Anal Bioanal Chem (2015) 407:4473–4484 DOI 10.1007/s00216-015-8573-x
RESEARCH PAPER
Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry Mickael Doué & Gaud Dervilly-Pinel & Karinne Pouponneau & Fabrice Monteau & Bruno Le Bizec
Received: 3 November 2014 / Revised: 12 February 2015 / Accepted: 17 February 2015 / Published online: 4 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultrahigh-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS– MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC–MS–MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL −1 and 0.5 ng mL−1 for sulfate and glucuronide steroids, respectively)
Published in the topical collection on Hormone and Veterinary Drug Residue Analysis with guest editors Siska Croubels, Els Daeseleire, Sarah De Saeger, Peter Van Eenoo, and Lynn Vanhaecke. M. Doué : G. Dervilly-Pinel (*) : K. Pouponneau : F. Monteau : B. Le Bizec École Nationale Vétérinaire, Agroalimentaire et de l’alimentation Nantes-Atlantique, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), LUNAM Université, Oniris, Atlanpole-La Chantrerie, CS 50707, 44307 Nantes, France e-mail: [email protected]
and reliable quantitative performance (R2 >0.995, RSD < 20 %, and bias97.0 367.2>97.0 370.2>98.0 367.2>97.0 370.2>98.0 367.2>97.0 349.2>269.2 371.2>97.0 351.2>271.2 351.2>271.2 351.2>97.0
369.2>97.0
465.3>85.0 463.3>85.0 465.3>85.0 466.3>113.0 463.3>85.0 445.3>269.2 466.3>113.0 463.3>85.0 447.3>85.0 447.3>271.2
Transition quantification
48 45 55 45 55 45 50 55 47 47 55
48
50 50 50 45 50 35 45 50 50 40
Cone energy (V)
35 30 37 30 37 30 30 40 35 35 35
35
30 30 30 25 30 40 25 30 25 45
Collision energy (eV)
97.0>80.0 97.0>80.0 – 97.0>80.0 – 97.0>80.0 269.2>145.1 97.0>80.0 351.2>145.1 271.2>183.0 97.0>80.0
97.0>80.0
465.3>113.0 463.3>113.0 465.3>113.0 – 463.3>113.0 445.3>113.0 – 463.3>113.0 447.3>113.0 447.3>113.0
Transition identification
95 95 – 95 – 95 80 95 45 90 93
95
50 50 50 – 50 35 – 50 45 45
Cone energy (V)
18 18 – 18 – 18 35 18 55 40 15
18
30 30 30 – 30 20 – 30 23 23
Collision energy (eV)
EpiT 17S d3 EpiT 17S d3 – EpiT 17S d3 – T 17S d3 T 17S d3 EpiT 17S d3 T 17S d3 T 17S d3 T 17S d3
EpiT 17S d3
EpiT 17G d3 EpiT 17G d3 EpiT 17G d3 – EpiT 17G d3 T 17G d3 – T 17G d3 T 17G d3 T 17G d3
Internal standard
Table 1 Names, abbreviations, retention times, identification and quantification transitions, and corresponding internal standards for glucuronide and sulfate steroids analyzed in this study. Glucuronidesteroid separation on Acquity UPC2 BEH column, and sulfate separation on Acquity UPC2 2-EP column
Conjugated steroids analysis by UHPSFC-MS/MS 4475
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M. Doué et al.
