Analytica Chimica Acta 853 (2015) 637–646
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Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions Lucie Nováková a , Alexandre Grand-Guillaume Perrenoud b , Raul Nicoli c, Martial Saugy c , Jean-Luc Veuthey b , Davy Guillarme b, * a
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland c Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, West Switzerland, Chemin des Croisettes 22, 1066 Epalinges, Switzerland b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Sensitivities and peak shapes were compared in UHPLC–MS/MS and UHPSFC–MS/MS. 10 mM ammonium formate can be considered a suitable mobile phase additive for UHPSFC–MS/MS operation. For interfacing UHPSFC and MS/MS, ethanol can be used as generic makeup solvent. The impact of operating parameters on MS sensitivity was different with acidic and basic compounds.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 June 2014 Received in revised form 22 August 2014 Accepted 6 October 2014 Available online 14 October 2014
The conditions for the analysis of selected doping substances by UHPSFC–MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10 mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the makeup solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC–MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC–MS/MS. Sensitivity was improved by 5–100-fold in UHPSFC–MS/MS vs. UHPLC–MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC–MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC–MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC–MS/MS. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Ultra high performance supercritical fluid chromatography Ultra high performance liquid chromatography Doping agents Mobile phase Make-up solvent
* Corresponding author. Tel.: +41 22 379 34 63; fax: +41 22 379 68 08. E-mail address: [email protected] (D. Guillarme). http://dx.doi.org/10.1016/j.aca.2014.10.004 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction The list of doping agents prohibited by the World Anti-Doping Agency (WADA) in the practice of sport includes a wide range of structurally diverse substances categorized in ten major different classes (S0–S9). In order to get uniform reporting of prohibited substances between laboratories, minimum routine detection and identification capabilities for drug testing methods have been established by WADA. In this context, minimum required performance levels (MRPL) were defined as minimal concentrations of non-threshold substances that all WADA-accredited laboratories shall be able to detect in routine testing [1,2]. Due to the high number of samples to be analyzed in a short period of time and the diversity of physico-chemical properties of substances present in the prohibited list, a two-steps approach is generally implemented in anti-doping analysis, including screening and selective confirmation steps. High throughput methods as well as sufficient sensitivity and specificity are needed to avoid false positive/negative results, especially in the screening phase. Both liquid chromatography (LC) and gas chromatography (GC) coupled to mass spectrometry (MS) are considered as reference methods. Regarding LC, ultra-high performance liquid chromatography mass spectrometry (UHPLC–MS) represents nowadays the technique of choice for the analysis of the majority of substances present in the prohibited list [3,5,7,9]. This technique is becoming much more popular than GC, especially for polar compounds due to simpler sample preparation which does not require a derivatization step [3–7]. Fast UHPLC–MS screening of urine samples is often performed with a simple “dilute and shoot” approach as a sample treatment. The latter is compatible with a high throughput strategy, decreasing costs and minimizing possible errors occurring during the sample preparation step [8]. MS detection is performed using either low resolution mass spectrometry (e.g., triple quadrupole, quadrupole linear ion trap) [4] or high resolution mass spectrometry [10] (e.g., QqTOF [3] or orbitrapbased mass analyzers [5,7,9]). Supercritical fluid chromatography (SFC) has become competitive with current LC approaches only recently, with the introduction of modern SFC instruments showing sufficient stability of backpressure regulation, improved pumping and injection capabilities and thus resulting in a good robustness of the technique [11–13]. The advantages of SFC attributed to the properties of supercritical fluid (i.e., low viscosity, high diffusivity) have already been widely demonstrated. Using high flow-rates, the analysis time can be substantially decreased in comparison with LC procedures, while maintaining or increasing the separation efficiency, especially when using sub-2-mm particles [11,12,14– 16], known as ultra-high performance supercritical fluid chromatography (UHPSFC). The coupling of SFC with MS using atmospheric pressure ionization (API) sources should be even easier to achieve compared to LC–MS due to the high proportion of volatile CO2 contained in the mobile phase, which enhances the evaporation step during the ionization process [17–19]. Similarly to LC–MS, mobile phase conditions in SFC–MS might alter both chromatographic performance and ionization efficiency and the addition of a protondonating organic modifier such as MeOH to the CO2-based mobile phase is often necessary to generate ions. Various interfacing approaches including pre-BPR (back-pressure regulator) flow splitting, total flow introduction-pressure-regulating fluid interface, total flow introduction with mechanical BPR and total flow introduction with passive BPR, have been described in the literature for routing the supercritical effluent toward the API source [19,26]. It is worth mentioning that several works have already employed SFC– MS for analyzing biological samples, with CO2/MeOH mobile phases with or without splitting and make-up flow [20–22].
However, until now, there is no systematic study describing the influence of volatile mobile phase additives on the ionization process in SFC–MS. Similarly, an evaluation of various make-up solvents is missing. Therefore, the aim of this study was to provide a deep insight into the influence of mobile phase and make-up solvent composition on ionization process in UHPSFC–MS/MS using a representative set of doping agents ionized both in positive and negative electrospray modes (ESI). Finally, optimal conditions for both UHPLC–MS/MS and UHPSFC–MS/MS were determined in order to evaluate and compare the sensitivity of the two techniques on a set of 31 doping agents. 2. Experimental 2.1. Chemicals and reagents Reference standards of doping substances including aminoglutethimide, amiphenazone, amphetamine, benzoylecgonine, buprenorphine hydrochloride, carphedon, cyclazodone, ephedrine, etilefrine, fenproporex, methadone, methamphetamine hydrochloride, methylecgonine, modafinil, oxymorphone, pemoline and sibutramine were kindly provided by the Swiss Laboratory for Doping Analyses (Epalinges, Switzerland). Acetazolamide, bendroflumethiazide, bumetanide, bupropion, caffeine, chlortalidone, chlorthiazide, clopamide, hydrochlorthiazide, ethacrynic acid, furosemide, indapamide, metolazone and probenecide were purchased from Sigma–Aldrich (Buchs, Switzerland). Methanol, ethanol (EtOH), isopropanol (IpOH), acetonitrile (ACN), formic acid (FA) and acetic acid ULC/MS grade were provided by Biosolve (Chemie Brunschwig, Basel, Switzerland). Ammonium hydroxide, ammonium acetate and ammonium formate were provided by Sigma–Fluka (Buchs, Switzerland). Pressurized liquid CO2 3.0 grade (99.9%) was purchased from PanGas (Dagmerstellen, Switzerland). Ultra-pure water was provided by a Milli-Q system from Millipore (Bedford, MA, USA). 2.2. Standard solutions The stock standard solutions of doping agents were prepared in MeOH at a concentration of 1 mg mL 1. These solutions were further diluted with water and ACN for UHPLC–MS/MS and UHPSFC–MS/MS respectively, to obtain a final concentration of 1 mg mL 1. For the sensitivity and linearity tests, these solutions were further serially diluted until achieving MS response with S/N 10. 2.3. UHPSFC–MS/MS instrumentation and analysis The supercritical fluid chromatography system was an Acquity UPC2 (Waters, Milford, MA, USA), which consisted of a binary pump, a fixed-loop autosampler, a column thermostat, a 2-stages backpressure regulator (BPR, active + passive) and a PDA detector. The system was coupled to a triple quadrupole mass spectrometer, namely Waters TQD (Waters, Manchester, UK) via commercial SFC– MS dedicated splitter device (Waters) and auxiliary pump (Waters 515 pump) delivering a make-up solvent flow at 0.3 mL min 1. The separation was achieved on two SFC-dedicated stationary phases, namely Acquity UPC2 BEH and Acquity UPC2 BEH 2-EP (100 3.0 mm, 1.7 mm). Gradient elution was performed using CO2 and MeOH as organic modifier at a flow-rate of 1.5 mL min 1. Various additives in MeOH were tested, including ammonium hydroxide (10 mM), ammonium acetate (10 mM), ammonium formate (10 mM) and a combination of ammonium hydroxide and formic acid at 0.05%, corresponding to 7.5 mM of ammonia and 13 mM of formic acid, respectively. Gradient elution program started with 1 min isocratic step at 2% of organic modifier in CO2 and was linearly increased up to 40% within 4 min. The final
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isocratic step at 40% of organic modifier was maintained for 1 min, followed by a 1 min column equilibration, providing a total analysis time of 7 min. The column temperature and BPR (back-pressure regulator) were set at 40 C and 120 bar, respectively. The partial loop with needle overfill mode was used to inject 2 mL of sample. Methanol was selected as needle wash solvent. The MS conditions were tuned in both ESI+ and ESI modes as follows: capillary voltage: +1000 V/ 1000 V, ion source temperature: 130 C, extractor: 2.0 V, RF lens: 0.5 V. The desolvation gas was nitrogen at a flow of 900 L h 1 and at a temperature of 450 C. Nitrogen was also used as cone gas (100 L h 1), while argon was employed as a collision gas. Analyses were performed in SRM (selected reaction monitoring) mode using the precursor ions (e.g., [M + H]+ or [M H] in ESI+ and ESI , respectively) and the most intense product ions. The cone voltages (5–60 V), collision energies (5–40 eV), and dwell times were optimized for each SRM transition (Table 1). MassLynx v4.1 software was used for MS control and data acquisition. QuanLynx software was used for data processing and peak integration. During the method optimization, MeOH delivered at 0.3 mL min 1 was used as a make-up solvent. Other solvents including EtOH, IpOH and MeOH with water (5, 10 and 20%) or with various additives (0.1% formic acid, 10 mM ammonium formate and 10 mM ammonium hydroxide) were further tested as alternative make-up solvents.
The separation was achieved using an Acquity BEH C18 analytical column (50 2.1 mm, 1.7 mm). Mobile phase was composed of ACN and water containing 0.1% formic acid. The initial isocratic step at 2% ACN was held for 1 min and thereafter the percentage was increased to 98% ACN within 4 min followed by a 2 min final equilibration, providing a total analysis time of 7 min, similar to the one achieved in UHPSFC–MS/MS. Mobile phase flow-rate was fixed at 0.5 mL min 1. The partial loop with needle overfill mode was employed to inject 5 mL. The analytical column was thermostated at 40 C. ACN was employed as strong wash solvent, while 10% ACN in water was used as weak wash solvent. Various mobile phase additives were tested to improve sensitivity and peak shape: formic acid (25 mM, corresponding to 0.1%), ammonium formate (10 mM at pH 3.5 and 9.0) and ammonium acetate (10 mM at pH 5.0). MS conditions were tuned in both ESI+ and ESI modes as follows: capillary voltage: +1000 V/ 1000 V, ion source temperature: 130 C, extractor: 2.0 V, RF lens: 0.2 V. The desolvation gas was nitrogen at a flow of 900 L h 1 and at a temperature of 450 C. Nitrogen was also used as a cone gas (100 L h 1). Argon was used as a collision gas. The same SRM transitions as in Section 2.3 were used (Table 1). The cone voltages and collision energies were tuned in both UHPSFC–MS/MS and UHPLC–MS/MS conditions and were found to be equivalent. Similarly, MassLynx 4.1 and QuanLynx softwares were used for data acquisition and processing, respectively.
