC-MSD analysis AAccepted:ccepted: 09.12.2009 and an Agilent 1100 HPLC with API-3200 Triple quadrupole mass spectrometer for the LC-MS/MS analysis. Validation of both methods was performed using six replicates at DDOI:OI: 10.4103/0253-7613.59928 different concentrations. RResultesult andand Discussion:Discussion: The results show that adrafinil, modafinil and their major metabolite CCorrespondenceorrespondence to:to: modafinilic acid could be detected as a single artifact without differentiation under EI-GC- Dr. A. Beotra MSD analysis. However, all drugs could be detected and differentiated under ESI-LCMS/ E-mail: [email protected] MS analysis without any artifaction. The GC-MSD analysis gives a single artifact for both the drugs without differentiation and thus can be used as a marker for screening purposes. Further, the Multiple Reaction Monitoring (MRM) method developed under LC-MS/MS is fit for the purpose for confirmation of suspicious samples in routine sports testing and in forensic and clinical analysis.

KKEYEY WWORDS:ORDS: Adrafinil, EI-GC-MSD, ESI-LC-MS/MS, modafinil, modafinilic acid

Introduction modafinil and adrafinil in clinical, forensic and dope testing was being conducted by gas chromatograph–mass spectrometer Adrafinil and modafinil are clinically used in the treatment [2] of narcolepsy, obstructive sleep apnea and idiopathic (GC-MSD) and high-performance liquid chromatography [3,4] hypersomnia. Modafinil is a central nervous system stimulant, (HPLC). However, during the analysis of all the three drugs, which possess wake-promoting actions like symathommimetic viz. adrafinil, modafinil and modafinilic acid, it was not possible agents, including amphetamine and methylphenidate. Adrafinil to differentiate them on GC-MSD since they give a single is a prodrug of modafinil and is readily converted into modafinil peak. Moreover, HPLC analysis possesses the shortcoming and its metabolite modafinilic acid after intake. These drugs of quantitation analysis for all the three compounds. Hence, have received wide publicity and become controversial in the liquid chromatograph–tandem mass spectrometer (LC-MS/ sporting world when several athletes were discovered allegedly MS) becomes the method of choice for detection of either using them as doping agents. By acknowledging the facts, of the drugs or metabolites. The present study explores the the World Anti-Doping Agency (WADA) banned these drugs in possibility of differentiating adrafinil, modafinil and their sports from 2004.[1] The minimum required performance limit major metabolites by LC-MS/MS. A method was developed for both modafinil and adrafinil is 500 ng/ml.[1] The testing for for screening on GC-MS and confirmation on LC-MS/MS for

278 Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 278-283 Dubey, et al.: Analysis of adraﬁ nil, modaﬁ nil and their metabolite by LCMS/MS and GCMS adrafinil, modafinil and modafinilic acid and validated as per LC-MS/MS the international guidelines.[5,6] An Aglient 1100 series LC system coupled with API 3200 Materials and Methods Triple quadrupole instrument equipped with a pneumatically assisted electrospray ion source (Applied-Biosystem-Sciex Reference standards Concord, Canada) was used. The entire system was controlled The reference standards of modafinil and adrafinil were using the Analyst 1.4.1® software. The main working parameters purchased from Sigma (St-Louis, MO, USA). The organic of the mass spectrometer are summarized in Table 2. solvents and reagents were of HPLC grade. The Amberlite XAD-2 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the Method development on GC-MSD and LC-MS/MS derivatizing reagent, iodomethane was purchased from Acros The method development was initiated by direct derivatization Organics (New Jersey, USA). Acetonitrile and ethyl acetate were and injection of the reference standards of both adrafinil and obtained from Qualigens (Mumbai, India), methanol from J.T modafinil on GC-MSD. For modafinilic acid, the excretion study Baker (Phillipsburg, NJ USA), tertiary butyl methyl ether (TBME) sample was processed and injected in scan mode and the method from Acros Organics and formic acid from Merck (Mumbai, was developed. The scan range was 40-550 with a scan rate of India). deionised water was prepared on a Milli Q laboratory 2.8 scan/second with an injection volume of 2 µl. plant (Millipore, Bedford, MA, USA). For the LC-MS/MS analysis, direct infusion of individual Sample extraction procedure for GC-MSD analysis reference standards dissolved in ethanol was performed using The sample extraction procedure for GC-MS analysis different collision energies. Ionization in the turbospray source involves solid phase extraction. was performed in positive mode scanning masses from m/z 100 Two milliliters of urine was applied onto the pre-prepared to 500 with a 0.2 µ step size. Nitrogen was used as nebulising