Fig. 1 Conjugated steroids of the study; isobaric compounds are presented in the same square
CH3
O
CH3
CH3
OR
CH3
CH3
OR
Glucuronide M = 463.3 g mol-1
CH3
Sulfate M = 367.2 g mol-1 RO
O
O
EpiT 17S EpiT 17G
T 17S T 17G
DHEA 3S DHEA 3G CH3
O
CH3
CH3
O
CH3
RO
Sulfate M = 369.2 g mol-1
RO H
H
Epiandro 3S Epiandro 3G
Etio 3G CH3
O
Glucuronide M = 465.3 g mol-1
CH3
RO H
O
CH3
Andro 3S
Glucuronide M = 445.3 g mol-1
CH3
OR
CH3
Sulfate M = 349.2 g mol-1
Sulfate M = 349.2 g mol-1
RO
HO H
E1 3S E1 3G
5a-ab 17S CH3
OR
CH3
OH
Glucuronide M = 447.3 g mol-1 Sulfate M = 351.2 g mol-1
RO
COOH O OH OH
E2 3S / E2 17S E2 3G / E2 17G
two days (D2), and three days (D3) after treatment. All urine samples were stored at −20 °C until analysis. The animal experiment was conducted in agreement with the animalwelfare rules currently in force in Oniris, France. Sample preparation All urine samples were unfrozen at room temperature, homogenized, and centrifuged (4000 rpm, 5 °C for 20 min). The urinary specific gravity (SG) was measured by refractometry and recorded. Solution (50 μL) containing four internal standards (IS), i.e. EpiT 17G d3, T 17G d3, EpiT 17S d3, and T 17S d3, at 10 ng μL−1 in ethanol was evaporated to dryness under a gentle stream of nitrogen and 2 mL urine sample was added. A liquid–liquid extraction with ethyl acetate (1:1, v/v) was then performed. Glucuronide and sulfate steroids present in the aqueous phase were separated by the strong-anionexchanger SPE method (SAX cartridges) described by Anizan et al. [3]. The two collected fractions were evaporated, and reconstituted in 200 μL MeOH.
-E2 3S
HO
OR = G: CH3 Glucuronide
OH O
RO
O
S
CH3
OR = S: Sulfate
O
O
Ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry Target compounds were analyzed using an Acquity ultraperformance convergence chromatography (UPC2®) system from Waters (Waters, Milford, MA, USA). The UPC2 system was equipped with a binary solvent delivery pump, an autosampler, a column oven, and a back-pressure regulator (BPR). Four stationary phases were screened for their ability to separate phase II steroids, namely: 1. Acquity UPC2 BEH (3.0×100 mm, 1.7 μm); 2. Acquity UPC2 BEH 2-ethyl-pyridine (BEH 2-EP, 3.0× 100 mm, 1.7 μm); 3. Acquity UPC2 HSS C18 SB (3.0×100 mm, 1.8 μm); and 4. Acquity UPC 2 CSH Fluorophenyl (3.0 × 100 mm, 1.7 μm). Two different analytical conditions were used to separate sulfate and glucuronide steroids.
Conjugated steroids analysis by UHPSFC-MS/MS
Sulfate-steroid separation was achieved on an Acquity UPC2 BEH 2-EP. A mixture of MeOH–H2O–ammonium formate (95:5:30, v/v/mmol L−1) (B) was used as co-solvent with CO2 (A), in gradient mode (A:B): 95:5 during 0–1 min, 82:18 at 4 min, 80:20 at 6 min, 78:22 at 7 min, 77:23 at 8.5 min, 70:30 during 10.5–13 min, and 95:5 during 13.1– 15 min. The back pressure was set at 130 bar. The flow was 2 mL min−1, the column temperature was 50 °C, and the injection volume was 2 μL. Glucuronide steroids were separated on an Acquity UPC2 BEH. A mixture of MeOH–H 2 O–ammonium formate (95:5:30, v/v/mmol L−1) (B) was used as co-solvent with CO2 (A), in gradient mode (A:B): 95:5 during 0–1 min, 72:28 during 4–7 min, 70:30 during 7.5–9.5 min, 95:5 during 9.6–12 min. The back pressure was 130 bar. The flow was 2 mL min−1, the column temperature was 40 °C, and the injection volume was 2 μL. To avoid any carry-over, 600 μL MeOH was used as weak wash solvent and 200 μL MeOH–H2O (90:10) was used as strong wash solvent between each injection. The Acquity UPC2 system was hyphenated with tandem mass spectrometry (UHPSFC–MS–MS). The coupling with MS consisted of a double T with PEEK and PEEKsil tubing of fixed dimension, and was placed after the BPR. The double T enabled the addition of make-up solvent to the column effluent to bring the compounds into the ion source of the mass spectrometer and to enhance ionization. The make-up solvent was a mixture of H2O–MeOH (50:50, v/v) with 0.1 % formic acid and was delivered by the 515 HPLC pump (Waters) at a constant flow of 0.15 mL min−1. Data were acquired on a XEVO TQ-S mass spectrometer (Waters) operating in ESI− mode. The optimized settings were: capillary voltage: 2.3 kV; source offset: 40 V; desolvation temperature: 500 °C; source temperature: 150 °C; desolvation flow: 500 L h−1; cone flow: 100 L h−1; and nebulizer: 6 bar. Argon was used as collision gas at 0.15 mL min−1 for selected reaction monitoring (SRM); collision energy and cone voltage were previously optimized for each compound (two transitions: one for quantification and one for identification) [3] and are reported in Table 1.