2.4. UHPLC–MS/MS instrumentation and analysis
3. Results and discussion
The system employed for UHPLC–MS/MS experiments was an Acquity UPLC (Waters) composed of a binary pump, a fixed-loop autosampler, a column thermostat and the same triple quadrupole mass spectrometer, namely TQD (Waters, Manchester, UK).
Doping agents reported in the WADA prohibited list include more than 200 compounds of various structures and physicochemical properties. For the optimization of both chromatographic and MS conditions, some representative analytes belonging to
Table 1 Physico-chemical properties of tested compounds and MS/MS parameters. Physico-chemical properties Name
Formula
Acetazolamide Bendroflumethiazide Bumetanide Ethacrynic acid Furosemide Hydrochlorothiazide Chlorothiazide Chlorthalidone Indapamide Metolazone Probenecid Aminoglutethimide Amiphenazole Amphetamine Benzoylecgonine Buprenorphine Bupropion Caffeine Carphedon Clopamide Cyclazodone Ephedrine Etilefrine Fenproporex Methadone Methamphetamine Methylecgonine Modafinil Oxymorphone Pemoline Sibutramine
C4H6N4O3S2 C15H14F3N3O4S2 C17H20N2O5S C13H12Cl2O4 C12H11ClN2O5S C7H8ClN3O4S2 C7H6ClN3O4S2 C14H11ClN2O4S C16H16ClN3O3S C16H16ClN3O3S C13H19NO4S C13H16N2O2 C9H9N3S C9H13N C16H19NO4 C29H41NO4 C13H18ClNO C8H10N4O2 C12H14N2O2 C14H20ClN3O3S C12H12N2O2 C10H15NO C10H15NO2 C12H16N2 C21H27NO C10H15N C10H17NO3 C15H15NO2S C17H19NO4 C9H8N2O2 C17H26ClN
Log P 0.260 1.292 2.883 2.840 2.304 0.021 0.150 0.701 1.964 3.163 2.514 0.542 1.167 1.789 2.263 2.826 2.323 0.628 0.703 2.082 1.184 1.079 0.627 2.043 3.930 2.202 0.157 1.183 1.148 0.425 5.467
0.296 0.313 0.363 0.431 0.475 0.260 0.285 0.539 0.468 0.812 0.305 0.352 0.258 0.197 0.397 0.595 0.266 0.753 0.525 0.626 0.452 0.266 0.269 0.256 0.350 0.211 0.435 0.453 0.555 0.449 0.269
pKa (acidic)
pKa (basic)
7.44 8.63 3.18 2.81 3.04 8.95 9.62 9.57 9.35 10.00 3.69 11.60 – – 3.35 9.47 – – 15.67 9.41 – 13.96 9.81 – – – 14.12 14.88 9.17 – –
3.37 4.21 4.48 – 2.49 4.08 – 3.85 1.87 2.79 5.36 4.41 5.12 9.94 10.83 8.31 7.16 0.52 0.80 3.58 2.76 9.38 9.22 7.90 9.05 10.38 9.57 1.07 7.58 0.27 9.69
Log D pH 3
Log D pH 5
Log D pH 9
0.26 1.29 2.05 2.43 2.01 0.02 0.15 0.70 1.93 3.16 2.43 0.79 0.81 1.31 0.46 0.27 0.74 0.63 0.70 1.42 0.99 2.02 2.74 1.05 0.83 0.90 3.26 1.18 1.94 0.42 2.37
0.26 1.29 1.73 0.66 0.34 0.02 0.15 0.70 1.96 3.16 1.19 0.46 0.80 1.31 0.24 0.07 0.21 0.63 0.70 2.07 1.18 2.00 2.44 0.64 0.88 0.90 3.25 1.18 1.30 0.42 2.38
1.90 0.68 0.29 0.91 0.88 0.44 1.52 0.60 1.77 3.12 0.63 0.54 1.17 0.80 0.25 2.60 2.32 0.63 0.70 1.92 1.18 0.50 0.14 2.01 3.60 0.81 0.90 1.18 0.84 0.42 4.70
H H ESI donor acceptor
Precursor (m/z)
Fragment (m/z)
CV (V)
CE (eV)
3 4 4 1 4 4 3 4 3 3 1 3 4 2 1 2 1 0 2 3 1 2 3 1 0 1 1 2 2 2 0
220.8 420.2 363.1 301.0 329.1 295.9 293.3 337.0 364.0 364.0 284.0 233.0 191.9 136.2 290.1 468.5 240.0 194.9 219.1 346.2 216.9 166.1 182.0 188.8 310.2 150.0 199.9 166.9 302.1 176.9 280.1
83.2 289.2 319.3 243.3 285.2 269.2 214.3 190.1 189.2 257.1 240.4 146.2 164.2 91.0 168.2 414.2 184.1 138.1 174.2 250.2 146.2 148.1 164.2 91.0 265.3 91.1 182.2 152.0 284.3 117.1 125.0
25 40 35 20 30 40 45 35 40 40 30 25 30 15 30 55 20 30 15 40 25 15 20 20 25 20 30 35 30 20 20
18 20 14 10 14 16 28 16 24 20 14 20 22 14 18 14 12 18 12 22 14 10 12 16 14 16 16 20 18 12 20
7 7 7 4 7 7 7 6 6 6 5 4 3 1 5 5 2 6 4 6 4 2 3 2 2 1 4 3 5 4 1
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS
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various doping agents categories and covering a broad range of physico-chemical properties (i.e., pKa, log P, molecular weight, log D, H-bond donor and acceptor groups, as shown in Table 1) were selected. The MS optimization was performed in ESI and ESI+ modes, for 11 and 20 compounds, respectively. The generic UHPLC–MS/MS and UHPSFC–MS/MS methods were set-up as described in Sections 2.3 and 2.4, based on the state-ofthe-art instrument and column technologies, with the aim to obtain high throughput screening methods. Fast generic gradient elution runs, covering a wide range of analyte polarities, were employed in UHPLC–MS/MS and UHPSFC–MS/MS, providing a total analysis time of 7 min including equilibration step in both cases. While C18 is unequivocally considered to be the stationary phase of choice in UHPLC–MS/MS, two chemistries, including hybrid silica (BEH) and hybrid silica bonded with 2-ethylpyridine (BEH 2-EP) were tested and compared in UHPSFC–MS/MS. Subsequently, the tuning of ESI source parameters was performed under UHPLC and UHPSFC conditions, in both positive and negative polarity modes. Similar results were obtained in UHPLC and UHPSFC using both positive and negative ionization modes: elevated desolvation temperature (450 C) and desolvation gas flow (900 L h 1) were required, while low capillary voltage (about 1 kV) provided the best response. The cone voltage values were also identical in UHPSFC and UHPLC. Thus, MS tuning parameters were found to be easily transferable between the two separation modes. 3.1. Selection of generic mobile phase additive for anti-doping screening in UHPLC–MS/MS Mobile phase composition in LC–MS is crucial for both chromatographic peak shape and mass spectrometry response. Therefore, several volatile additives can be used to enhance ionization of acids (e.g., basic buffers) and bases (e.g., acidic buffers). Conversely, compounds are well retained in reversedphase chromatography when they are under their neutral form. Therefore, finding a generic additive providing overall good chromatographic features and mass spectrometry response for a wide range of ionizable compounds might be challenging. With the
aim to choose the most generic additive for screening doping agents, a detailed study dealing with the evaluation of the influence of selected volatile additives on both chromatography and mass spectrometry response was accomplished. The separation was performed using the generic gradient described in Section 2.4, but with different mobile phase additives, including formic acid (0.1% corresponding to 25 mM), ammonium formate pH 3.5 and pH 9.0 (10 mM) and ammonium acetate pH 5.0 (10 mM). To show a fair comparison of the influence of mobile phase additives, eight compounds ionized in ESI and eight compounds ionized in ESI+ were selected for graphical presentation (Fig. 1). The MS responses for all additives were scaled for each compound relatively to the response obtained in presence of 25 mM formic acid, which was considered as the reference additive (response of 100%). The graphical presentation easily reveals a limited interest for ammonium acetate pH 5.0, as it provided worse results compared to 25 mM formic acid for almost all compounds. Ammonium formate at pH 9.0 offers a significant ionization enhancement for a few compounds in ESI+ mode (e.g., sibutramine, methadone, and amiphenazole). This behavior was attributed to the higher proportion of ACN required to elute these compounds from the column under their neutral form at pH 9, offering a better desolvation in ESI+, as reported elsewhere [23]. On the other hand, ammonium formate buffer at pH 3.5 generally provided low sensitivities, probably due to the presence of ammonia that competed with basic compounds in ESI+. Because the chromatographic performance (peak widths, asymmetry) was similar for all of the tested additives (Fig. 2), 25 mM formic acid was selected as the most generic additive for UHPLC–MS/MS analysis of doping agents, since it provided the best sensitivity. 3.2. Selection of generic mobile phase additive for anti-doping screening in UHPSFC–MS/MS Similarly to LC–MS, the proper selection of volatile mobile phase additive(s) is also considered important to obtain good chromatographic and MS performance capabilities in SFC–MS.
ES I −
E SI +
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE METOLAZONE
METHADONE
CAFFEINE
CHLORTHALIDONE
BUPROPION
0%
HYDROCHLOROTHIAZIDE
50% 100% 150%
ETHACRYNIC ACID
BENZOYLECGONINE
OXYMORFONE
BUMETANIDE
AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 1. Influence of selected additives on ESI–MS response under UHPLC–MS/MS conditions (Acquity BEH C18 column, experimental conditions described in Section 2.4) using various additives/buffers: 0.1% formic acid (gray surface), 10 mM ammonium formate pH 3.5 (blue dotted line), 10 mM ammonium formate pH 9.0 (purple full line) and 10 mM ammonium acetate pH 5.0 (red dashed line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPLC–MS/MS conditions using 10 mM ammonium formate pH 9.0 (A), 10 mM ammonium acetate pH 5.0 (B), 10 mM ammonium formate pH 3.5 (C) and 0.1% formic acid (D).