XAD2 column. Mefruside (2 µg/ml) was added as the internal and curtain gas. For modafinilic acid, the excretion study sample standard. Washing was performed with 2 ml of water to eliminate was processed and injected through LC in scan mode and the most of the water-soluble urinary constituents that had not been method was optimized. absorbed on the solid support. The drugs were then eluted with Method validation 2 ml of methanol. The entire effluent was evaporated under nitrogen stream at 60°C and the residue was dissolved in 200 µl The analytical method was validated as per the requirement acetone and 50 mg K2CO3 was added to make the reaction mixture of WADA ISL (version 6.0) keeping in view linearity, accuracy, alkaline. Then, derivatization was performed using iodomethane precision, specificity, recovery, limit of detection (LOD) and limit for 3 h at 60°C. The acetone layer was then cooled and dried under of quantitation (LOQ). nitrogen evaporator at 60°C and the sample was reconstituted in 50 µl of ethyl acetate and injected on GC-MSD. Table 1 Sample extraction procedure for LC-MS/MS analysis Analytical parameters of GC-MSD The sample extraction procedure used for LC-MS/MS involves liquid-liquid extraction. Two/four milliliters of the urine sample Injection mode Automatic split based on specific gravity was taken and 500 ng/ml of methyl Split ratio 11:1 testosterone was added as the internal standard. One milliliter Injection volume 2 µl of phosphate buffer was added to adjust the pH to 7.0. Hydrolysis Injection port temperature 280°C was performed by addition of ß-glucuronidase enzyme (E. coli) Carrier ﬂ ow 135 kpa helium (constant to the sample. The sample was incubated at 60°C for 1 h and pressure) 250 µl of K CO (pH 9-10) was added. Liquid-liquid extraction Oven program 150°C for 1 min, 19.5°C/min, 2 3 300°C at 7 min, ﬁ nal hold for was performed by addition of TBME and the organic layer was 2 min separated in another test tube. To the remaining aqueous layer, Column Ultra 2, fused silica, 0.2 mm x 150 µl of 6N HCl (pH 2-3) and 4 ml of ethyl acetate were added. 12.5 m x 0.11 µm A second liquid-liquid extraction was performed and the organic layer was separated into the initial test tube. The collective Table 2 organic layers of both the extraction steps were evaporated to dryness. Finally, the dry extract was reconstituted in 100 µl of Analytical parameters of LC-MS/MS the mobile phase (50:50, V/V) injected on LC-MS/MS. Column Automatic Split with Intersilr Instrumentation and conditions C-18 column (3.0 µm X 50 mm GC-MSD X 4.6 mm) Flow 700 µl/min Gas chromatography mass spectrometric analysis was Mobile phase A: 1% formic acid; B: acetonitrile carried out on an Agilent 6890N Network GC system with Gradient 0-5 min B 15%, 5-6 min B 60%, 5973 Network mass selective detector, equipped with 7683 6-7 min B 100%, 7-11 min B 15% series automated liquid sampler (Agilent Technologies Inc., Polarity Positive Wilmington, DE, USA). The entire system is controlled by Source 550°C the Chemstation® Software (Agilent Technologies Inc.). The Curtain gas 15 psi instrumental conditions are shown in Table 1. CAD 3 psi

Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 278-283 279 Dubey, et al.: Analysis of adraﬁ nil, modaﬁ nil and their metabolite by LCMS/MS and GCMS

