Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control

Analytica Chimica Acta 700 (2011) 126–136

Contents lists available at ScienceDirect

Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control Yves Moulard ∗ , Ludovic Bailly-Chouriberry, Sophie Boyer, Patrice Garcia, Marie-Agnès Popot, Yves Bonnaire L.C.H., Laboratoire des Courses Hippiques, 15 rue de Paradis, 91370 Verrières le Buisson, France

a r t i c l e

i n f o

Article history: Received 31 August 2010 Received in revised form 1 December 2010 Accepted 2 January 2011 Available online 14 January 2011 Keywords: Screening Equine Urine Mass spectrometry LC–MS High resolution Mass accuracy

a b s t r a c t Liquid chromatography–mass spectrometry (LC–MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50 000) and high mass accuracy (<5 ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5 mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC–MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive–negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive–negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Currently, in doping, forensic or food safety control laboratories, liquid chromatography–mass spectrometry (LC–MS) is going to be the most important technique used routinely for the screening and confirmation of prohibited substances in biological samples. Furthermore, the recent applications of LC–MS fitted with electrospray ionization (ESI) probe for almost all the classes of compounds complement effectively gas chromatography–mass spectrometry (GC/MS) and sometimes provide further aid in the interpretation of analytical findings [1]. Since the increase in terms of sensitivity of new generation of mass spectrometers, new molecules can now be confirmed in biological samples such as protein based drugs, particularly recombinant human erythropoietin (EPO) [2–4] and recombinant equine [5] or bovine growth hormone [6]. Nevertheless, due to the constant release of new drugs on the market, the screening must

∗ Corresponding author. Tel.: +33 1 69 75 28 28; fax: +33 1 69 75 28 29. E-mail address: [email protected] (Y. Moulard). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.01.006

be improved in order to fulfill the detection of whole molecules. Indeed, the screening previously carried out by ELISA has been discontinued in several laboratories because of its restrictive analysis capability and now is replaced by mass spectrometry-based methods, decreasing the level of false negative results. To date, the application range of multidimensional MS is extremely wide, allowing the selection of different scan modes for screening of multiple target analytes in a single analysis. Using a LC system coupled to a triple-quadrupole MS, high-throughput LC–MS methods have been developed for fast screening in horse urine of either basic drugs and corticosteroids [7], corticosteroids and acid drugs [8] or drugs in horse plasma [9]. A similar approach for anabolic steroids has also been proposed for screening in horse [10] and in bovine urine [11]. On the other hand, fewer numbers of screening methods based on ion trap or linear ion trap instruments have been reported. Indeed, the major drawback of this technology is its restricted number of MS/MS events that can be monitored at the same time which is not conducive to high-throughput analysis. However, attempts to employ scan to scan polarity switching for the detection of a limited number of diuretics and beta-blockers in human urine by ion trap have been reported [12].

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Other multi-residues screening methods have been developed employing hybrid tandem mass spectrometry (Q-Trap) allowing the production of full scan MS/MS spectra after the detection of specific transitions (MRM). This technology was successfully applied for the simultaneous screening of more than 250 basic drugs in equine urine [13]. Besides, using the same technology and due to the availability of new softwares for mass spectrometry data processing, creation of libraries containing a large number of target compounds allowed a comprehensive screening of toxic substances and drugs in plasma, urine and other biological fluids [14]. In the same manner, compounds identification was obtained from QqQ [15]. To date, the number of target molecules analyzed simultaneously is restricted by the minimum scan time available for these mass analyzers. Consequently, loss of sensitivity is observed when too many transitions are selected. In LC–MS/MS analysis, the identification of molecules is only performed starting from previous knowledge of the target compounds which do not allow the identification of unknown compounds. In order to fill this gap, new possibilities are offered through high-resolution LC–MS instruments allowing identification by exact mass and isotopic peaks ratio. According to Nielen et al. [16] and Vonaparti et al. [17] the accurate mass capability of LC–TOF/MS allows the reconstruction of extracted exact mass chromatograms for compounds of interest in biological samples. Moreover, a UPLC–TOF/MS method was set up for the multi-residue analysis of corticosteroids in human and animal urine [18]. In addition, LC–TOF/MS allows retrospective analysis but its sensitivity is still controversial. Since the recent development of the orbitrap technology [19–24], which provides high mass resolution, high mass accuracy and good sensitivity with possible retrospective analysis, a new tool for the improvement of multi-residue screening is now available. Thus, a rapid screening method for 510 pesticides have been developed by Zhang et al. [25] on the exactive benchtop mass spectrometer. In the case of doping control analysis, it has been recently reported a screening for known and unknown compounds which was performed from human plasma samples [26]. In this article, a method has been developed on the exactive, a new benchtop mass spectrometer equipped with the orbitrap technology, for the detection of more than 235 drug targets in horse urine samples. Identification of compounds of interest was achieved using the exact mass in positive–negative ionization mode at their expected retention times. The aim of this paper is to present the method we have developed, illustrated with some examples of data obtained by the application of this innovative technology in horse doping control analysis and validated with 20 target compounds from 16 different urine samples.

2. Materials and methods 2.1. Chemicals and reagents HPLC-grade water was obtained by purifying water in filtration system (Millipore, Bedford, MA, USA). HPLC-grade methanol, nhexane and acetonitrile were purchased from Carlo Erba (Milan, Italy), ethanol, formic acid, ammonium dihydrogen phosphate and ammonium sulfate from VWR (Paris, France) and methylene chloride from JT Baker (Deventer, Netherland). ␤-Glucuronidase from E. coli (152 U mL−1 solution) was obtained from Roche Diagnostics (Meylan, France). Bond Elut C18 HF (3 cc, 500 mg) solid phase extraction cartridges were purchased from Varian (Palo Alto, CA, USA). Hydrocortisone d3, used as internal standard, was obtained from CIL (Andover, MA, USA). Standards stock solutions were prepared in methanol and stored for one year at 4 ◦ C because of isomerization of some compounds like triamcinolone.