Ultra-high-performance liquid chromatography hyphenated tandem mass spectrometry (UHPLC–MS–MS) UHPLC–MS–MS analysis was performed on an Acquity UPLC (Waters, Milford, MA, USA) coupled to a Xevo TQS mass spectrometer (Waters) operating in ESI− mode. The liquid-chromatography separation was developed and described elsewhere [3]. Regarding mass-spectrometry acquisition, the optimized conditions were the same as those used for UHPSFC–MS–MS analysis, enabling comparison of the methods.
4477
Data processing MassLynx (Waters) software was used for the data acquisition, data handling, and instrument control, whereas TargetLynx (Waters) was used for the integration and assignment of all chromatographic peaks acquired in SRM mode. Urinary specific gravity (SG) was used as a normalization strategy for quantification purposes. Indeed, using the specific density of the samples, inter-sample variability in the measured concentration caused by differences in density can be corrected. Correction was performed using the average value of 1.033 for the SG of the urine samples, according to the equation: Final concentration ¼
1:033−1 SG sample−1 Sample concentration
ð1Þ
The unambiguous identification of target compounds was on the basis of their retention time and their transition ratios, according to the criteria of 2002/657/EC Decision [24].
Results and discussion Method development The efficiency of UHPSFC for reliable separation of conjugated steroids, including steroid isomers (Fig. 1), was evaluated under different chromatographic conditions. On the basis of a recently published comprehensive tutorial [18], all important conditions of UHPSFC analysis, including stationary phase, modifier composition and percentage, dilution solvent, back pressure, column temperature, and composition and flow of make-up solvent, were investigated. Stationary phase, including modifier composition and percentage SFC is regarded as a unified separation method because it enables the use of both non-polar and polar stationary phases (SP) with the same mobile phase [25]. Thus four SP, namely Waters Acquity UPC2 BEH, BEH 2-EP, HSS C18 SB, and CSH fluorophenyl, with different selectivity properties and therefore suitable for a first screening step [26], were evaluated for their ability to provide good peak shape, resolution, and sensitivity for each conjugated steroid. For all four SP, methanol, ethanol, and acetonitrile were used as co-solvent (B) with CO2 (A), in a gradient from 5 to 30 % B in 20 min. First the development of a unique method for both glucuronide and sulfate forms was attempted. Preliminary results revealed that glucuronide and sulfate steroids were eluted