Working solutions for calibration and quality control samples to lack of high temperature programming these compounds are were prepared from 1 mg/ml of stock solution by dilution not degraded and therefore show good separation and resolution using ethanol for GC-MS and LC-MS/MS, separately. Quality with different retention times [Figure 3]. The comparative result control samples (spiked) were prepared alike in six replicates for the analysis of modafinil, adrafinil and modafinilic acid under at four concentrations, i.e. 100 ng/ml, 250 ng/ml, 500 ng/ EI-GC-MS and ESI-LC-MS/MS are tabulated in Table 3. ml and 1000 ng/ml for LC-MS/MS analysis and six replicates Limit of detection at three concentrations, i.e. 250 ng/ml, 500 ng/ml and 1000 To measure the limit of detection for adrafinil and modafinil, ng/ml for GC-MS analysis. The concentrations of the direct six aliquots of negative urine samples were spiked with 100 ng/ calibration standard were 100 ng/ml, 250 ng/ml, 500 ng/ml ml and 250 ng/ml of certified reference standard for GC-MS and 1000 ng/ml. analysis and 25 ng/ml, 50 ng/ml and 100 ng/ml of certified Excretion study of modaﬁ nil reference standard for LC-MS/MS analysis. The LOD for modafinil and adrafinil was found to be 250 ng/ml on GC-MS Two healthy male volunteers aged 25 ± 3 years were given and 100 ng/ml on LC-MS/MS [Figure 4]. one single dose of modafinil (100 mg; Sun Pharmaceuticals, Vapi, Gujarat, India) as per the approval of the Ethics Committee. Calibration curve Urine samples were collected for 72 h and were stored at –20°C. A calibration curve was constructed with different concentrations ranging between 100 ng/ml and 1000 ng/ml Results on GC-MSD and LC-MS/MS for both modafinil and adrafinil [Figure 5]. Linearity was assessed by a weighted (1/x) least Behavior of modafinil, adrafinil and modafinilic acid under squares regression analysis. The calibration curve had a EI-GC-MS and ESI-LCMS/MS: correlation coefficient (r2) of ≥0.99 of both the drugs on the 2-[(diphenylmethyl) sulfinyl] acetamide (modafinil) and GC-MSD and LC-MS/MS instruments. The acceptance criterion 2-[(diphenylmethyl) sulfinyl] hydroxamide (adrafinil) and their for each calculated standard concentration was 15% deviation carboxylic metabolite modafilinic acid are severely degraded from the nominal value. during EI-GC-MS analysis. Due to the sterically rigid and strong electronegative structure, the only possible site of ionization Precision and accuracy is the diphenylmethyl sulfinyl linkage Hence, as expected from The batch accuracy and precision were determined by the structures of adrafinil, modafinil and their metabolite analyzing six sets of quality control samples. The accuracy of modafinilic acid as shown in Figure 1, the main fragment comes detection of modafinil and adrafinil were in the range of 86- from diphenylmethyl sulfinyl linkage giving m/z 167 as the most 88% and 94-104% on GC-MS and LC-MS/MS, respectively. The prominent ion. Further, demethylation occurs from this fragment precision of the method was expressed as CV%. Tables 4 and 5 giving m/z 152 as second main fragment. The high temperature summarize the calculated concentrations, recovery percentage, programming used in gas chromatography leads to the formation accuracy and precision of the method for spiked samples of of a single artifact of modafinil, adrafinil and modafinilic acid adrafinil and modafinil in human urine on GC-MS and LC-MS/MS. under which they elute as a single peak at the same retention Specificity and matrix effect time [Figure 2]. Whereas, in liquid chromatography condition, due The specificity and matrix effect of the method was

Table 3

Retention time, mass fragments (m/z) and MRM transitions of adrafi nil, modafi nil and modafi nilic acid on GC-MSD and LC-MS/MS

Compound Retention time (min) MRM transitions Fragmentation ions (GC-MSD) (LC-MS/MS) (LC-MS/MS) (GC-MSD) Adrafi nil 7.2 4.9 288–104 167 Modafi nil 5.1 274-167 165 Modafi nilic 5.8 273-167 acid 152

Figure 1: Chemical structure of (a) adrafi nil, (b) modafi nil and (c) modafi nilic acid

(a) (b) (c)

280 Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 278-283 Dubey, et al.: Analysis of adraﬁ nil, modaﬁ nil and their metabolite by LCMS/MS and GCMS