127

Table 1 Liquid chromatography gradient for the separation of the 235 target ions. Time (min) 0 5 20 25 25.2 30

Flow rate (mL min−1 )

0.300

Water, 0.1% FA 80 80 50 0 80 80

Acetonitrile, 0.1% FA 20 20 50 100 20 20

Calibration mixtures comprised the following: MSCAL5-1EA (caffeine, tetrapeptide “Met-Arg-Phe-Ala”, ultramark for positive ion mode) and MSCAL6-1EA (sodium dodecylsulfate, taurocholic acid sodium salt, ultramark for negative ion mode) were purchased from SUPELCO (Bellefonte, PA, USA). 2.2. Sample preparation One gram of ammonium sulfate and 25 ␮L of internal standard solution (d3-hydrocortisone, 10 ␮g mL−1 ) were added to 5 mL urine sample. A milliliter of 1 M ammonium dihydrogenophosphate (pH 5.8) and 50 ␮L ␤-glucuronidase were added. After enzymatic hydrolysis for 1 hour at 55 ◦ C and centrifugation at 4000 rpm (2862 × g) during 30 min, the supernatant was adjusted to pH 8 and then diluted with 5 mL of water. The extraction was then performed on Bond Elut C18-HF cartridge (3cc, 500 mg) on XL4 automated system (Gilson, Villiers le Bel, France). The sample was then loaded onto the cartridge after conditioning (3 mL of methanol followed by 3 mL of water). The cartridge was washed with 3 mL water and 3 mL hexane and the compounds of interest were eluted with 3 mL of a mixture dichloromethane/ethanol (99/1, v/v). After evaporation using water-bath at 60 ◦ C, the extracts were reconstituted in 100 ␮L of a mixture of water/acetonitrile (80/20, v/v) before injection for LC–MS analysis. 2.3. Instrument parameters The liquid chromatography system selected for this purpose was a Prominence UFLCXR (Shimadzu, Japan) with a binary pump, an on-line vacuum degasser, a thermostated autosampler and a heated column compartment. The LC system was equipped with a Sunfire ˚ from C18 column (2.1 mm i.d. × 150 mm, 3.5 ␮m particle size, 80 A) Waters (Saint-Quentin en Yvelines, France) and a Sunfire C18 precolumn (2.1 mm i.d. × 10 mm, 3.5 ␮m particle size) maintained at 35 ◦ C. The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The chromatographic flow was 300 ␮L min−1 . The LC gradient employed is shown in Table 1. A 20 ␮L of each sample was injected for analysis. Mass analyses have been performed on the exactive mass spectrometer (Thermo Fisher Scientific, San Jose, USA) equipped with an electrospray ionization (ESI) probe. The instrument was operated in positive–negative polarity switching mode. Parameters were set at 300 ◦ C for ion transfer capillary temperature and 4.0 kV or −4.0 kV for ESI needle spray voltage in positive or negative ion modes, respectively. Nitrogen sheath gas and auxiliary gas were maintained at 45 and 3 arbitrary units, respectively. The automatic gain control (AGC) and resolution settings were scaled to 500 000 and 50 000, respectively. MS data were acquired using an external calibration in the scan range of m/z 70–650 and processed using Xcalibur software version 2.1. The orbitrap was calibrated every 15 days with MSCAL5-1EA and MSCAL6-1EA for positive and negative ion modes, respectively. The calibration was performed when the daily results of the deviation mass accuracy reached ±2.5 ppm. Data processing was performed with the help of ToxID software (version 2.1.1, Thermo Fisher Scientific, San Jose, CA, USA).

128

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Table 2 Mass list of target compound used daily in routine screening analysis in horse urine sample. Index

Compound Name

Elemental Composition

Polarity

Exact Mass (m/z)

Expected RT (min)

Therapeutic Class

1

11-nor-9-COOHTetrahydrocannabinol

C21 H28 O4

−

343.19148

25.1

Sedative

+

345.20604

C22 H30 O6

−

389.19696

C21 H32 O5 C22 H34 O7

+ −

365.23225 409.22318

10.8

Glucocorticoid

5

11-nor-9-COOHTetrahydrocannabinol + HCOOH 20Beta-dihydro-hydrocortisone 20Beta-dihydrohydrocortisone + HCOOH 5’ Hydroxy Omeprazole

C17 H19 N3 O4 S

− +

360.10235 362.11690

2.7

Antiulcerative

6

Acefylline

C9 H10 N4 O4

− +

237.06293 239.07748

2.42

Bronchodilatator

7

Acepromazine

C19 H22 N2 OS

+

327.15256

8.7

Tranquilizer

8

Acetazolamide

C4 H6 N4 O3 S2

− +

220.98085 222.99541

2.3

Diuretic

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Althiazide Altrenogest Ambroxol Amcinonide-C2 H2 O + HCOOH Amcinonide-C2 H2 O Aminorex Amitriptyline Amphetamine Atipamezole Bamifylline Beclometasone Beclometasone + HCOOH Bendroflumethiazide Benzocaine Benzoylecgonine

C11 H14 ClN3 O4 S3 C21 H26 O2 C13 H18 Br2 N2 O C27 H35 FO8 C26 H33 FO6 C9 H10 N2 O C20 H23 N C9 H13 N C14 H16 N2 C20 H27 N5 O3 C22 H29 ClO5 C23 H31 ClO7 C15 H14 F3 N3 O4 S2 C9 H11 NO2 C16 H19 NO4

− + + − + + + + + + + − − + +

381.97622 311.20056 376.98586 505.22432 461.23339 163.08659 278.19033 136.11208 213.13863 386.21867 409.17763 453.16855 420.03051 166.08626 290.13868

14.5 22.1 2.2 19.7

Diuretic Progestogen Expectorant Glucocorticoid

1.3 10.3 1.3 3.0 1.5 15.9

Anorexic Antidepressant Psychostimulant Alpha-2 adrenergic antagonist Bronchodilatator Glucocorticoid

18.5 12.3 2.6

Diuretic Local anesthesic Anesthesic

24

Benzthiazide

C15 H14 ClN3 O4 S3

− +

429.97622 431.99077

16.7

Diuretic

25 26 27 28

Benzydamine Betamethasone Betamethasone + HCOOH Bromazepam

C19 H23 N3 O C22 H29 FO5 C23 H31 FO7 C14 H10 BrN3 O

+ + − +

310.19139 393.20718 437.19810 316.00800

9.9 15.1

NSAID Glucocorticoid

10.9

29 30 31

Budesonide Budesonide + HCOOH Buflomedil

C25 H34 O6 C26 H36 O8 C17 H25 NO4

+ − +

431.24282 475.23374 308.18563

20.1 20.1 2.0

Anxiolytic, anticonvulsant, sedative-hypnotic Glucocorticoid

32

Bumetanide

C17 H20 N2 O5 S

− +

363.10202 365.11657

20.7

Diuretic

33 34 35 36

Bupivacaine Butorphanol Butylscopolamine Caffeine

C18 H28 N2 O C21 H29 NO2 C21 H30 NO4 C8 H10 N4 O2

+ + + +

289.22744 328.22711 360.21693 195.08765

2.9 2.5 2.3 2.4

Local anesthesic Analgesic, antitussive Antispasmodic, anticholinergic Cardiac and respiratory stimulant, diuretic

