Generic sample preparation combined with high-resolution liquid chromatography-time-of-flight mass spectrometry for unification of urine screening in doping-control laboratories

Abstract

A unification of doping-control screening procedures of prohibited small molecule substances--including stimulants, narcotics, steroids, beta2-agonists and diuretics--is highly urgent in order to free resources for new classes such as banned proteins. Conceptually this may be achieved by the use of a combination of one gas chromatography-time-of-flight mass spectrometry method and one liquid chromatography-time-of-flight mass spectrometry method. In this work a quantitative screening method using high-resolution liquid chromatography in combination with accurate-mass time-of-flight mass spectrometry was developed and validated for determination of glucocorticosteroids, beta2-agonists, thiazide diuretics, and narcotics and stimulants in urine. To enable the simultaneous isolation of all the compounds of interest and the necessary purification of the resulting extracts, a generic extraction and hydrolysis procedure was combined with a solid-phase extraction modified for these groups of compounds. All 56 compounds are determined using positive electrospray ionisation with the exception of the thiazide diuretics for which the best sensitivity was obtained by using negative electrospray ionisation. The results show that, with the exception of clenhexyl, procaterol, and reproterol, all compounds can be detected below the respective minimum required performance level and the results for linearity, repeatability, within-lab reproducibility, and accuracy show that the method can be used for quantitative screening. If qualitative screening is sufficient the instrumental analysis may be limited to positive ionisation, because all analytes including the thiazides can be detected at the respective minimum required levels in the positive mode. The results show that the application of accurate-mass time-of-flight mass spectrometry in combination with generic extraction and purification procedures is suitable for unification and expansion of the window of screening methods of doping laboratories. Moreover, the full-scan accurate-mass data sets obtained still allow retrospective examination for emerging doping agents, without re-analyzing the samples.

Fig. 1

(Cortico)steroids involved in this study: budesonide (a), prednisolone (b), prednisone (c), desonide (d), methylprednisolone (e), cortisone (f), cortisol (g), flumethasone (h), flucortisone (i), fluocortolone (j), fluniside (k), triamcinolone acetonide (l), dexamethasone (m), betamethasone (n), triamcinolone (o), gestrinone (p), tetrahydrogestrinone (THG) (q), trenbolone (r), epitrenbolone (s)

Fig. 2

β2-Agonists involved in this study: clenbuterol (a), clenproperol (b), clenpenterol (c), clencyclohexerol (d), brombuterol (e), mapenterol (f), salbutamol (g), cimaterol (h), cimbuterol (i), mabuterol (j), salmeterol (k), zilpaterol (l), carbuterol (m), terbutaline (n), clenhexyl (o), isoxsuprine (p), procaterol (q), fenoterol (r), ractopamine (s), tulobuterol (t), reproterol (u), formoterol (v)

Fig. 3

Thiazide diuretics involved in this study: chlorothiazide (a), hydrochlorothiazide (b), hydroflumethiazide (c), benzthiazide (d), bendroflumethiazide (e), althiazide (f), trichlormethiazide (g), methyclothiazide (h), cyclothiazide (i), polythiazide (j)

Fig. 4

Narcotics and stimulants involved in this study; sydnocarb (a), oxycodone (b), phenylephrine (c), mephentermine (d), methoxyphenamine (e)

Fig. 5

Total ion and extracted ion chromatograms (TIC and EIC) of urine samples and individual analytes analysed in the positive mode. EICs of individual analytes are representative for all analytes and are extracted using a mass window of 5 mDa. From the top down the chromatograms shown are: TIC of a blank urine (A); TIC of a solvent standard of the analytes (B); TIC of a blank urine fortified at the 1.0 × VL level (C); EIC of phenylephrine (split peak just before 3 min), 30 μg L⁻¹ (D); EIC of cimbuterol, 50 μg L⁻¹ (E); EIC of clenbuterol, 2 μg L⁻¹ (F); EIC of mapenterol, 50 μg L⁻¹ (G); EIC of cortisone, >30 μg L⁻¹ (H); EIC of beclomethasone, 30 μg L⁻¹ (I); EIC of trenbolone, 30 μg L⁻¹ (J); EIC of gestrinone, 30 μg L⁻¹ (K); EIC of tetrahydrogestrinone (THG), 30 μg L⁻¹ (L).

Fig. 6

Total ion and extracted ion chromatograms (TIC and EIC) of urine samples and individual analytes analysed in negative-ion mode. EICs of individual compounds are representative for all analytes and are extracted using a mass window of 5 mDa. From the top down the chromatograms shown are: TIC of a blank urine (A); TIC of a solvent standard of the analytes (B); TIC of a blank urine fortified at 1.0 × the VL level (C); EIC of chlorothiazide, 100 μg L⁻¹ (D); EIC of hydrochlorothiazide, 100 μg L⁻¹ (E); EIC of hydroflumethiazide, 100 μg L⁻¹ (F); EIC of trichloromethiazide, 100 μg L⁻¹ (G); EIC of methyclothiazide, 100 μg L⁻¹ (H); EIC of althiazide, 100 μg L⁻¹ (I); EIC of benzthiazide, 100 μg L⁻¹ (J); EIC of cyclothiazide, 100 μg L⁻¹ (K); EIC of polythiazide, 100 μg L⁻¹ (L)

Fig. 7

Deviations in RT (ΔRT), the mass error (Δm/z) and SigmaFit show some positive correlation