Figure 2: Total ion chromatogram of adrafi nil, modafi nil and modafi nilic examined on both the instruments by analyzing the drug free acid showing a single artifact under GC-MS analysis urine processed through the sample procedure as applied for the quality control samples. No major interference peaks were observed on either of the instruments. Selectivity The selectivity of the method was examined by analyzing the drug free urines fortified with the drugs having properties similar to adrafinil and modafinil at various concentrations. No compound has shown any co-elution or interference with mass spectra of the analyte of interest on both the instruments. Recovery Recoveries of adrafinil, modafinil and internal standard were evaluated by comparing the mean peak areas of the processed samples (spiked) with the mean peak areas of the unprocessed direct reference standard solutions of the same concentration. Recoveries of all compounds were found to be within the acceptable range [Tables 4 and 5]. Internal standards are good and acceptable for both the methods. Excretion study sample Modafinil and its main metabolite, modafinilic acid were identified in all the excretion study samples. But, the metabolite could not be quantitated due to unavailability of the pure standard. A graph was plotted for area of abundance against excretion time to explore the excretion profile of the parent and metabolite. Parent drug could be traced till eighty hours post drug administration, whereas the peak concentration was eliminated within 6 h [Figure 6]. Discussion The present method developed shows that adrafinil,

Figure 3: Total ion chromatogram of an excretion study sample of adrafi nil analyzed on ESI-LC-MS/MS in MRM mode showing presence of adrafi nil, modafi nil and modafi nilic acid

5.74 3.4e6 3.3e6 3.2e6 3.1e6 3.0e6 2.9e6 2.8e6 2.7e6 2.6e6 MODAFINILIC ACID 2.5e6 2.4e6 2.3e6 2.2e6 2.1e6 2.0e6 1.9e6 1.8e6 Intensity, cps 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 MODAFINIL 5.0e5 4.0e5 3.0e5 5.05 2.0e5 1.0e5 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Time, min

Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 278-283 281 Dubey, et al.: Analysis of adraﬁ nil, modaﬁ nil and their metabolite by LCMS/MS and GCMS

Figure 4 : Total ion chromatogram of a negative urine sample analyzed on ESI-LC-MS/MS in MRM mode showing absence of adrafi nil, modafi nil and modafi nilic acid

3.6e5 3.5e5 3.4e5 3.3e5 3.2e5 3.1e5 3.0e5 2.9e5 2.8e5 2.7e5 2.6e5 2.5e5 2.4e5 2.3e5 2.2e5 2.1e5

2.0e5 1.9e5 1.8e5 1.7e5

Intensity, cps 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 Time, min

Table 4

Recovery percentage, accuracy and precision of modaﬁ nil and adraﬁ nil on GC-MS

Compound Expected concentration Calculated concentration Recovery percentage CV% Accuracy % (ng/ml) (ng/ml) (mean ± SD) (mean ± SD) Modaﬁ nil (n = 6) 250 219 ± 22.4 80.4 ± 8.5 10.2 87.6 500 422 ± 32.4 69.2 ± 5.3 7.6 84.4 1000 868 ± 100.4 97.7 ± 11.2 11.5 86.8 Adraﬁ nil (n = 6) 250 219 ± 22.4 68.6 ± 6.5 10.2 87.6 500 422 ± 32.4 71.2 ± 4.6 7.6 84.4 1000 868 ± 100.4 81.6 ± 9.4 11.5 86.8

Table 5

Recovery percentage, accuracy and precision of modaﬁ nil and adraﬁ nil on LC-MS/MS

Compound Expected concentration Calculated concentration Recovery percentage CV% Accuracy % (ng/ml) (ng/ml) (mean ± SD) (mean ± SD) Modaﬁ nil (n = 6) 100 101 ± 5.4 101.2 ± 5.4 5.3 101 250 240 ± 16.8 102.3 ± 7.1 7.0 96.5 500 519 ± 24.0 106.4 ± 4.9 4.6 103.8 1000 1026 ± 39.9 99.6 ± 3.8 3.8 102.6 Adraﬁ nil (n = 6) 100 88 ± 4.5 89.2 ± 4.5 5.1 88.6 250 236 ± 10.8 100.7 ± 4.6 4.6 94.4 500 483 ± 16.4 97.9 ± 3.3 3.3 96.6 1000 1042±40.8 101.7±3.9 3.9 104.2