37

Canrenoic acid

C22 H30 O4

− +

357.20713 359.22169

16.1

Diuretic

38

Canrenone

C22 H28 O3

− +

339.19657 341.21112

22.0

Diuretic

2 3 4

Vasodilator

39

Capsaicin

C18 H27 NO3

+

306.20637

22.6

Topical analgesic

40

Carbazochrome

C10 H12 N4 O3

− +

235.08366 237.09822

1.7

Antihemorrhagic

41 42 43 44 45 46

Carbazochrome + HCOOH Carbetapentane Cetirizine Chlorothiazide Chlorpheniramine Chlorpromazine

C11 H14 N4 O5 C20 H31 NO3 C21 H25 ClN2 O3 C7 H6 ClN3 O4 S2 C16 H19 ClN2 C17 H19 ClN2 S

− + + − + +

281.08914 334.23767 389.16265 293.94155 275.13095 319.10302

10.0 13.1 2.9 2.2 11.5

47 48 49 50 51 52

Chlortalidone Cimetidine Clenbuterol Clidinium Clobetasol propionate Clobetasol propionate + HCOOH

C14 H11 ClN2 O4 S C10 H16 N6 S C12 H18 Cl2 N2 O C22 H26 NO3 C25 H32 ClFO5 C26 H34 ClFO7

− + + + + −

337.00553 253.12299 277.0869 352.19072 467.19951 511.19043

Antitussive Antihystaminic Diuretic Antihystaminic Antiemetic, tranquilizer, antipsychotic Diuretic Antiulcerative Bronchodilatator Antispasmodic, anticholinergic Glucocorticoid

8.0 1.2 1.9 3.4 24.0

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

129

Table 2 (Continued) Index

Compound Name

Elemental Composition

Polarity

Exact Mass (m/z)

Expected RT (min)

Therapeutic Class

53

Clonazepam

C15 H10 ClN3 O3

+

316.04835

16.8

54 55 56 57 58 59

Cortivazol –C2 H2 O Cortivazol –C2 H2 O + HCOOH Cyclothiazide Dantrolene Dehydronorketamine Dembrexine

C30 H36 N2 O4 C31 H38 N2 O6 C14 H16 ClN3 O4 S2 C14 H10 N4 O5 C12 H16 ClNO C13 H17 Br2 NO2

+ − − − + +

489.27478 533.26571 388.01980 313.05784 226.09932 377.96988

24.5

Anticonvulsant, sedative-hypnotic Glucocorticoid

17.2 15.5 1.3 1.9

Diuretic Muscle relaxant Anesthesic Mucolytic

60

Deracoxib

C17 H14 F3 N3 O3 S

− +

396.06352 398.07807

21.8

NSAID

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81

Deracoxib + HCOOH Desonide Desonide + HCOOH Desoximetasone Desoximetasone + HCOOH Dexamethasone Dexamethasone + HCOOH Diazepam Diazoxide Dichlorisone Dichlorisone + HCOOH Diclorphenamide Diphenhydramine Domperidone Dyphylline Edrophonium Eplerenone Etamiphyllin Ethacrynic Acid Ethacrynic Acid –CH2 CO2 Etofylline

C18 H16 F3 N3 O5 S C24 H32 O6 C25 H34 O8 C22 H29 FO4 C23 H31 FO6 C22 H29 FO5 C23 H31 FO7 C16 H13 ClN2 O C8 H7 ClN2 O2 S C21 H26 Cl2 O4 C22 H28 Cl2 O6 C6 H6 Cl2 N2 O4 S2 C17 H21 NO C22 H24 ClN5 O2 C10 H14 N4 O4 C10 H16 NO C24 H30 O6 C13 H21 N5 O2 C13 H12 Cl2 O4 C11 H10 Cl2 O2 C9 H12 N4 O3

− + − + − + − + − + − − + + + + + + − − +

442.06900 417.22717 461.21809 377.21226 421.20319 393.20718 437.19810 285.07892 228.98440 413.12809 457.11902 302.90733 256.16959 426.16913 255.10878 166.12264 415.21152 280.17680 301.00399 242,99851 225.09822

16.5

Glucocorticoid

17.6

Glucocorticoid

15.3

Glucocorticoid

19.6 8.6 16.9

Anxiolytic, muscle relaxant Antihypertensive Antipruritic

7.7 5.1 3.1 1.9 1.2 15.1 1.2 22.9

Diuretic Antihystaminic Antiemetic, antidopaminergic Bronchodilatator Cholinergic, muscle stimulant Diuretic Bronchodilatator Diuretic

2.0

Bronchodilatator

82

Etoricoxib

C18 H15 ClN2 O2 S

− +

357.04700 359.06155

6.4

NSAID

83 84 85 86 87 88 89

Famprofazone Fenspiride Firocoxib Fludrocortisone Fludrocortisone + HCOOH Flurandrenolide Flurandrenolide + HCOOH

C24 H31 N3 O C15 H20 N2 O2 C17 H20 O5 S C21 H29 FO5 C22 H31 FO7 C24 H33 FO6 C25 H35 FO8

+ + + + − + −

378.25399 261.15975 337.11042 381.20718 425.19810 437.23339 481.22432

10.8 1.3 20.4 12.7

Analgesic Bronchodilatator NSAID Glucocorticoid

17.4

Glucocorticoid

90

Flufenamic acid

C14 H10 F3 NO2

+ −

282.07364 280.05909

24.4

NSAID

91 92 93 94

Flumethasone Flumethasone + HCOOH Flunisolide Flunisolide + HCOOH

C22 H28 F2 O5 C23 H30 F2 O7 C24 H31 FO6 C25 H33 FO8

+ − + −

411.19776 455.18868 435.21774 479.20867

15.4

Glucocorticoid

16.8

Glucocorticoid

95

Flunixin

C14 H11 F3 N2 O2

+ −

297.08454 295.06999

19.5

NSAID

96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

Fluocinolone acetonide Fluocinolone acetonide + HCOOH Fluocinonide Fluocinonide + HCOOH Fluorometholone Fluorometholone + HCCOH Fluprednisolone Fluprednisolone + HCOOH Fluticasone propionate Fluticasone propionate + HCOOH FormylAminoAntipyrine Furosemide Glycopyrrolate Guaifenesin Halcinonide Halcinonide + HCOOH Hydrochlorothiazide Hydrocortisone Hydrocortisone + HCOOH Hydroxy dantrolene Hydroflumethiazide Hydroxy fenspiride Hydroxy Lidocaine