However, so far no systematic study is available in the literature describing the influence of various additives on SFC–MS response. Therefore, a detailed investigation of various volatile additives including 10 mM ammonium formate, 10 mM ammonium acetate, 10 mM ammonium hydroxide and finally a mixture of 7.5 mM ammonium hydroxide and 13 mM formic acid was performed
ESI +
using two stationary phase chemistries, namely BEH and BEH 2-EP. The same data presentation as in 3.1 was used and the MS response achieved in presence of 10 mM ammonium formate was taken as a reference value for the scaling of other additives. Similar trends were observed on both stationary phases for individual additives (Figs. 3 and 4). The most uniform MS response was observed with
ES I −
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE
METHADONE
METOLAZONE
CHLORTHALIDONE
CAFFEINE
BUPROPION
0%
HYDROCHLOROTHIAZIDE
50% 100% 150%
BENZOYLECGONINE
ETHACRYNIC ACID
OXYMORFONE
BUMETANIDE
AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 3. Influence of selected additives on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3) using various additives: 10 mM ammonium formate (light blue surface), 10 mM ammonium acetate (red dashed line), 10 mM ammonium hydroxide (green dotted line), 13 mM formic acid + 7.5 mM ammonium hydroxide (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ESI +
E SI − ACETAZOLAMIDE INDAPAMIDE
AMIPHENAZOLE METHADONE
METOLAZONE
CHLORTHALIDONE
CAFFEINE
BUPROPION
HYDROCHLOROTHIAZIDE
0%
50% 100% 150%
ETHACRYNIC ACID
BENZOYLECGONINE
BUMETANIDE
OXYMORFONE AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 4. Influence of selected additives on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH 2-EP column, experimental conditions described in Section 2.3) using various additives: 10 mM ammonium formate (light blue surface), 10 mM ammonium acetate (red dashed line), 10 mM ammonium hydroxide (green dotted line), 13 mM formic acid + 7.5 mM ammonium hydroxide (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ammonium acetate and ammonium formate, providing high signal intensity for most of the compounds. Ammonium hydroxide was found to be less convenient for UHPSFC–MS operation, especially in negative ionization mode, since the intensity was sometimes less than 50% of the reference value. A mixture of ammonium hydroxide and formic acid appeared as a promising combination for several analytes, such as sibutramine, oxymorphone, caffeine or bupropion. However, at the same time, much lower response was obtained for other analytes, such as amiphenazole, ethacrynic acid or furosemide (only on BEH column), compared to the reference. Therefore, this combination of additives was found to be less generic. The final decision on additive and stationary phase choice was based on the evaluation of peak shapes when using individual additives on both stationary phases (Figs. 5 and 6). For several compounds ionized in ESI+, such as sibutramine, carphedon, modafinil, pemoline or caffeine, and for the majority of compounds ionized in ESI , the choice of the additive was not critical, as very narrow and symmetrical peaks were obtained at any mobile phase and stationary phase conditions. The choice of a suitable additive became much more critical for a few basic compounds, including oxymorphone, methylecgonine, ephedrine, etilefrine, metamphetamine and fenproporex. In these cases, ammonium hydroxide was found to be the less convenient additive providing wider peaks and increased tailing (Figs. 5 and 6) for the investigated set of compounds. This observation may be related to the importance of ion-pairing retention mechanisms occurring between additives and compounds but also between additives and stationary phases. Both ammonium formate and acetate additives provided good chromatographic performance, with slightly better peak shapes for ammonium formate (i.e., oxymorphone and methylecgonine). This could be related to the higher acidity of formic vs. acetic acid. In ESI mode, the peak shape was only an issue for bumetanide and furosemide, providing peak tailing when ammonium hydroxide or mixed additive (formic acid + ammonium hydroxide) were used. This effect was stronger on BEH 2-EP stationary phase, probably because this phase possesses a positive charge on the 2-EP moiety
that can interact with these two acidic molecules. On the contrary, these peaks remained always symmetrical with ammonium formate. Due to the better peak symmetry of several critical compounds on BEH stationary phase, the latter was used for further experiments with ammonium formate as additive (Fig. 5). In LC–MS, it is well known that the MS response might be significantly improved when decreasing additive concentration [24]. To evaluate the influence of its concentration on MS response under UHPSFC–MS/MS conditions, some experiments were conducted with ammonium formate at different concentrations (1, 5, 10 and 20 mM). The data reported in Fig. 7 were scaled considering the reference MS response obtained with 10 mM ammonium formate (100%). As expected, the response was increased in UHPSFC–MS when decreasing additive concentration, especially at 1 mM ammonium formate (1.5-fold increase in MS signal). However, this low buffer concentration compromised peak shapes. Therefore, less than 10 mM is not recommended for UHPSFC–MS/ MS operation, if the quality of chromatography is judged important. Recently, water has also been discussed as an interesting additive in SFC, due to its positive effect on peak shapes and better repeatability of retention times [25]. The influence of 1, 2 and 5% water on MS response was therefore evaluated. The corresponding MS responses are reported in Fig. 8 and compared with a reference condition in absence of water. A significant increase in signal intensity was observed with increasing water concentration, for all the compounds in ESI+ mode (i.e., >150% of the response), except methadone. The behavior of acids analyzed in ESI was opposite, and a substantial decrease in signal intensity was noticed for all tested compounds, particularly at high water proportion, namely 5% (Fig. 8). Because the gain in sensitivity was significant with basic drugs (ESI+) and since the sensitivity reduction was reasonable in ESI in the presence of 2% of water, we decided to add this limited amount of water in the mobile phase. Furthermore, as described elsewhere [25], this could also contribute to better retention times repeatability, which is an important feature of anti-doping screening method.
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Fig. 5. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPSFC–MS/MS conditions using 13 mM formic acid + 7.5 mM ammonium hydroxide (A), 10 mM ammonium hydroxide (B), 10 mM ammonium acetate (C) and 10 mM ammonium formate (D), all of them in MeOH. Separation was accomplished on BEH stationary phase.
3.3. The selection of optimal make-up conditions for interfacing UHPSFC with MS/MS The hyphenation of UHPSFC and MS can be accomplished using several types of interfaces, as described elsewhere [19]. In our work, the pre-BPR flow-splitting with addition of make-up solvent was used, as recently described by Grand-Guillaume Perrenoud et al. [26]. However, to date, there is no systematic study showing the influence of make-up conditions on MS sensitivity. Then, we decided to investigate the impact of various organic solvents (i.e., MeOH, EtOH, IpOH), water proportion (i.e., from 5 to 20%) and volatile additives (i.e., formic acid, ammonium formate and ammonium hydroxide) on MS signal. To draw reliable conclusions, four compounds ionized in ESI+ and four compounds ionized in ESI were selected. The achieved responses with different makeup conditions were scaled based on the reference response achieved with pure MeOH. A great difference in the influence of make-up conditions on individual doping agents was observed between ESI+ and ESI , as illustrated in Fig. 9. When MeOH was replaced by EtOH as make up solvent, the MS response was generally increased in ESI (typically from 10 to 50%), while a signal reduction was observed (typically ranging from 10 to 50%) in ESI+. More deleterious effects were observed when IpOH was used as a make-up solvent. Indeed, IpOH lead to a slight increase or no change in the signal intensity (typically from 10 to 25%) in ESI+, while the signal was completely lost in ESI for all tested analytes. Because the ESI sensitivity was slightly reduced when adding 2% water into the mobile phase (see Section 3.2 and Fig. 8), we decided to use ethanol as make-up solvent, since it enhanced sensitivities in ESI compared to MeOH and provided only a moderate reduction of MS response in ESI+. Another benefit of ethanol is its limited environmental impact as it can be considered as a green solvent.
The addition of water to the make-up was also tested at concentrations ranging between 5 and 20%. In ESI , a significant loss of signal intensity (>50% of the reference) was observed, while an enhancement (>150% of the reference) occurred in ESI+. However, highest proportion of water (e.g., 20%) lead to lower sensitivity. With this elevated water amount, it is probable that the desolvation step became more critical (hard to eliminate water even with pneumatically-assisted ESI). Because water was not beneficial for sensitivity in both ESI+ and ESI , it was not added to the make-up for our doping agents screening. Surprisingly, the addition of acidic or basic additives to the make-up did not provide any improvement of the MS signal. Very limited increase (up to 15%) or even decrease of signal was observed in ESI+ when 25 mM formic acid was added to the makeup, while sensitivity was systematically decreased in ESI . With an addition of 10 mM ammonium formate in the make-up solvent, the gain in sensitivity in ESI+ and ESI was quite negligible (up to 15% on the maximum for only 3 compounds), while the loss was severe for sibutramine (about 10-fold lower response in presence of ammonium formate). With 10 mM ammonium hydroxide, a slight increase of MS signal (between 5 and 40%) was observed in ESI+ and ESI for the majority of the compounds, but a huge loss of intensity was again observed for sibutramine (compound 5). Thus, no additive was useful in the make-up solvent. 3.4. Comparison of UHPLC–MS/MS and UHPSFC–MS/MS as screening strategies for doping control analysis Using the generic UHPLC–MS/MS and UHPSFC–MS/MS methods described in Sections 3.1–3.3, the sensitivities were evaluated and compared for a representative set of doping agents. The concentrations providing S/N ratios 10 were taken into consideration as
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Fig. 6. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPSFC–MS/MS conditions using 13 mM formic acid + 7.5 mM ammonium hydroxide (A), 10 mM ammonium hydroxide (B), 10 mM ammonium acetate (C) and 10 mM ammonium formate (D), all of them in MeOH. Separation was accomplished on BEH-2EP stationary phase.