282 Indian J Pharmacol | Dec 2009 | Vol 41 | Issue 6 | 278-283 Dubey, et al.: Analysis of adraﬁ nil, modaﬁ nil and their metabolite by LCMS/MS and GCMS

Figure 5: Calibration curves of modaﬁ nil and adraﬁ nil on GC-MS and LC-MS/MS

MODA+ADRA.rdb (ADRAFINIL-2): "Linear Through Zero" Regression ("1 / x" weighting): y = 4.04e-005 x (r = 0.9988) MODA+ADRA.rdb (MODAFINIL-1): "Linear Through Zero" Regression ("1 / x" weighting): y = 0.00203 x (r = 0.9985) 0.044 0.042 2.2 2.1 0.040 2.0 0.038 1.9 0.036 1.8 0.034 1.7 0.032 1.6 0.030 1.5 0.028 1.4 0.026 1.3 0.024 1.2 0.022 1.1 0.020 1.0 0.018 A nalyte A / rea IS A rea 0.9 0.016 0.8 0.014 A nalyte A / rea IS A rea 0.7 0.012 0.6 0.010 0.5 8.000e-3 0.4 6.000e-3 0.3 4.000e-3 0.2 2.000e-3 100 200 300 400 500 600 700 800 900 1000 100 200 300 400 500 600 700 800 900 1000 Analyte Conc. / IS Conc. Analyte Conc. / IS Conc. Modaﬁ nil- LC-MS/MS Adraﬁ nil-LC-MS/MS

Adraﬁ nil-GC/MS Aﬁ nil GC/MS Mod

Figure 6: Excretion profi les of modafi nil and modafi nilic acid in a detecting and differentiating modafinil, adrafinil and modafinilic human male volunteer acid. It is free from interference by methylphenidate, cocaine, amphetamines and other drugs of abuse. The quantitation of excretion study samples shows that peak levels of modafinil could be found at 10 h both in GC-MS and LC-MS/MS analysis, which proves the authenticity of the method. The pharmacological properties of modafinil and adrafinil are similar to amphetamine but without some of the side- effects associated with amphetamine like stimulants. Hence, both modafinil and adrafinil have very good potential to be abused, thereby necessitating the need to have screening and confirmation method. Further work is in progress to differentiate enantiomers of modafinil on LC-MS/MS. References 1. The World anti Doping Code. The 2008 prohibited list international standard. modafinil and their major metabolite, modafinilic acid could Available from: http://www.wada.ama.org/rtecontent/document/list_2008.pdf. be detected as a single artifact without differentiation under [01 August 2009]. EI-GC-MSD analysis. However, all drugs could be detected 2. Tseng YL, Uralets V, Lin CT, Kuo FH. Detection of modafi nil in human urine by and differentiated under ESI-LC-MS/MS analysis without any gas chromatography-mass spectrometry. J Pharm Biomed Anal 2005;39:1042-5. artifaction at different retention times. The GC-MSD analysis 3. Moachon G, Matinier D. Simultaneous determination of modafi nil and its acid metabolite by high-performance liquid chromatography in human plasma. J gives a single artifact and thus can be used as a marker Chromatogr B Biomed Appl 1994;654:91-6. for screening purposes. Further, ESI-LC-MS/MS allows the 4. Schwertner HA, Kong SB. Determination of modafi nil in plasma and urine by differentiation of adrafinil, modafinil and modafinilic acid; hence reversed phase high-performance liquid –chromatography. J Pharm Biomed the method is appropriate for the purpose for confirmation of Anal 2005;37:475-9. suspicious samples in routine sports testing and in forensic 5. The World Anti Doping Code, International Standard for Laboratories. Version 6, 2009. 6. Eurachem guidelines for Analytical Method validation. and clinical analysis. Modafinil and adrafinil are the stimulants 7. Cass QB, Ferreira GT. A method for the determination of the plasma levels of [1] with the potential of abuse in sports. Modafinil is approved modafi nil enantiomers, (+/-)-modafi nilic acid and modafi nil sulfone by direct by the FDA for use in the management of excessive sleepiness human plasma injection and bidimensional achiral-chiral chromatography. J associated with narcolepsy.[7] This method can be of utility for Pharm Biomed Anal 2007;37:475-9.

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