C24 H30 F2 O6 C25 H32 F2 O8 C26 H32 F2 O7 C27 H34 F2 O9 C22 H29 FO4 C23 H31 FO6 C21 H27 FO5 C22 H29 FO7 C25 H31 F3 O5 S C26 H33 F3 O7 S C12 H13 N3 O2 C12 H11 ClN2 O5 S C19 H28 NO3 C10 H14 O4 C24 H32 ClFO5 C25 H34 ClFO7 C7 H8 ClN3 O4 S2 C21 H30 O5 C22 H32 O7 C14 H10 N4 O6 C8 H8 F3 N3 O4 S2 C15 H21 N2 O3 C14 H22 N2 O2

+ − + − + − + − + − + − + + + − − + − − − + +

453.20832 497.19925 495.21889 539.20981 377.21226 421.20319 379.19153 423.18245 501.19171 545.18263 232.10805 329.00044 318.20637 199.09649 455.19951 499.19043 295.95720 363.21660 407.20753 329.05276 329.98356 278.16249 251.17540

17.2

Glucocorticoid

22.9

Glucocorticoid

17.6

Glucocorticoid

12.4

Glucocorticoid

24.1

Glucocorticoid

2.6 14.9 4.2 5.0 23.7

Analgesic, antispasmodic Diuretic Preanesthesic, antimuscarinic Expectorant, muscle relaxant Glucocorticoid

3.2 12.6

Diuretic Glucocorticoid

15.2 5.5 1.3 1.3

Muscle relaxant Diuretic Bronchodilatator Local anesthesic

130

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Table 2 (Continued) Index

Compound Name

Elemental Composition

Polarity

Exact Mass (m/z)

Expected RT (min)

Therapeutic Class

119

Hydroxy Meloxicam

C14 H13 N3 O5 S2

+ −

368.03694 366.02239

13.6

NSAID

120

Hydroxy Mepivacaine

C15 H22 N2 O2

+

263.17540

1.5

Local anesthesic

121

Hydroxy Piroxicam

C15 H13 N3 O5 S

− +

346.05031 348.06487

15.8

NSAID

122

Hydroxy sildenafil

C22 H30 N6 O5 S

−

489.19256

6.5

Phosphodiesterase type 5 inhibitor

123

Hydroxy Tenoxicam

C13 H11 N3 O5 S2

− +

352.00674 354.02129

9.6

NSAID

124

C24 H31 FO7

+

451.21266

11.1

Glucocorticoid

C25 H33 FO9

−

495.20358

126 127 128

Hydroxy Triamcinolone acetonide Hydroxy Triamcinolone acetonide + HCOOH Hydroxy-Chlorpheniramine Hydroxyzine Imipramine

C16 H19 ClN2 O C21 H27 ClN2 O2 C19 H24 N2

+ + +

291.12587 375.18338 281.20123

1.8 10.1 9.6

Antihystaminic Antihystaminic Antidepressant

129

Indapamide

C16 H16 ClN3 O3 S

− +

364.05281 366.06737

15.8

Diuretic

130 131 132 133

Ipratropium Isoflupredone Isoflupredone + HCOOH Isopropamide

C20 H30 NO3 C21 H27 FO5 C22 H29 FO7 C23 H33 N2 O

+ + − +

332.22202 379.19153 423.18245 353.25874

1.4 12.4

Bronchodilatator Glucocorticoid

3.4

Antiemetic, antidarrheal, anticholinergic

134

Isoxicam

C14 H13 N3 O5 S

− +

334.05031 336.06487

21.4

NSAID

135

Ketamine

C13 H16 ClNO

+

238.09932

1.8

Anesthesic

136

Ketoprofen

C16 H14 O3

− +

253.08702 255.10157

19.3

NSAID

137

Ketorolac

C15 H13 NO3

− +

254.08227 256.09682

16.3

NSAID

138 139 140

Levamisole Lidocaine Medetomidine

C11 H12 N2 S C14 H22 N2 O C13 H16 N2

+ + +

205.07940 235.18049 201.13863

1.3 1.8 2.8

Anthelmintic Local anesthesic Sedative-analgesic

141

Meloxicam

C14 H13 N3 O4 S2

+ −

352.04202 350.02747

20.4

NSAID

142 143 144 145

Mepenzolate Mepivacaine Meprednisone Meprednisone + HCOOH

C21 H26 NO3 C15 H22 N2 O C22 H28 O5 C23 H30 O7

+ + + −

340.19072 247.18049 373.20095 417.19188

3.0 1.8 15.1

Antispasmodic Local anesthesic Glucocorticoid

146

Methazolamide

C5 H8 N4 O3 S2

− +

234.99650 237.01106

3.5

Diuretic

147 148 149 150 151 152 153 154

Methocarbamol Methylaminoantypirine Methylanisotropine Methylphenidate Methylprednisolone Methylprednisolone + HCOOH Methylscopolamine Metoclopramide

C11 H15 NO5 C12 H15 N3 O C17 H32 NO2 C14 H19 NO2 C22 H30 O5 C23 H32 O7 C18 H24 NO4 C14 H22 ClN3 O2

+ + + + + − + +

242.10230 218.12879 282.24276 234.14886 375.21660 419.20753 318.16998 300.14733

6.4 2.6 7.9 1.8 14.6

Muscle relaxant NSAID Antispasmodic, antimuscarinic CNS stimulant Glucocorticoid

1.3 1.2

Antispasmodic, anticholinergic Antiemetic

155

Metolazone

C16 H16 ClN3 O3 S

− +

364.05281 366.06737

14.0

Diuretic

156 157 158 159 160 161 162 163

Morphine Nafronyl Nefopam Neostigmine Nikethamide Nimesulide Nimesulide reduced (NH2 ) Nordazepam