ESI +
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE
METHADONE
ESI −
METOLAZONE
CAFFEINE
CHLORTALIDONE
BUPROPION
HYDROCHLORTHIAZIDE
0% 50% 100% 150% 200%
BENZOYLECGONINE
ETHACRYNIC ACID
OXYMORPHONE
BUMETANIDE FUROSEMIDE
AMPHETAMINE SIBUTRAMINE
Fig. 7. Influence of additive concentration on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3). Experiments were performed with ammonium formate 20 mM (red dashed line), 10 mM (light blue surface), 5 mM (green dotted blue) and 1 mM (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ACETAZOLAMIDE
ESI +
AMIPHENAZOLE
E SI −
INDAPAMIDE
METHADONE
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METOLAZONE
CAFFEINE
CHLORTALIDONE
HYDROCHLORTHIAZIDE
BUPROPION
0% 50% 100% 150% 200%
BENZOYLECGONINE
ETHACRYNIC ACID
BUMETANIDE
OXYMORPHONE AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 8. Influence of the addition of water in mobile phase on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3). Experiments were performed with 10 mM ammonium formate without water addition (light blue surface), with an addition of 1% water (red dashed line), 2% water (green dotted line) and 5% water (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
a comparison criterion. The values obtained for both methods are provided in Table 2. As evidenced, UHPSFC–MS/MS appears as a suitable strategy for the screening of selected doping agents, providing enhanced sensitivities compared to UHPLC–MS/MS. A
significant gain (5–100-fold better MS response in UHPSFC–MS/MS vs. UHPLC–MS/MS) was noticed for 56% of compounds, while the sensitivity was not really different (up to a factor 2) between the two analytical strategies for 41% of the doping agents. Finally, a
ESI-
ESI+
300
250
200
150
100
50
0
1
2
3
4
5
6
7
8
Fig. 9. Influence of various make-up solvents on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3) with 2% water and ammonium formate 10 mM as a mobile phase additive. Make-up solvents were delivered at 0.3 mL min 1 using MeOH (black), EtOH (gray), isopropanol (yellow), MeOH + 5% H2O (white), MeOH + 10% H2O (light blue), MeOH + 20% H2O (dark blue), MeOH + 13 mM formic acid (red), MeOH + 10 mM ammonium formate (orange) and MeOH + 10 mM ammonium hydroxide (green). Following compounds were selected for data presentation: ESI negative: (1) acetazolamide, (2) indapamide, (3) hydrochlorthiazide, (4) ethacrynic acid; ESI positive: (5) sibutramine, (6) amphetamine, (7) oxymorphone, (8) benzoylecgonine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 A comparison of UHPLC–MS/MS and UHPSFC–MS/MS methods: linearity and sensitivity data. The values of LOQ were obtained under optimized conditions for each method. UHPLC–MS
UHPSFC–MS
Acetazolamide Bendroflumetziazide Indapamide Metolazone Probenecid Chlortalidon Hydrochlorthiazide Chlorothiazide Ethacrynic acid Bumetanide Furosemide Sibutramine Carphedone Buprenorphine Clopamide Methamphetamine Amphetamine Ephedrine Oxymorphone Methylecgonine Benzoylecgnine Buproprion Fenproporex Caffeine Aminoglutethimide Cyclazodone Modafinil Pemoline Methadone Etilefrine Amiphenazole
Linearity
LOQ (ng mL
0.9929 0.9969 0.9945 0.9892 0.9990 0.9991 0.9990 0.9750 0.9991 NA NA 0.9997 0.9758 0.9957 0.9919 0.9995 0.9710 0.9834 0.9983 0.9993 0.9994 0.9992 0.9861 0.9995 0.9984 0.9925 0.9996 0.9898 0.9982 0.9992 0.9976
5.0 1.0 5.0 5.0 5.0 5.0 5.0 1.0 5.0 100.0 50.0 1.0 0.5 5.0 0.5 0.5 0.5 0.5 1.0 1.0 0.5 0.5 0.5 0.5 1.0 0.1 0.5 0.5 5.0 0.5 10.0
1
)
Linearity
LOQ (ng mL
0.9990 0.9759 0.9973 0.9965 0.9994 0.9986 0.9990 0.9900 0.9991 0.9993 0.9990 NA NA NA 0.9986 0.9857 0.9931 0.9851 0.9902 0.9517 0.9981 0.9984 0.9997 0.9982 0.9997 0.9998 0.9997 0.9968 0.9676 0.9798 0.9906
50.0 50.0 25.0 25.0 10.0 50.0 50.0 10.0 25.0 25.0 25.0 50.0 50.0 500.0 5.0 0.5 1.0 1.0 10.0 1.0 0.5 1.0 1.0 5.0 5.0 1.0 5.0 1.0 10.0 5.0 5.0
1
)
NA: data not available as less than five calibration points were obtained due to low sensitivity.
better MS response (higher than 2-fold) was only obtained for bumetanide in UHPLC–MS/MS (about 4). Finally, even if a quantification of these non-threshold substances (except for ephedrine) is not formally required for screening purposes, performance of both techniques was also studied in terms of linearity, for concentrations ranging between 0.05 ng mL 1 and 1 mg mL 1. At least five concentration levels were considered for the construction of calibration curves. The results shown in Table 2 demonstrate that both methods provided very good linearity with correlation coefficients higher than 0.990 for most compounds under both conditions. Lower correlation coefficients were obtained for 7 and 6 compounds under UHPSFC–MS/MS and UHPLC–MS/MS conditions, respectively. 4. Conclusion The goal of this study was to determine the best conditions for UHPLC–MS/MS and UHPSFC–MS/MS analysis of doping agents, taking into account the MS responses and peak shapes. For this purpose, 31 representative doping agents detected in ESI+ or ESI were evaluated. Various mobile phase conditions were tested in both chromatographic modes. In the case of UHPLC–MS/MS, 0.1% (25 mM) formic acid in water/ACN was found to be the most generic mobile phase additive and provided the best MS response in ESI+ and ESI and acceptable peak shapes. In UHPSFC–MS/MS, the best compromise between peak shapes and MS responses was achieved when adding 2% water and 10 mM
ammonium formate into the CO2/MeOH mobile phase. Under these conditions, a very homogeneous MS response was obtained in both ESI+ and ESI , while the peaks remained symmetrical whatever the physico-chemical properties of the drugs. Furthermore, the addition of water also contributed to obtain better retention times repeatability, which is an important feature for anti-doping screening methods. Except mobile phase, the make-up solvent added for interfacing UHPSFC with MS was also optimized. Ethanol appears as the best candidate since it provides a suitable MS response for all doping agents and is considered as a green solvent. Finally, sensitivities were compared for the set of 31 doping agents in UHPLC–MS/MS and UHPSFC–MS/MS. Sensitivity was improved by 5–100-fold in UHPSFC–MS/MS vs. UHPLC–MS/MS for 56% of the tested drugs. For 41% of the doping agents, the sensitivity was not significantly different (up to a factor 2) between the two analytical strategies, while a better MS response was only attained with bumetanide (about 4) in UHPLC–MS/MS. In the second paper of this series, these optimal mobile phase conditions for UHPLC–MS/MS and UHPSFC–MS/MS will be employed for the screening of more than 100 doping agents in urine matrix. Acknowledgement The authors gratefully acknowledge research projects of Charles University in Prague UNCE 204026/2012. References [1] World Anti-Doping Agency (WADA), The World Anti-Doping Code, The 2008 Prohibited List, Montreal, 2008, www.wada-ama.org. [2] World Anti-doping Agency (WADA), The World Anti-Doping Code, Minimal Required Performance Limits, Technical Document TD2004MRPL, Montreal, 2004, www.wada-ama.org. [3] F. Badoud, E. Grata, L. Perrenoud, L. Avois, M. Saugy, S. Rudaz, J.-L. Veuthey, J. Chromatogr. A 1216 (2009) 4423–4433. [4] M. Mazzarino, I. Fiacco, X. De la Torre, F. Botrè, J. Chromatogr. A 1218 (2011) 8156–8167. [5] A. Mussenga, D.A. Cowan, J. Chromatogr. A 1288 (2013) 82–95. [6] P. Van Eeno, W. Van Gansbeke, N. De Brabanter, K. Deventer, F.T. Delbeke, J. Chromatogr. A 1218 (2011) 3306–3316. [7] A. Jiménez Girón, K. Deventer, K. Roels, P. Van Eenoo, Anal. Chim. Acta 721 (2012) 137–146. [8] K. Deventer, O.J. Pozo, A.G. Verstraete, P. Van Eenoo, Trends Anal. Chem. 55 (2014) 1–13. [9] M. Thevis, A. Thomas, V. Pop, W. Schänzer, J. Chromatogr. A 1292 (2013) 38–50. [10] R.J.B. Peters, A.A.M. Stolker, J.G.J. Mol, A. Lommen, E. Lyris, Y. Angelis, A. Vonaparti, M. Stamou, C. Georgakopoulos, M.W.F. Nielen, Trends Anal. Chem. 29 (2010) 1250. [11] A. Grand-Guillaume Perrenoud, J.-L. Veuthey, D. Guillarme, J. Chromatogr. A 1266 (2012) 158–167. [12] A. Grand-Guillaume Perrenoud, M. Ch Hamman, Goel, J.-L. Veuthey, D. Guillarme, S. Fekete, J. Chromatogr. A 1314 (2013) 288–297. [13] M. Saito, J. Biosci. Bioeng. 115 (2013) 590–599. [14] L. Nováková, P. Chocholouš, P. Solich, Talanta 121 (2014) 178–186. [15] A. Grand-Guillaume Perrenoud, J. Boccard, J.-L. Veuthey, D. Guillarme, J. Chromatogr. A 1262 (2012) 205–213. [16] A. Dispas, P. Lebrun, E. Ziemons, R. Marini, E. Rozet, P. Hubert, J. Chromatogr. A 1353 (2014) 78–88. [17] F. Li, Y. Hsieh, J. Sep. Sci. 31 (2008) 1231–1237. [18] R. Chen, Chromatogr. Today, February/March, 2009, 11–15. [19] J.D. Pinkston, Eur. J. Mass Spectrom. 11 (2005) 189–197. [20] Y. Hsieh, L. Favreau, J. Schwerdt, K.-C. Cheng, J. Pharm. Biomed. Anal. 40 (2006) 799–804. [21] R.A. Coe, J.O. Rathe, J.W. Lee, J. Pharm. Biomed Anal. 42 (2006) 573–580. [22] Y. Hsieh, F. Li, Ch J. Duncan, Anal. Chem. 79 (2007) 3856–3861. [23] J. Schappler, R. Nicoli, D. Nguyen, S. Rudaz, J.-L. Veuthey, D. Guillarme, Talanta 78 (2009) 377–387. [24] M. Hol9 capek, K. Volná, P. Jandera, L. Kolárová, K. Lemr, M. Exner, A. Církva, J. Mass Spectrom. 39 (2004) 43. [25] M. Ashraf-Khorassani, L.T. Taylor, J. Sep. Sci. 33 (2010) 1682–1691. [26] A. Grand-Guillaume Perrenoud, J.L. Veuthey, D. Guillarme, J. Chromatogr. A 1339 (2014) 174–184.