C17 H19 NO3 C24 H33 NO3 C17 H19 NO C12 H19 N2 O2 C10 H14 N2 O C13 H12 N2 O5 S C13 H14 N2 O3 S C15 H11 ClN2 O

+ + + + + − + +

286.14377 384.25332 254.15394 223.14410 179.11789 307.03942 279.07979 271.06327

1.2 11.1 2.9 1.2 2.4 21.9 8.7 15.9

Analgesic Vasodilator Analgesic Cholinergic Respiratory stimulant NSAID

164

Norketamine

C12 H14 ClNO

+

224.08367

1.4

Anxiolytic, anticonvulsant, muscle relaxant Anesthesic

165

Omeprazole

C17 H19 N3 O3 S

− +

344.10744 346.12199

4.2

Antiulcerative

125

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

131

Table 2 (Continued) Therapeutic Class

Index

Compound Name

Elemental Composition

Polarity

Exact Mass (m/z)

Expected RT (min)

166

C17 H19 N3 O5 S

+

378.11182

5.4

167

Omeprazole metab hydroxy + oxyde Oxazepam

C15 H11 ClN2 O2

+

287.05818

15.6

168 169 170 171 172

Oxybuprocaine Oxyphenbutazone Oxyphenonium Paramethasone acetate Paramethasone acetate + HCOOH

C17 H28 N2 O3 C19 H20 N2 O3 C21 H34 NO3 C24 H31 FO6 C25 H33 FO8

+ + + + −

309.21727 325.15467 348.25332 435.21774 479.20867

4.2 19.8 9.0 20.2

Anxiolytic, anticonvulsant, muscle relaxant Local anesthesic NSAID Antispasmodic, antimuscarinic Glucocorticoid

173

Parecoxib

C19 H18 N2 O4 S

− +

369.09145 371.10600

21.8

NSAID

174 175 176 177 178 179 180 181

Pemoline Pentoxiphylline Pethidine Phenazone Phenobarbital Phenylbutazone Phenytoin Pipenzolate

C9 H8 N2 O2 C13 H18 N4 O3 C15 H21 NO2 C11 H12 N2 O C12 H12 N2 O3 C19 H20 N2 O2 C15 H12 N2 O2 C22 H28 NO3

+ + + + + + + +

177.06585 279.14517 248.16451 189.10224 233.09207 309.15975 253.09715 354.20637

3.4 5.4 2.2 4.1 4.9 24.0 14.7 3.4

CNS stimulant Vasodilatator Analgesic Analgesic Anticonvulsant NSAID Anticonvulsant Antispasmodic, antimuscarinic

182

Piroxicam

C15 H13 N3 O4 S

− +

330.05540 332.06995

15.2

NSAID

183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198

Prednisolone Prednisolone + HCOOH Prednisone Prednisone + HCOOH Prifinium Primidone Probenicid Procainamide Prolintane Promazine Propantheline Propentofylline Pseudo capsaicin Pyridostigmine Pyrilamine Ranitidine

C21 H28 O5 C22 H30 O7 C21 H26 O5 C22 H28 O7 C22 H28 N C12 H14 N2 O2 C13 H19 NO4 S C13 H21 N3 O C15 H23 N C17 H20 N2 S C23 H30 NO3 C15 H22 N4 O3 C17 H27 NO3 C9 H13 N2 O2 C17 H23 N3 O C13 H22 N4 O3 S

+ − + − + + − + + + + + + + + +

361.20095 405.19188 359.18530 403.17623 306.22163 219.11280 284.09620 236.17574 218.19033 285.14200 368.22202 307.17647 294.20637 181.09715 286.19139 315.14854

12.3

Glucocorticoid

12.4

Glucocorticoid

9.6 4.3 21.3 0.9 2.9 8.8 9.8 12.1 22.4 1.2 2.0 1.1

Antispasmodic Anticonvulsant Urisosuric Antiarrhytmic CNS stimulant Antipsychotic Antispasmodic, antimuscarinic Vasodilator Topical analgesic Cholinergic Antihystaminic Antiulcerative

199

Rofecoxib

C17 H14 O4 S

− +

313.05400 315.06856

16.8

NSAID

200

Sildenafil

C22 H30 N6 O4 S

− +

473.19765 475.21220

8.2

Vasodilatator

201 202 203

Spironolactone Sulindac Sulindac –CO2

C24 H32 O4 S C20 H17 FO3 S C19 H17 FOS

+ + −

417.20941 357.09552 311.09114

22.0 17.0

Diuretic NSAID

204

Tadalafil

C22 H19 N3 O4

− +

388.13028 390.14483

17.4

Vasodilatator

205

Temazepam

C16 H13 ClN2 O2

+

301.07383

18.2

Sedative, hypnotic

206

Tenoxicam

C13 H11 N3 O4 S2

− +

336.01182 338.02637

9.0

NSAID

207 208 209 210 211 212 213

Tetracaine Tetrahydrogestrinone Tetrazepam Theobromine Theophylline Tiemonium Timolol

C15 H24 N2 O2 C21 H28 O2 C16 H17 ClN2 O C7 H8 N4 O2 C7 H8 N4 O2 C18 H24 NO2 S C13 H24 N4 O3 S

+ + + + + + +

265.19105 313.21621 289.11022 181.07200 181.07200 318.15223 317.16419

4.9 22.9 15.3 2.0 1.9 2.2 1.8

214 215

Tixocortol pivalate Tixocortol pivalate + HCOOH

C26 H38 O5 S C27 H40 O7 S

+ −

463.25127 507.24220

24.0

Local anesthesic Anabolic steroid Muscle relaxant Diuretic, vasodilatator Bronchodilatator Antispasmodic, antimuscarinic Antihypertensive, antiarrhytmic Glucocorticoid

216

Torsemide

C16 H20 N4 O3 S

− +

347.11833 349.13289

8.5

Diuretic

217 218 219 220 221

Tramadol Triamcinolone Triamcinolone + HCOOH Triamcinolone acetonide Triamcinolone acetonide + HCOOH

C16 H25 NO2 C21 H27 FO6 C22 H29 FO8 C24 H31 FO6 C25 H33 FO8

+ + − + −

264.19581 395.18644 439.17737 435.21774 479.20867

1.9 8.9

Analgesic Glucocorticoid

16.4

Glucocorticoid

132

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Table 2 (Continued) Index

Compound Name

Elemental Composition

Polarity

Exact Mass (m/z)

Expected RT (min)

Therapeutic Class

222 223

C30 H41 FO7 C31 H43 FO9

+ −

533.29091 577.28183

25.5

Glucocorticoid

224 225 226 227

Triamcinolone hexacetonide Triamcinolone hexacetonide + HCOOH Triamterene Trichlormethiazide Tripelennamine Tropicamide