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions Lucie Nováková a , Alexandre Grand-Guillaume Perrenoud b , Raul Nicoli c, Martial Saugy c , Jean-Luc Veuthey b , Davy Guillarme b, * a
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d’Yvoy 20, 1211 Geneva 4, Switzerland c Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, West Switzerland, Chemin des Croisettes 22, 1066 Epalinges, Switzerland b
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
Sensitivities and peak shapes were compared in UHPLC–MS/MS and UHPSFC–MS/MS. 10 mM ammonium formate can be considered a suitable mobile phase additive for UHPSFC–MS/MS operation. For interfacing UHPSFC and MS/MS, ethanol can be used as generic makeup solvent. The impact of operating parameters on MS sensitivity was different with acidic and basic compounds.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 June 2014 Received in revised form 22 August 2014 Accepted 6 October 2014 Available online 14 October 2014
The conditions for the analysis of selected doping substances by UHPSFC–MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10 mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the makeup solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC–MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC–MS/MS. Sensitivity was improved by 5–100-fold in UHPSFC–MS/MS vs. UHPLC–MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC–MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC–MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC–MS/MS. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Ultra high performance supercritical fluid chromatography Ultra high performance liquid chromatography Doping agents Mobile phase Make-up solvent
* Corresponding author. Tel.: +41 22 379 34 63; fax: +41 22 379 68 08. E-mail address: [email protected] (D. Guillarme). http://dx.doi.org/10.1016/j.aca.2014.10.004 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
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1. Introduction The list of doping agents prohibited by the World Anti-Doping Agency (WADA) in the practice of sport includes a wide range of structurally diverse substances categorized in ten major different classes (S0–S9). In order to get uniform reporting of prohibited substances between laboratories, minimum routine detection and identification capabilities for drug testing methods have been established by WADA. In this context, minimum required performance levels (MRPL) were defined as minimal concentrations of non-threshold substances that all WADA-accredited laboratories shall be able to detect in routine testing [1,2]. Due to the high number of samples to be analyzed in a short period of time and the diversity of physico-chemical properties of substances present in the prohibited list, a two-steps approach is generally implemented in anti-doping analysis, including screening and selective confirmation steps. High throughput methods as well as sufficient sensitivity and specificity are needed to avoid false positive/negative results, especially in the screening phase. Both liquid chromatography (LC) and gas chromatography (GC) coupled to mass spectrometry (MS) are considered as reference methods. Regarding LC, ultra-high performance liquid chromatography mass spectrometry (UHPLC–MS) represents nowadays the technique of choice for the analysis of the majority of substances present in the prohibited list [3,5,7,9]. This technique is becoming much more popular than GC, especially for polar compounds due to simpler sample preparation which does not require a derivatization step [3–7]. Fast UHPLC–MS screening of urine samples is often performed with a simple “dilute and shoot” approach as a sample treatment. The latter is compatible with a high throughput strategy, decreasing costs and minimizing possible errors occurring during the sample preparation step [8]. MS detection is performed using either low resolution mass spectrometry (e.g., triple quadrupole, quadrupole linear ion trap) [4] or high resolution mass spectrometry [10] (e.g., QqTOF [3] or orbitrapbased mass analyzers [5,7,9]). Supercritical fluid chromatography (SFC) has become competitive with current LC approaches only recently, with the introduction of modern SFC instruments showing sufficient stability of backpressure regulation, improved pumping and injection capabilities and thus resulting in a good robustness of the technique [11–13]. The advantages of SFC attributed to the properties of supercritical fluid (i.e., low viscosity, high diffusivity) have already been widely demonstrated. Using high flow-rates, the analysis time can be substantially decreased in comparison with LC procedures, while maintaining or increasing the separation efficiency, especially when using sub-2-mm particles [11,12,14– 16], known as ultra-high performance supercritical fluid chromatography (UHPSFC). The coupling of SFC with MS using atmospheric pressure ionization (API) sources should be even easier to achieve compared to LC–MS due to the high proportion of volatile CO2 contained in the mobile phase, which enhances the evaporation step during the ionization process [17–19]. Similarly to LC–MS, mobile phase conditions in SFC–MS might alter both chromatographic performance and ionization efficiency and the addition of a protondonating organic modifier such as MeOH to the CO2-based mobile phase is often necessary to generate ions. Various interfacing approaches including pre-BPR (back-pressure regulator) flow splitting, total flow introduction-pressure-regulating fluid interface, total flow introduction with mechanical BPR and total flow introduction with passive BPR, have been described in the literature for routing the supercritical effluent toward the API source [19,26]. It is worth mentioning that several works have already employed SFC– MS for analyzing biological samples, with CO2/MeOH mobile phases with or without splitting and make-up flow [20–22].
However, until now, there is no systematic study describing the influence of volatile mobile phase additives on the ionization process in SFC–MS. Similarly, an evaluation of various make-up solvents is missing. Therefore, the aim of this study was to provide a deep insight into the influence of mobile phase and make-up solvent composition on ionization process in UHPSFC–MS/MS using a representative set of doping agents ionized both in positive and negative electrospray modes (ESI). Finally, optimal conditions for both UHPLC–MS/MS and UHPSFC–MS/MS were determined in order to evaluate and compare the sensitivity of the two techniques on a set of 31 doping agents. 2. Experimental 2.1. Chemicals and reagents Reference standards of doping substances including aminoglutethimide, amiphenazone, amphetamine, benzoylecgonine, buprenorphine hydrochloride, carphedon, cyclazodone, ephedrine, etilefrine, fenproporex, methadone, methamphetamine hydrochloride, methylecgonine, modafinil, oxymorphone, pemoline and sibutramine were kindly provided by the Swiss Laboratory for Doping Analyses (Epalinges, Switzerland). Acetazolamide, bendroflumethiazide, bumetanide, bupropion, caffeine, chlortalidone, chlorthiazide, clopamide, hydrochlorthiazide, ethacrynic acid, furosemide, indapamide, metolazone and probenecide were purchased from Sigma–Aldrich (Buchs, Switzerland). Methanol, ethanol (EtOH), isopropanol (IpOH), acetonitrile (ACN), formic acid (FA) and acetic acid ULC/MS grade were provided by Biosolve (Chemie Brunschwig, Basel, Switzerland). Ammonium hydroxide, ammonium acetate and ammonium formate were provided by Sigma–Fluka (Buchs, Switzerland). Pressurized liquid CO2 3.0 grade (99.9%) was purchased from PanGas (Dagmerstellen, Switzerland). Ultra-pure water was provided by a Milli-Q system from Millipore (Bedford, MA, USA). 2.2. Standard solutions The stock standard solutions of doping agents were prepared in MeOH at a concentration of 1 mg mL 1. These solutions were further diluted with water and ACN for UHPLC–MS/MS and UHPSFC–MS/MS respectively, to obtain a final concentration of 1 mg mL 1. For the sensitivity and linearity tests, these solutions were further serially diluted until achieving MS response with S/N 10. 2.3. UHPSFC–MS/MS instrumentation and analysis The supercritical fluid chromatography system was an Acquity UPC2 (Waters, Milford, MA, USA), which consisted of a binary pump, a fixed-loop autosampler, a column thermostat, a 2-stages backpressure regulator (BPR, active + passive) and a PDA detector. The system was coupled to a triple quadrupole mass spectrometer, namely Waters TQD (Waters, Manchester, UK) via commercial SFC– MS dedicated splitter device (Waters) and auxiliary pump (Waters 515 pump) delivering a make-up solvent flow at 0.3 mL min 1. The separation was achieved on two SFC-dedicated stationary phases, namely Acquity UPC2 BEH and Acquity UPC2 BEH 2-EP (100 3.0 mm, 1.7 mm). Gradient elution was performed using CO2 and MeOH as organic modifier at a flow-rate of 1.5 mL min 1. Various additives in MeOH were tested, including ammonium hydroxide (10 mM), ammonium acetate (10 mM), ammonium formate (10 mM) and a combination of ammonium hydroxide and formic acid at 0.05%, corresponding to 7.5 mM of ammonia and 13 mM of formic acid, respectively. Gradient elution program started with 1 min isocratic step at 2% of organic modifier in CO2 and was linearly increased up to 40% within 4 min. The final
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isocratic step at 40% of organic modifier was maintained for 1 min, followed by a 1 min column equilibration, providing a total analysis time of 7 min. The column temperature and BPR (back-pressure regulator) were set at 40 C and 120 bar, respectively. The partial loop with needle overfill mode was used to inject 2 mL of sample. Methanol was selected as needle wash solvent. The MS conditions were tuned in both ESI+ and ESI modes as follows: capillary voltage: +1000 V/ 1000 V, ion source temperature: 130 C, extractor: 2.0 V, RF lens: 0.5 V. The desolvation gas was nitrogen at a flow of 900 L h 1 and at a temperature of 450 C. Nitrogen was also used as cone gas (100 L h 1), while argon was employed as a collision gas. Analyses were performed in SRM (selected reaction monitoring) mode using the precursor ions (e.g., [M + H]+ or [M H] in ESI+ and ESI , respectively) and the most intense product ions. The cone voltages (5–60 V), collision energies (5–40 eV), and dwell times were optimized for each SRM transition (Table 1). MassLynx v4.1 software was used for MS control and data acquisition. QuanLynx software was used for data processing and peak integration. During the method optimization, MeOH delivered at 0.3 mL min 1 was used as a make-up solvent. Other solvents including EtOH, IpOH and MeOH with water (5, 10 and 20%) or with various additives (0.1% formic acid, 10 mM ammonium formate and 10 mM ammonium hydroxide) were further tested as alternative make-up solvents.
The separation was achieved using an Acquity BEH C18 analytical column (50 2.1 mm, 1.7 mm). Mobile phase was composed of ACN and water containing 0.1% formic acid. The initial isocratic step at 2% ACN was held for 1 min and thereafter the percentage was increased to 98% ACN within 4 min followed by a 2 min final equilibration, providing a total analysis time of 7 min, similar to the one achieved in UHPSFC–MS/MS. Mobile phase flow-rate was fixed at 0.5 mL min 1. The partial loop with needle overfill mode was employed to inject 5 mL. The analytical column was thermostated at 40 C. ACN was employed as strong wash solvent, while 10% ACN in water was used as weak wash solvent. Various mobile phase additives were tested to improve sensitivity and peak shape: formic acid (25 mM, corresponding to 0.1%), ammonium formate (10 mM at pH 3.5 and 9.0) and ammonium acetate (10 mM at pH 5.0). MS conditions were tuned in both ESI+ and ESI modes as follows: capillary voltage: +1000 V/ 1000 V, ion source temperature: 130 C, extractor: 2.0 V, RF lens: 0.2 V. The desolvation gas was nitrogen at a flow of 900 L h 1 and at a temperature of 450 C. Nitrogen was also used as a cone gas (100 L h 1). Argon was used as a collision gas. The same SRM transitions as in Section 2.3 were used (Table 1). The cone voltages and collision energies were tuned in both UHPSFC–MS/MS and UHPLC–MS/MS conditions and were found to be equivalent. Similarly, MassLynx 4.1 and QuanLynx softwares were used for data acquisition and processing, respectively.