C12 H11 N7 C8 H8 Cl3 N3 O4 S2 C16 H21 N3 C17 H20 N2 O2

+ − + +

254.11487 377.89490 256.18082 285.15975

2.9 11.3 1.9 1.3

Diuretic Diuretic Antihystaminic Mydriatic, anticholinergic

228

Valdecoxib

C16 H14 N2 O3 S

− +

313.06524 315.07979

18.5

NSAID

229

Vardenafil

C23 H32 N6 O4 S

− +

487.21330 489.22785

3.4

Vasodilatator

230

Verapamil

C27 H38 N2 O4

+

455.29043

10.6

231

Xipamide

C15 H15 ClN2 O4 S

− +

353.03683 355.05138

19.5

Antihypertensive, antiarrhytmic Diuretic

232

Xylazine

C12 H16 N2 S

+

221.11070

1.8

233 234 235

Benzyldimethylphenylammonium Hydrocortisone-d3 Hydrocortisone-d3 + HCOOH

C15 H18 N C21 D3 H27 O5 C22 D3 H29 O7

+ + −

212,14392 366.23543 410.22636

1.5 12.5 12.5

Sedative, analgesic, muscle relaxant Internal Standard Internal Standard Internal Standard

3. Results and discussion

3.2. Case of isomeric compounds

3.1. List of masses of target compounds screened

In horse doping control, during screening unambiguous discrimination of epimeric molecules such as betamethasone and dexamethasone is essential in order to carry out their respective confirmation analysis. Regarding the fact that both molecules are epimers, their only difference is their tridimensional structure. In this specific case, high resolution and high mass accuracy cannot assist in the discrimination of both compounds, thus leading to the need for a good chromatographic resolution as previously reported by Luo et al. [28]. Fig. 1 showed the chromatographic separation of betamethasone at 15.1 min and dexamethasone at 15.3 min in horse negative urine sample spiked at 500 pg mL−1 of each molecule. The extracted ion chromatograms for betamethasone and dexamethasone, for 12 C, 13 C isotope in both ionization modes showed the same pattern. The comparison between experimental and theoretical m/z ratio in negative ionization mode resulted in 1.7 ppm and 2.3 ppm mass error for 12 C and 13 C, respectively. Moreover, in positive ionization mode, 0.6 ppm and 1.7 ppm mass error were obtained for 12 C and 13 C, respectively. This result demonstrated good correlation between the experimental mass obtained after a full scan MS analysis and the theoretical mass calculated from the molecular elemental composition. This isotopic pattern is the chemical signature of the compound. Indeed, the natural occurrence of 13 C is 1.1% of 12 C. In the case of betamethasone molecule which is composed of 23 carbons in negative and 22 carbons in positive ionization mode, the experimental results obtained, approximately 20% of 13 C form is in good correlation with the theory. The 13 C/12 C ratio is important to check compound identification during an analysis in order to be sure that there is no coelution. It is important to note that the results presented in this paper were obtained without any lock mass and with only external calibration.

According to the International Federation of Horseracing Authorities (IFHA), in horse doping control, there is no exhaustive list of prohibited substances compared to human doping control (list of World Anti-Doping Agency, WADA [27]). Indeed, doping control in the horse focuses on all substances capable at any time of causing an action or effect on the mammalian body, thus, leading to doping control without target molecule limitation. Following the constant increase of samples to be analyzed, associated with the constant release of new drugs on the market, the screening step must now be ready to screen for hundreds of different target molecules in the same run and if possible their metabolites in order to prove the administration of the substances to the horse. With the introduction of high resolution mass spectrometers, it is now possible to use their narrow mass-extraction capability combined with very good mass accuracy (<5 ppm) to extract the chromatogram of a target ion after full-scan MS analysis. Routinely, part of the screening that we conduct is based on the orbitrap technology through the use of benchtop exactive high resolution–high mass accuracy mass spectrometer. Two hundred and thirty five (235) target ions (Table 2) are searched daily in urine samples that are screened for with a continuous and unlimited addition of new compounds to the method. Starting from previous knowledge of ionization and possible adduct formation, the elemental composition of each target compound was calculated in order to obtain their theoretical mass for the different species detected by electrospray in negative ionization mode ([M−H]− , [M+HCOO]− ) and in positive ionization mode ([M+H]+ , [M+H−C2 H2 O]+ ). Following the determination of monoisotopic patterns of each molecule, a mixture of standards was analyzed by LC–MS in order to obtain their respective retention time. All data were tabulated showing compound name, elemental composition, exact ion mass and retention time (Table 2), which was used by ToxID in creating a summary report containing all extracted ion chromatograms in the narrow window of each molecule m/z ratio (typically with ±2.5 ppm mass error) at each specific retention time. Final results are summarized in a list of detected compounds which contains detected m/z ratio mass deviation expressed in ppm, expected and detected retention times.

3.3. Validation, mass accuracy and stability The screening validation was performed for 20 selected compounds analyzed routinely in doping control laboratory and spiked at different levels in 16 horse urine samples randomly selected (Table 3). These 16 urine samples have been analyzed according

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

133

Fig. 1. Extracted ion chromatograms of betamethasone (RT: 15.1 min) and dexamethasone (RT: 15.3 min) epimers in a spiked horse urine sample at 500 pg mL−1 using 5 ppm mass accuracy (a) for 12 C isotope of the formate adduct ion ([M+HCOO]− , m/z 437.19810) in negative ion mode, (b) for 13 C isotope of the formate adduct ion ([M+HCOO]− , m/z 438.20146) in negative ion mode, (c) for 12 C isotope of the protonated species ([M+H]+ , m/z 393.20718) in positive ion mode, and (d) for 13 C isotope of the protonated species ([M+H]+ , m/z 394.21053) in positive ion mode. Their respective experimental mass spectrum with a 50,000 resolution is presented (e) in negative and (g) in positive ion mode. Their respective theoretical mass spectrum is presented (f) in negative and (h) in positive ion mode.