2.4. UHPLC–MS/MS instrumentation and analysis
3. Results and discussion
The system employed for UHPLC–MS/MS experiments was an Acquity UPLC (Waters) composed of a binary pump, a fixed-loop autosampler, a column thermostat and the same triple quadrupole mass spectrometer, namely TQD (Waters, Manchester, UK).
Doping agents reported in the WADA prohibited list include more than 200 compounds of various structures and physicochemical properties. For the optimization of both chromatographic and MS conditions, some representative analytes belonging to
Table 1 Physico-chemical properties of tested compounds and MS/MS parameters. Physico-chemical properties Name
Formula
Acetazolamide Bendroflumethiazide Bumetanide Ethacrynic acid Furosemide Hydrochlorothiazide Chlorothiazide Chlorthalidone Indapamide Metolazone Probenecid Aminoglutethimide Amiphenazole Amphetamine Benzoylecgonine Buprenorphine Bupropion Caffeine Carphedon Clopamide Cyclazodone Ephedrine Etilefrine Fenproporex Methadone Methamphetamine Methylecgonine Modafinil Oxymorphone Pemoline Sibutramine
C4H6N4O3S2 C15H14F3N3O4S2 C17H20N2O5S C13H12Cl2O4 C12H11ClN2O5S C7H8ClN3O4S2 C7H6ClN3O4S2 C14H11ClN2O4S C16H16ClN3O3S C16H16ClN3O3S C13H19NO4S C13H16N2O2 C9H9N3S C9H13N C16H19NO4 C29H41NO4 C13H18ClNO C8H10N4O2 C12H14N2O2 C14H20ClN3O3S C12H12N2O2 C10H15NO C10H15NO2 C12H16N2 C21H27NO C10H15N C10H17NO3 C15H15NO2S C17H19NO4 C9H8N2O2 C17H26ClN
Log P 0.260 1.292 2.883 2.840 2.304 0.021 0.150 0.701 1.964 3.163 2.514 0.542 1.167 1.789 2.263 2.826 2.323 0.628 0.703 2.082 1.184 1.079 0.627 2.043 3.930 2.202 0.157 1.183 1.148 0.425 5.467
0.296 0.313 0.363 0.431 0.475 0.260 0.285 0.539 0.468 0.812 0.305 0.352 0.258 0.197 0.397 0.595 0.266 0.753 0.525 0.626 0.452 0.266 0.269 0.256 0.350 0.211 0.435 0.453 0.555 0.449 0.269
pKa (acidic)
pKa (basic)
7.44 8.63 3.18 2.81 3.04 8.95 9.62 9.57 9.35 10.00 3.69 11.60 – – 3.35 9.47 – – 15.67 9.41 – 13.96 9.81 – – – 14.12 14.88 9.17 – –
3.37 4.21 4.48 – 2.49 4.08 – 3.85 1.87 2.79 5.36 4.41 5.12 9.94 10.83 8.31 7.16 0.52 0.80 3.58 2.76 9.38 9.22 7.90 9.05 10.38 9.57 1.07 7.58 0.27 9.69
Log D pH 3
Log D pH 5
Log D pH 9
0.26 1.29 2.05 2.43 2.01 0.02 0.15 0.70 1.93 3.16 2.43 0.79 0.81 1.31 0.46 0.27 0.74 0.63 0.70 1.42 0.99 2.02 2.74 1.05 0.83 0.90 3.26 1.18 1.94 0.42 2.37
0.26 1.29 1.73 0.66 0.34 0.02 0.15 0.70 1.96 3.16 1.19 0.46 0.80 1.31 0.24 0.07 0.21 0.63 0.70 2.07 1.18 2.00 2.44 0.64 0.88 0.90 3.25 1.18 1.30 0.42 2.38
1.90 0.68 0.29 0.91 0.88 0.44 1.52 0.60 1.77 3.12 0.63 0.54 1.17 0.80 0.25 2.60 2.32 0.63 0.70 1.92 1.18 0.50 0.14 2.01 3.60 0.81 0.90 1.18 0.84 0.42 4.70
H H ESI donor acceptor
Precursor (m/z)
Fragment (m/z)
CV (V)
CE (eV)
3 4 4 1 4 4 3 4 3 3 1 3 4 2 1 2 1 0 2 3 1 2 3 1 0 1 1 2 2 2 0
220.8 420.2 363.1 301.0 329.1 295.9 293.3 337.0 364.0 364.0 284.0 233.0 191.9 136.2 290.1 468.5 240.0 194.9 219.1 346.2 216.9 166.1 182.0 188.8 310.2 150.0 199.9 166.9 302.1 176.9 280.1
83.2 289.2 319.3 243.3 285.2 269.2 214.3 190.1 189.2 257.1 240.4 146.2 164.2 91.0 168.2 414.2 184.1 138.1 174.2 250.2 146.2 148.1 164.2 91.0 265.3 91.1 182.2 152.0 284.3 117.1 125.0
25 40 35 20 30 40 45 35 40 40 30 25 30 15 30 55 20 30 15 40 25 15 20 20 25 20 30 35 30 20 20
18 20 14 10 14 16 28 16 24 20 14 20 22 14 18 14 12 18 12 22 14 10 12 16 14 16 16 20 18 12 20
7 7 7 4 7 7 7 6 6 6 5 4 3 1 5 5 2 6 4 6 4 2 3 2 2 1 4 3 5 4 1
NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS POS
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various doping agents categories and covering a broad range of physico-chemical properties (i.e., pKa, log P, molecular weight, log D, H-bond donor and acceptor groups, as shown in Table 1) were selected. The MS optimization was performed in ESI and ESI+ modes, for 11 and 20 compounds, respectively. The generic UHPLC–MS/MS and UHPSFC–MS/MS methods were set-up as described in Sections 2.3 and 2.4, based on the state-ofthe-art instrument and column technologies, with the aim to obtain high throughput screening methods. Fast generic gradient elution runs, covering a wide range of analyte polarities, were employed in UHPLC–MS/MS and UHPSFC–MS/MS, providing a total analysis time of 7 min including equilibration step in both cases. While C18 is unequivocally considered to be the stationary phase of choice in UHPLC–MS/MS, two chemistries, including hybrid silica (BEH) and hybrid silica bonded with 2-ethylpyridine (BEH 2-EP) were tested and compared in UHPSFC–MS/MS. Subsequently, the tuning of ESI source parameters was performed under UHPLC and UHPSFC conditions, in both positive and negative polarity modes. Similar results were obtained in UHPLC and UHPSFC using both positive and negative ionization modes: elevated desolvation temperature (450 C) and desolvation gas flow (900 L h 1) were required, while low capillary voltage (about 1 kV) provided the best response. The cone voltage values were also identical in UHPSFC and UHPLC. Thus, MS tuning parameters were found to be easily transferable between the two separation modes. 3.1. Selection of generic mobile phase additive for anti-doping screening in UHPLC–MS/MS Mobile phase composition in LC–MS is crucial for both chromatographic peak shape and mass spectrometry response. Therefore, several volatile additives can be used to enhance ionization of acids (e.g., basic buffers) and bases (e.g., acidic buffers). Conversely, compounds are well retained in reversedphase chromatography when they are under their neutral form. Therefore, finding a generic additive providing overall good chromatographic features and mass spectrometry response for a wide range of ionizable compounds might be challenging. With the
aim to choose the most generic additive for screening doping agents, a detailed study dealing with the evaluation of the influence of selected volatile additives on both chromatography and mass spectrometry response was accomplished. The separation was performed using the generic gradient described in Section 2.4, but with different mobile phase additives, including formic acid (0.1% corresponding to 25 mM), ammonium formate pH 3.5 and pH 9.0 (10 mM) and ammonium acetate pH 5.0 (10 mM). To show a fair comparison of the influence of mobile phase additives, eight compounds ionized in ESI and eight compounds ionized in ESI+ were selected for graphical presentation (Fig. 1). The MS responses for all additives were scaled for each compound relatively to the response obtained in presence of 25 mM formic acid, which was considered as the reference additive (response of 100%). The graphical presentation easily reveals a limited interest for ammonium acetate pH 5.0, as it provided worse results compared to 25 mM formic acid for almost all compounds. Ammonium formate at pH 9.0 offers a significant ionization enhancement for a few compounds in ESI+ mode (e.g., sibutramine, methadone, and amiphenazole). This behavior was attributed to the higher proportion of ACN required to elute these compounds from the column under their neutral form at pH 9, offering a better desolvation in ESI+, as reported elsewhere [23]. On the other hand, ammonium formate buffer at pH 3.5 generally provided low sensitivities, probably due to the presence of ammonia that competed with basic compounds in ESI+. Because the chromatographic performance (peak widths, asymmetry) was similar for all of the tested additives (Fig. 2), 25 mM formic acid was selected as the most generic additive for UHPLC–MS/MS analysis of doping agents, since it provided the best sensitivity. 3.2. Selection of generic mobile phase additive for anti-doping screening in UHPSFC–MS/MS Similarly to LC–MS, the proper selection of volatile mobile phase additive(s) is also considered important to obtain good chromatographic and MS performance capabilities in SFC–MS.
ES I −
E SI +
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE METOLAZONE
METHADONE
CAFFEINE
CHLORTHALIDONE
BUPROPION
0%
HYDROCHLOROTHIAZIDE
50% 100% 150%
ETHACRYNIC ACID
BENZOYLECGONINE
OXYMORFONE
BUMETANIDE
AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 1. Influence of selected additives on ESI–MS response under UHPLC–MS/MS conditions (Acquity BEH C18 column, experimental conditions described in Section 2.4) using various additives/buffers: 0.1% formic acid (gray surface), 10 mM ammonium formate pH 3.5 (blue dotted line), 10 mM ammonium formate pH 9.0 (purple full line) and 10 mM ammonium acetate pH 5.0 (red dashed line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPLC–MS/MS conditions using 10 mM ammonium formate pH 9.0 (A), 10 mM ammonium acetate pH 5.0 (B), 10 mM ammonium formate pH 3.5 (C) and 0.1% formic acid (D).
However, so far no systematic study is available in the literature describing the influence of various additives on SFC–MS response. Therefore, a detailed investigation of various volatile additives including 10 mM ammonium formate, 10 mM ammonium acetate, 10 mM ammonium hydroxide and finally a mixture of 7.5 mM ammonium hydroxide and 13 mM formic acid was performed
ESI +
using two stationary phase chemistries, namely BEH and BEH 2-EP. The same data presentation as in 3.1 was used and the MS response achieved in presence of 10 mM ammonium formate was taken as a reference value for the scaling of other additives. Similar trends were observed on both stationary phases for individual additives (Figs. 3 and 4). The most uniform MS response was observed with
ES I −
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE
METHADONE
METOLAZONE
CHLORTHALIDONE
CAFFEINE
BUPROPION
0%
HYDROCHLOROTHIAZIDE
50% 100% 150%
BENZOYLECGONINE
ETHACRYNIC ACID
OXYMORFONE
BUMETANIDE
AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 3. Influence of selected additives on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3) using various additives: 10 mM ammonium formate (light blue surface), 10 mM ammonium acetate (red dashed line), 10 mM ammonium hydroxide (green dotted line), 13 mM formic acid + 7.5 mM ammonium hydroxide (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ESI +
E SI − ACETAZOLAMIDE INDAPAMIDE
AMIPHENAZOLE METHADONE
METOLAZONE
CHLORTHALIDONE
CAFFEINE
BUPROPION
HYDROCHLOROTHIAZIDE
0%
50% 100% 150%
ETHACRYNIC ACID
BENZOYLECGONINE
BUMETANIDE
OXYMORFONE AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 4. Influence of selected additives on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH 2-EP column, experimental conditions described in Section 2.3) using various additives: 10 mM ammonium formate (light blue surface), 10 mM ammonium acetate (red dashed line), 10 mM ammonium hydroxide (green dotted line), 13 mM formic acid + 7.5 mM ammonium hydroxide (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
ammonium acetate and ammonium formate, providing high signal intensity for most of the compounds. Ammonium hydroxide was found to be less convenient for UHPSFC–MS operation, especially in negative ionization mode, since the intensity was sometimes less than 50% of the reference value. A mixture of ammonium hydroxide and formic acid appeared as a promising combination for several analytes, such as sibutramine, oxymorphone, caffeine or bupropion. However, at the same time, much lower response was obtained for other analytes, such as amiphenazole, ethacrynic acid or furosemide (only on BEH column), compared to the reference. Therefore, this combination of additives was found to be less generic. The final decision on additive and stationary phase choice was based on the evaluation of peak shapes when using individual additives on both stationary phases (Figs. 5 and 6). For several compounds ionized in ESI+, such as sibutramine, carphedon, modafinil, pemoline or caffeine, and for the majority of compounds ionized in ESI , the choice of the additive was not critical, as very narrow and symmetrical peaks were obtained at any mobile phase and stationary phase conditions. The choice of a suitable additive became much more critical for a few basic compounds, including oxymorphone, methylecgonine, ephedrine, etilefrine, metamphetamine and fenproporex. In these cases, ammonium hydroxide was found to be the less convenient additive providing wider peaks and increased tailing (Figs. 5 and 6) for the investigated set of compounds. This observation may be related to the importance of ion-pairing retention mechanisms occurring between additives and compounds but also between additives and stationary phases. Both ammonium formate and acetate additives provided good chromatographic performance, with slightly better peak shapes for ammonium formate (i.e., oxymorphone and methylecgonine). This could be related to the higher acidity of formic vs. acetic acid. In ESI mode, the peak shape was only an issue for bumetanide and furosemide, providing peak tailing when ammonium hydroxide or mixed additive (formic acid + ammonium hydroxide) were used. This effect was stronger on BEH 2-EP stationary phase, probably because this phase possesses a positive charge on the 2-EP moiety
that can interact with these two acidic molecules. On the contrary, these peaks remained always symmetrical with ammonium formate. Due to the better peak symmetry of several critical compounds on BEH stationary phase, the latter was used for further experiments with ammonium formate as additive (Fig. 5). In LC–MS, it is well known that the MS response might be significantly improved when decreasing additive concentration [24]. To evaluate the influence of its concentration on MS response under UHPSFC–MS/MS conditions, some experiments were conducted with ammonium formate at different concentrations (1, 5, 10 and 20 mM). The data reported in Fig. 7 were scaled considering the reference MS response obtained with 10 mM ammonium formate (100%). As expected, the response was increased in UHPSFC–MS when decreasing additive concentration, especially at 1 mM ammonium formate (1.5-fold increase in MS signal). However, this low buffer concentration compromised peak shapes. Therefore, less than 10 mM is not recommended for UHPSFC–MS/ MS operation, if the quality of chromatography is judged important. Recently, water has also been discussed as an interesting additive in SFC, due to its positive effect on peak shapes and better repeatability of retention times [25]. The influence of 1, 2 and 5% water on MS response was therefore evaluated. The corresponding MS responses are reported in Fig. 8 and compared with a reference condition in absence of water. A significant increase in signal intensity was observed with increasing water concentration, for all the compounds in ESI+ mode (i.e., >150% of the response), except methadone. The behavior of acids analyzed in ESI was opposite, and a substantial decrease in signal intensity was noticed for all tested compounds, particularly at high water proportion, namely 5% (Fig. 8). Because the gain in sensitivity was significant with basic drugs (ESI+) and since the sensitivity reduction was reasonable in ESI in the presence of 2% of water, we decided to add this limited amount of water in the mobile phase. Furthermore, as described elsewhere [25], this could also contribute to better retention times repeatability, which is an important feature of anti-doping screening method.
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Fig. 5. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPSFC–MS/MS conditions using 13 mM formic acid + 7.5 mM ammonium hydroxide (A), 10 mM ammonium hydroxide (B), 10 mM ammonium acetate (C) and 10 mM ammonium formate (D), all of them in MeOH. Separation was accomplished on BEH stationary phase.
3.3. The selection of optimal make-up conditions for interfacing UHPSFC with MS/MS The hyphenation of UHPSFC and MS can be accomplished using several types of interfaces, as described elsewhere [19]. In our work, the pre-BPR flow-splitting with addition of make-up solvent was used, as recently described by Grand-Guillaume Perrenoud et al. [26]. However, to date, there is no systematic study showing the influence of make-up conditions on MS sensitivity. Then, we decided to investigate the impact of various organic solvents (i.e., MeOH, EtOH, IpOH), water proportion (i.e., from 5 to 20%) and volatile additives (i.e., formic acid, ammonium formate and ammonium hydroxide) on MS signal. To draw reliable conclusions, four compounds ionized in ESI+ and four compounds ionized in ESI were selected. The achieved responses with different makeup conditions were scaled based on the reference response achieved with pure MeOH. A great difference in the influence of make-up conditions on individual doping agents was observed between ESI+ and ESI , as illustrated in Fig. 9. When MeOH was replaced by EtOH as make up solvent, the MS response was generally increased in ESI (typically from 10 to 50%), while a signal reduction was observed (typically ranging from 10 to 50%) in ESI+. More deleterious effects were observed when IpOH was used as a make-up solvent. Indeed, IpOH lead to a slight increase or no change in the signal intensity (typically from 10 to 25%) in ESI+, while the signal was completely lost in ESI for all tested analytes. Because the ESI sensitivity was slightly reduced when adding 2% water into the mobile phase (see Section 3.2 and Fig. 8), we decided to use ethanol as make-up solvent, since it enhanced sensitivities in ESI compared to MeOH and provided only a moderate reduction of MS response in ESI+. Another benefit of ethanol is its limited environmental impact as it can be considered as a green solvent.
The addition of water to the make-up was also tested at concentrations ranging between 5 and 20%. In ESI , a significant loss of signal intensity (>50% of the reference) was observed, while an enhancement (>150% of the reference) occurred in ESI+. However, highest proportion of water (e.g., 20%) lead to lower sensitivity. With this elevated water amount, it is probable that the desolvation step became more critical (hard to eliminate water even with pneumatically-assisted ESI). Because water was not beneficial for sensitivity in both ESI+ and ESI , it was not added to the make-up for our doping agents screening. Surprisingly, the addition of acidic or basic additives to the make-up did not provide any improvement of the MS signal. Very limited increase (up to 15%) or even decrease of signal was observed in ESI+ when 25 mM formic acid was added to the makeup, while sensitivity was systematically decreased in ESI . With an addition of 10 mM ammonium formate in the make-up solvent, the gain in sensitivity in ESI+ and ESI was quite negligible (up to 15% on the maximum for only 3 compounds), while the loss was severe for sibutramine (about 10-fold lower response in presence of ammonium formate). With 10 mM ammonium hydroxide, a slight increase of MS signal (between 5 and 40%) was observed in ESI+ and ESI for the majority of the compounds, but a huge loss of intensity was again observed for sibutramine (compound 5). Thus, no additive was useful in the make-up solvent. 3.4. Comparison of UHPLC–MS/MS and UHPSFC–MS/MS as screening strategies for doping control analysis Using the generic UHPLC–MS/MS and UHPSFC–MS/MS methods described in Sections 3.1–3.3, the sensitivities were evaluated and compared for a representative set of doping agents. The concentrations providing S/N ratios 10 were taken into consideration as
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Fig. 6. SRM chromatograms of selected compounds in ESI+ (metamphetamine, methylecgonine and oxymorphone) and in ESI (furosemide) modes under UHPSFC–MS/MS conditions using 13 mM formic acid + 7.5 mM ammonium hydroxide (A), 10 mM ammonium hydroxide (B), 10 mM ammonium acetate (C) and 10 mM ammonium formate (D), all of them in MeOH. Separation was accomplished on BEH-2EP stationary phase.