134

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Table 3 Method validation based on 20 compounds spiked in 16 different horse urine samples for screening analysis. RT SDb (%)

Spiked urine concentration (ng mL−1 )

Beclomethasone Betamethasone Budesonide Capsaïcine Cyclothiazide Ethacrynic acid Dexamethasone Flumethasone Furosemide Hydrochlorothiazide Meloxicam Methylprednisolone Prednisone Triamcinolone Triamcinolone acetonide Trichlormethiazide

1 0.5 1 0.5 50 50 0.5 0.5 50 50 10 20 20 1 0.5 50

0.5 0.05 0.5 0.1 20 5 0.05 0.05 10 5 0.1 2 1 0.5 0.05 10

15.9 15 20.1 22.6 17.1 22.8 15.2 15.4 14.7 2.9 20.2 14.6 12.3 8.6 16.4 10.9

1.83 1.50 1.43 1.40 1.29 1.03 1.19 1.40 1.17 1.20 1.41 1.07 1.71 2.00 1.54 0.87

Cafeine Methocarbamol Prednisolone Theophylline

50 50 20 100

5 5 1 10

2.1 5.9 12.2 1.6

0.93 1.67 0.91 0.88

a b c

Limit of detection (ng mL−1 )

RTa average (min)

Analytes

Concentration deviation for positive ion mode (%) 0.56 4.55 c

Concentration deviation for negative ion mode (%) c

4.52 5.14

2.05

c

c

1.76 2.37 2.99 0.35 1.94 1.37 9.89 6.02 8.75 0.19 0.73 1.23

c

4.83 0.34 c c

11.77 7.65 11.89 0.31 0.91 c

36.91 36.78 32.71 20.42

0–15%

c c

44.50

> 15%

c

RT: retention time. SD: Standard deviation. No response for this ionization mode.

to the protocol described in this paper. Regarding the 235 target compounds, no interferences were detected at their respective retention time leading to good method specificity. Thus, these urine samples have been considered as blank samples. Fortification of each of the 16 urine samples was performed with a pool of 20 analytes at defined levels above their limit of detection (Table 3). The study of each retention time shows a wide range from 1.6 to 22.8 min corresponding to a large range of chemical molecules. For each molecule detected in the 16 selected urine samples, the retention time standard deviation do not exceed 2%. These results fulfill our requirements regarding the horse doping control criteria (Guidelines by the Association of Official Racing Chemist (AORC)) [29]. Consequently, the chromatographic conditions used for this screening are robust. This chromatographic aspect is crucial because of the use of ToxID software for the data processing. Indeed, this software is able to extract specific ion chromatograms from the total ion current at unambiguous retention times. For 16 of the 20 compounds spiked in the different horse urine samples, the concentration standard deviation values were comprised between 0% and 15% as expected. For the 4 other analytes, this value is >15% with a maximum at 44.5% for prednisolone. Therefore, considering the limit of detection established for each molecule we prevent us against false negative case for this screening analysis in horse doping control. To appraise the robustness of the mass spectrometer, quality control must be used in order to assess mass accuracy and stability of the instrument calibration. The quality control mixture analyzed routinely is composed with the same 20 molecules used for the validation step (Table 3). The daily accuracy measurement of dexamethasone (m/z 437.19810 in negative and m/z 393.20718 in positive ionization mode), spiked at 500 pg mL−1 in horse blank urine sample, used in the quality control mixture is presented (Fig. 2). The data collected during a period of 5 weeks demonstrated a good stability either in positive or negative ionization mode at ±2.5 ppm mass error. The daily analysis of all the 20 quality control molecules (Table 3) from extracted urine samples, from the most hydrophilic substance (i.e. theophylline) to the most hydrophobic (i.e. ethacrynic acid), assesses the good mass spectrometer stability.

Fig. 2. Daily mass accuracy measurement (ppm) of dexamethasone (a) for 12 C isotope of the formate adduct ion ([M+HCOO]− , m/z 437.19810) in negative ion mode and (b) for 12 C isotope of the protonated species ([M+H]+ , m/z 393.20718) in positive ion mode. * Orbitrap calibration was performed every 15 days.

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

135

Fig. 3. Extracted ion chromatograms of 12 C and 13 C isotopes for triamcinolone acetonide (RT: 16.5 min) and hydroxy triamcinolone acetonide metabolite (RT: 11.0 min) in negative and positive ionization mode (a) in a blank urine sample, (b) in a post race urine sample and (c) in a spiked horse urine sample at 500 pg mL−1 using 5 ppm mass accuracy. The extracted ion chromatograms are reconstructed on the [M+HCOO]− formate adduct ion in negative ion mode (m/z 479.20867 for triamcinolone acetonide, m/z 495.20358 for the hydroxy metabolite and m/z 410.22636 for hydrocortisone d3) and on the [M+H]+ protonated species in positive ion mode (m/z 435.21774 for triamcinolone acetonide, m/z 451.21266 for the hydroxy metabolite and m/z 366.23543 for hydrocortisone d3).

136

Y. Moulard et al. / Analytica Chimica Acta 700 (2011) 126–136

Furthermore, the use of two internal standards (benzyldimethylphenyl ammonium chloride and hydrocortisone d3) spiked in each biological urine sample allowed us to appraise the mass deviation in positive and negative ionization mode by daily comparison of their experimental with the theoretical masses. Often, the mass of endogenous compounds such as hydrocortisone and its metabolite (dihydrocortisone) are checked to improve the mass accuracy and stability of the quality control sample.

rospectively the data at any time. In this case, by only adding the elemental composition of the retro-suspected compound in the ToxID mass list, the analyst can easily reanalyze the suspicious sample without re-extraction and new LC–MS sequence. This economical aspect is important for doping control laboratories. Currently, this LC–HRMS method has been spread to over 300 target compounds and could be extended to an unlimited number of compounds in the future.

3.4. LC–MS analysis of a positive sample

Acknowledgments

Consecutive to the screening obtained with the exactive mass spectrometer, a ‘hit’ for the presence of triamcinolone acetonide and its hydroxy metabolite was suspected in the sample. Confirmation for the presence of the suspect analytes was performed by triple quadrupole instrument in order to fulfill criteria for confirmation as provided for under the Guidelines by the Association of Official Racing Chemist or WADA. Starting from the same extracts, an attempt at confirmation by means of high mass accuracy was performed. The results presented in Fig. 3c show the presence of triamcinolone acetonide detected in the two ionization modes at 16.5 min in the spiked urine sample. Furthermore, the extracted ion chromatograms from the blank urine (Fig. 3a) did not show the presence of triamcinolone acetonide and its hydroxyl metabolite at their expected retention time, 16.5 min and 11.0 min, respectively. In the case of the post-race sample (Fig. 3b), the presence of triamcinolone acetonide and its hydroxyl metabolite, was well detected in the two ionization modes at 16.5 min and 11.0 min, respectively. The metabolite identification was possible because the elemental composition of this specific structure was previously entered in the target list used by ToxID. The retention time of this metabolite was obtained from the analysis of triamcinolone acetonide postadministration sample (data not shown). In this case, through the presence of both molecules in the target drug list, it was possible to use this technique as a confirmatory method. So far, no criteria are available for the use of this method as a confirmatory method in any official racing laboratory.

The authors are indebted to Solene Guegan, Pierre Remy and Julien Bouley for their skills and their technical assistance.

4. Conclusion A high throughput, sensitive, specific and robust LC–HRMS method has been developed for the simultaneous screening of 235 acidic, basic and neutral drugs in positive–negative ionization switching mode by the high resolution exactive benchtop LC–MS orbitrap mass spectrometer. The application of the method to screening has been extensively studied and was briefly demonstrated in this paper through the detection of prohibited substances such as the two epimers (betamethasone, dexamethasone) or triamcinolone acetonide and its metabolite in horse urine sample. This method has been validated in accordance with analytical criteria (i.e. AORC criteria) and prevents us against false negative case for horse doping control. In addition, the robustness of the instrument with the orbitrap technology has been demonstrated for applicability in routine analysis, probably due to its high stability in high mass accuracy. It should be emphasized, that stability of mass accuracy is the main reason for the success of this screening method. Nevertheless, from the low C-Trap dynamic range (1–1.2 × 104 ), chemical background present in the sample can affect the sensitivity of the method. The main advantage of this technology in screening analysis is the possibility after a full-scan MS acquisition to process ret-

References [1] E. Houghton, S. Maynard, Handb. Exp. Pharmacol. (2010) 369. [2] F. Guan, C. Uboh, L. Soma, E. Birks, J. Chen, J. Mitchell, Y. You, J. Rudy, F. Xu, X. Li, M. Mbuy, Anal. Chem. 79 (2007) 4627. [3] F. Guan, C. Uboh, L. Soma, E. Birks, J. Chen, Y. You, J. Rudy, L. Xiaoqing, Anal. Chem. 80 (2008) 3811. [4] N.H. Yu, E.N. Ho, T.S. Wan, A.S. Wong, Bioanal. Anal. Chem. 396 (2010) 2513. [5] L. Bailly-Chouriberry, G. Pinel, P. Garcia, M.A. Popot, B. Le Bizec, Y. Bonnaire, Anal. Chem. 80 (2008) 8340. [6] M.H. Le Breton, S. Rochereau-Roulet, G. Pinel, L. Bailly-Chouriberry, G. Rychen, S. Jurjanz, T. Goldmann, B. Le Bizec, Rapid Commun. Mass Spectrom. 22 (2008) 3130. [7] G.N. Leung, E.W. Chung, E.N. Ho, W.H. Kwok, D.K. Leung, F.P. Tang, T.S. Wan, N.H. Yu, J. Chromatogr. B. 825 (2005) 47. [8] E.N. Ho, D.K. Leung, T.S. Wan, N.H. Yu, J. Chromatogr. A 1120 (2006) 38. [9] W.H. Kwok, D.K. Leung, G.N. Leung, T.S. Wan, C.H. Wong, J.K. Wong, J. Chromatogr. A 1217 (2010) 3289. [10] N.H. Yu, E.N. Ho, F.P. Tang, T.S. Wan, A.S. Wong, J. Chromatogr. A 1189 (2008) 426. [11] C. Van Poucke, M. Van De Velde, C Van Peteghem, J. Mass Spectrom. 40 (2005) 731. [12] K. Deventer, P. Van Eenoo, F.T. Delbeke, Rapid. Commun. Mass Spectrom. 19 (2005) 90. [13] S.M. Stanley, H.C. Foo, J. Chromatogr. B 836 (2006) 1. [14] F.L. Sauvage, F. Saint-Marcoux, B. Duretz, D. Deporte, G. Lachatre, P. Marquet, Clin. Chem. 52 (2006) 1735. [15] M. Gergov, I. Ojanpera, E. Vuori, J. Chromatogr. B 795 (2003) 41. [16] M.W. Nielen, M.C. van Engelen, R. Zuiderent, R. Ramaker, Anal. Chim. Acta 586 (2007) 122. [17] A. Vonaparti, E. Lyris, Y.S. Angelis, I. Panderi, M. Koupparis, A. TsantiliKakoulidou, R.J. Peters, M.W. Nielen, C. Georgakopoulos, Rapid. Commun. Mass Spectrom. 24 (2010) 1595. [18] M.E. Touber, M.C. van Engelen, C. Georgakopoulus, J.A. van Rhijn, M.W. Nielen, Anal. Chim. Acta 586 (2007) 137. [19] A. Makarov, Anal. Chem. 72 (2000) 1156. [20] A. Makarov, E. Denisov, A. Kholomeev, W. Balschun, O. Lange, K. Strupat, S. Horning, Anal. Chem. 78 (2006) 2113. [21] A. Makarov, E. Denisov, O. Lange, J. Am. Soc. Mass Spectrom. 20 (2009) 1391. [22] A. Makarov, M. Scigelova, J. Chromatogr. A 1217 (2010) 3938. [23] M. Hardman, A.A. Makarov, Anal. Chem. 75 (2003) 1699. [24] Q. Hu, R.J. Noll, H. Li, A. Makarov, M. Hardman, R. Graham Cooks, J. Mass Spectrom. 40 (2005) 430. [25] A. Zhang, J.S. Chang, C. Gu, M. Sanders, Non-targeted Screening and Accurate Mass Confirmation of 510 Pesticides on the High Resolution Exactive Benchtop LC/MS Orbitrap Mass Spectrometer, Application note: 51878, Thermo Fisher Scientific, San Jose, CA, USA, 2010, p. 1. [26] A. Thomas, S. Guddat, M. Kohler, O. Krug, W. Schanzer, M. Petrou, M. Thevis, Rapid. Commun. Mass Spectrom. 24 (2010) 1124. [27] World Antidoping Agency (WADA), http://www.wada ama.org/Documents/ WADP-Prohibited-list/WADA Prohibited List World Anti-Doping Program 2010 EN.pdf. [28] Y. Luo, C.E. Uboh, L.R. Soma, F. Guan, J.A. Rudy, D.S. Tsang, Rapid Commun. Mass Spectrom. 19 (2005) 825. [29] Association of Official Racing Chemist (AORC), http://cobra. vdl.iastate.edu/aorc-2/AORC%20MS%20Criteria.pdf.