ESI +
ACETAZOLAMIDE AMIPHENAZOLE
INDAPAMIDE
METHADONE
ESI −
METOLAZONE
CAFFEINE
CHLORTALIDONE
BUPROPION
HYDROCHLORTHIAZIDE
0% 50% 100% 150% 200%
BENZOYLECGONINE
ETHACRYNIC ACID
OXYMORPHONE
BUMETANIDE FUROSEMIDE
AMPHETAMINE SIBUTRAMINE
Fig. 7. Influence of additive concentration on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3). Experiments were performed with ammonium formate 20 mM (red dashed line), 10 mM (light blue surface), 5 mM (green dotted blue) and 1 mM (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ACETAZOLAMIDE
ESI +
AMIPHENAZOLE
E SI −
INDAPAMIDE
METHADONE
645
METOLAZONE
CAFFEINE
CHLORTALIDONE
HYDROCHLORTHIAZIDE
BUPROPION
0% 50% 100% 150% 200%
BENZOYLECGONINE
ETHACRYNIC ACID
BUMETANIDE
OXYMORPHONE AMPHETAMINE
FUROSEMIDE SIBUTRAMINE
Fig. 8. Influence of the addition of water in mobile phase on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3). Experiments were performed with 10 mM ammonium formate without water addition (light blue surface), with an addition of 1% water (red dashed line), 2% water (green dotted line) and 5% water (purple full line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
a comparison criterion. The values obtained for both methods are provided in Table 2. As evidenced, UHPSFC–MS/MS appears as a suitable strategy for the screening of selected doping agents, providing enhanced sensitivities compared to UHPLC–MS/MS. A
significant gain (5–100-fold better MS response in UHPSFC–MS/MS vs. UHPLC–MS/MS) was noticed for 56% of compounds, while the sensitivity was not really different (up to a factor 2) between the two analytical strategies for 41% of the doping agents. Finally, a
ESI-
ESI+
300
250
200
150
100
50
0
1
2
3
4
5
6
7
8
Fig. 9. Influence of various make-up solvents on ESI–MS response under UHPSFC–MS/MS conditions (Acquity UPC2 BEH column, experimental conditions described in Section 2.3) with 2% water and ammonium formate 10 mM as a mobile phase additive. Make-up solvents were delivered at 0.3 mL min 1 using MeOH (black), EtOH (gray), isopropanol (yellow), MeOH + 5% H2O (white), MeOH + 10% H2O (light blue), MeOH + 20% H2O (dark blue), MeOH + 13 mM formic acid (red), MeOH + 10 mM ammonium formate (orange) and MeOH + 10 mM ammonium hydroxide (green). Following compounds were selected for data presentation: ESI negative: (1) acetazolamide, (2) indapamide, (3) hydrochlorthiazide, (4) ethacrynic acid; ESI positive: (5) sibutramine, (6) amphetamine, (7) oxymorphone, (8) benzoylecgonine. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Table 2 A comparison of UHPLC–MS/MS and UHPSFC–MS/MS methods: linearity and sensitivity data. The values of LOQ were obtained under optimized conditions for each method. UHPLC–MS
UHPSFC–MS
Acetazolamide Bendroflumetziazide Indapamide Metolazone Probenecid Chlortalidon Hydrochlorthiazide Chlorothiazide Ethacrynic acid Bumetanide Furosemide Sibutramine Carphedone Buprenorphine Clopamide Methamphetamine Amphetamine Ephedrine Oxymorphone Methylecgonine Benzoylecgnine Buproprion Fenproporex Caffeine Aminoglutethimide Cyclazodone Modafinil Pemoline Methadone Etilefrine Amiphenazole
Linearity
LOQ (ng mL
0.9929 0.9969 0.9945 0.9892 0.9990 0.9991 0.9990 0.9750 0.9991 NA NA 0.9997 0.9758 0.9957 0.9919 0.9995 0.9710 0.9834 0.9983 0.9993 0.9994 0.9992 0.9861 0.9995 0.9984 0.9925 0.9996 0.9898 0.9982 0.9992 0.9976
5.0 1.0 5.0 5.0 5.0 5.0 5.0 1.0 5.0 100.0 50.0 1.0 0.5 5.0 0.5 0.5 0.5 0.5 1.0 1.0 0.5 0.5 0.5 0.5 1.0 0.1 0.5 0.5 5.0 0.5 10.0
1
)
Linearity
LOQ (ng mL
0.9990 0.9759 0.9973 0.9965 0.9994 0.9986 0.9990 0.9900 0.9991 0.9993 0.9990 NA NA NA 0.9986 0.9857 0.9931 0.9851 0.9902 0.9517 0.9981 0.9984 0.9997 0.9982 0.9997 0.9998 0.9997 0.9968 0.9676 0.9798 0.9906
50.0 50.0 25.0 25.0 10.0 50.0 50.0 10.0 25.0 25.0 25.0 50.0 50.0 500.0 5.0 0.5 1.0 1.0 10.0 1.0 0.5 1.0 1.0 5.0 5.0 1.0 5.0 1.0 10.0 5.0 5.0
1
)
NA: data not available as less than five calibration points were obtained due to low sensitivity.
better MS response (higher than 2-fold) was only obtained for bumetanide in UHPLC–MS/MS (about 4). Finally, even if a quantification of these non-threshold substances (except for ephedrine) is not formally required for screening purposes, performance of both techniques was also studied in terms of linearity, for concentrations ranging between 0.05 ng mL 1 and 1 mg mL 1. At least five concentration levels were considered for the construction of calibration curves. The results shown in Table 2 demonstrate that both methods provided very good linearity with correlation coefficients higher than 0.990 for most compounds under both conditions. Lower correlation coefficients were obtained for 7 and 6 compounds under UHPSFC–MS/MS and UHPLC–MS/MS conditions, respectively. 4. Conclusion The goal of this study was to determine the best conditions for UHPLC–MS/MS and UHPSFC–MS/MS analysis of doping agents, taking into account the MS responses and peak shapes. For this purpose, 31 representative doping agents detected in ESI+ or ESI were evaluated. Various mobile phase conditions were tested in both chromatographic modes. In the case of UHPLC–MS/MS, 0.1% (25 mM) formic acid in water/ACN was found to be the most generic mobile phase additive and provided the best MS response in ESI+ and ESI and acceptable peak shapes. In UHPSFC–MS/MS, the best compromise between peak shapes and MS responses was achieved when adding 2% water and 10 mM
ammonium formate into the CO2/MeOH mobile phase. Under these conditions, a very homogeneous MS response was obtained in both ESI+ and ESI , while the peaks remained symmetrical whatever the physico-chemical properties of the drugs. Furthermore, the addition of water also contributed to obtain better retention times repeatability, which is an important feature for anti-doping screening methods. Except mobile phase, the make-up solvent added for interfacing UHPSFC with MS was also optimized. Ethanol appears as the best candidate since it provides a suitable MS response for all doping agents and is considered as a green solvent. Finally, sensitivities were compared for the set of 31 doping agents in UHPLC–MS/MS and UHPSFC–MS/MS. Sensitivity was improved by 5–100-fold in UHPSFC–MS/MS vs. UHPLC–MS/MS for 56% of the tested drugs. For 41% of the doping agents, the sensitivity was not significantly different (up to a factor 2) between the two analytical strategies, while a better MS response was only attained with bumetanide (about 4) in UHPLC–MS/MS. In the second paper of this series, these optimal mobile phase conditions for UHPLC–MS/MS and UHPSFC–MS/MS will be employed for the screening of more than 100 doping agents in urine matrix. Acknowledgement The authors gratefully acknowledge research projects of Charles University in Prague UNCE 204026/2012. References [1] World Anti-Doping Agency (WADA), The World Anti-Doping Code, The 2008 Prohibited List, Montreal, 2008, www.wada-ama.org. [2] World Anti-doping Agency (WADA), The World Anti-Doping Code, Minimal Required Performance Limits, Technical Document TD2004MRPL, Montreal, 2004, www.wada-ama.org. [3] F. Badoud, E. Grata, L. Perrenoud, L. Avois, M. Saugy, S. Rudaz, J.-L. Veuthey, J. Chromatogr. A 1216 (2009) 4423–4433. [4] M. Mazzarino, I. Fiacco, X. De la Torre, F. Botrè, J. Chromatogr. A 1218 (2011) 8156–8167. [5] A. Mussenga, D.A. Cowan, J. Chromatogr. A 1288 (2013) 82–95. [6] P. Van Eeno, W. Van Gansbeke, N. De Brabanter, K. Deventer, F.T. Delbeke, J. Chromatogr. A 1218 (2011) 3306–3316. [7] A. Jiménez Girón, K. Deventer, K. Roels, P. Van Eenoo, Anal. Chim. Acta 721 (2012) 137–146. [8] K. Deventer, O.J. Pozo, A.G. Verstraete, P. Van Eenoo, Trends Anal. Chem. 55 (2014) 1–13. [9] M. Thevis, A. Thomas, V. Pop, W. Schänzer, J. Chromatogr. A 1292 (2013) 38–50. [10] R.J.B. Peters, A.A.M. Stolker, J.G.J. Mol, A. Lommen, E. Lyris, Y. Angelis, A. Vonaparti, M. Stamou, C. Georgakopoulos, M.W.F. Nielen, Trends Anal. Chem. 29 (2010) 1250. [11] A. Grand-Guillaume Perrenoud, J.-L. Veuthey, D. Guillarme, J. Chromatogr. A 1266 (2012) 158–167. [12] A. Grand-Guillaume Perrenoud, M. Ch Hamman, Goel, J.-L. Veuthey, D. Guillarme, S. Fekete, J. Chromatogr. A 1314 (2013) 288–297. [13] M. Saito, J. Biosci. Bioeng. 115 (2013) 590–599. [14] L. Nováková, P. Chocholouš, P. Solich, Talanta 121 (2014) 178–186. [15] A. Grand-Guillaume Perrenoud, J. Boccard, J.-L. Veuthey, D. Guillarme, J. Chromatogr. A 1262 (2012) 205–213. [16] A. Dispas, P. Lebrun, E. Ziemons, R. Marini, E. Rozet, P. Hubert, J. Chromatogr. A 1353 (2014) 78–88. [17] F. Li, Y. Hsieh, J. Sep. Sci. 31 (2008) 1231–1237. [18] R. Chen, Chromatogr. Today, February/March, 2009, 11–15. [19] J.D. Pinkston, Eur. J. Mass Spectrom. 11 (2005) 189–197. [20] Y. Hsieh, L. Favreau, J. Schwerdt, K.-C. Cheng, J. Pharm. Biomed. Anal. 40 (2006) 799–804. [21] R.A. Coe, J.O. Rathe, J.W. Lee, J. Pharm. Biomed Anal. 42 (2006) 573–580. [22] Y. Hsieh, F. Li, Ch J. Duncan, Anal. Chem. 79 (2007) 3856–3861. [23] J. Schappler, R. Nicoli, D. Nguyen, S. Rudaz, J.-L. Veuthey, D. Guillarme, Talanta 78 (2009) 377–387. [24] M. Hol9 capek, K. Volná, P. Jandera, L. Kolárová, K. Lemr, M. Exner, A. Církva, J. Mass Spectrom. 39 (2004) 43. [25] M. Ashraf-Khorassani, L.T. Taylor, J. Sep. Sci. 33 (2010) 1682–1691. [26] A. Grand-Guillaume Perrenoud, J.L. Veuthey, D. Guillarme, J. Chromatogr. A 1339 (2014) 174–184.