4478
M. Doué et al.
almost all together over the same, relatively long period of time (more than 4 min), regardless of the SP or co-solvent used (data not shown). A combination of the limited solubility of the conjugated steroids in the mobile phase and the presence of residual silanol groups in the SPs probably results in the broad peaks observed. When the percentages of ethanol and acetonitrile exceeded 25 % (flow of 2.5 mL min−1) it resulted in high pressures which were incompatible with the Acquity UPC2 system (maximum pressure 413 bar). Because no differences between the co-solvents could be observed at this stage, only methanol was subsequently used. The use of low concentrations of additives, usually acids or bases, in the methanol increases the solubility of analytes and provides coverage of the active sites, resulting in a beneficial effect on the peak shape [27]. Several additives compatible with MS detection, including ammonia, formic acid, ammonium acetate, ammonium formate, and water, were therefore tested. Results obtained with BEH 2-EP SP for the eight selected glucuronide compounds are presented in Fig. 2. Data were acquired in scan mode, and the extracted ion chromatograms of m/z455±11 enabled the selective detection of all the glucuronide compounds on the basis of their [M−H]− forms (Table 1). The addition of formic acid decreased the retention time of all target compounds but had no effect on the peak shape and the separation (Fig. 2c). In contrast all bases, namely ammonia (Fig. 2b), ammonium acetate (Fig. 2d), and ammonium formate (Fig. 2e), used with MeOH resulted in improved Fig. 2 Extracted ion chromatograms of m/z455±11 obtained for the separation of the eight selected glucuronide steroids, detected on the basis of their [M−H]− forms, with BEH 2EP SP. Gradient of CO2 (A) and MeOH (B) from 5 to 30 % B in 20 min with different additives: (a) no additive; (b) 0.2 % ammonia (NH4OH); (c) 0.1 % formic acid (FA); (d) 20 mmol L−1 ammonium acetate (CH3COONH4); (e) 20 mmol L−1 ammonium formate (HCOONH4); (f) 5 % H2O; (g) 20 mmol L−1 HCOONH4 +5 % H2O
2a. No additive 2b. 0.2% NH4OH 2c. 0.1% FA 2d. 20 mM CH3COONH4 2e. 20 mM HCOONH4 2f. 5% H2O 2g. 20 mM HCOONH4 + 5% H2O
resolution of the conjugated steroids whilst reducing analysis time. A small amount of water added to MeOH also seemed to increase the efficiency of the separation (Fig. 2f). The reason for this is that the use of water enhanced the solvating power of the mobile phase and possibly introduced HILIC-like analyte partition [18]. On the basis of these results, a combination of water with ammonium formate as additive in MeOH was tested and, as illustrated in Fig. 2g, proved to be the best option. With regard to the stationary phases, the two polar SPs, i.e. BEH 2-EP and BEH, resulted in better resolution and peak shape for both sulfate and glucuronide compounds. These two SPs were therefore used. Additional tests were performed to establish the percentage of each additive; the best results were obtained with 5 % water and 30 mmol L−1 COONH4 in MeOH. Dilution solvent, back pressure, and column temperature The choice of the solvent in which the extract is finally reconstituted is as important in UHPSFC as it is for UHPLC applications because it affects the efficiency of the separation [28]. The sample should be dissolved in a solvent close to (or weaker than) the mobile phase in terms of polarity. Supercritical CO2 is regarded as having a polarity close to that of hexane or heptane. However, glucuronide and sulfate compounds cannot be solvated in such apolar solvents. Thus, several injection solvents enabling the dissolution of conjugated steroids were tested: MeOH; MeOH–H2O (95:5); acetonitrile;
Conjugated steroids analysis by UHPSFC-MS/MS
4479
and isopropanol. Surprisingly, the effect of the dissolution solvent was barely perceptible (data not shown); no differences in terms of separation efficiency and peak width could be observed. A possible explanation may be the presence of water in the mobile phase which, combined with the use of a polar dilution solvent, would not induce any peak distortions. Thus MeOH was retained as the dilution solvent for the final extract, on the basis of its compatibility with the samplepreparation procedure (see BSample preparation^ section). The back pressure and the column temperature directly affect the elution power of supercritical fluid by changing the density of supercritical CO2 [29]. Nevertheless, because the composition of the mobile phase is the most important factor in controlling SFC separation when a high percentage of co-solvent (5–30 %) is used, the back-pressure and temperature optimizations were used only for fine tuning [18, 30]. In this work, an optimum back pressure of 130 bar was chosen (tested values: from 100 to 150 bar in steps of 10 bar). Regarding the column temperature, better resolution was obtained at 50 °C for the sulfate forms, whereas 40 °C was the best compromise for glucuronides (tested values: from 30 °C to 50 °C in steps of 5 °C). Composition and flow rate of make-up solvent To bring the compounds into the ion source, a make-up solvent was added after the back-pressure regulator. In this case, ionization efficiency depends mainly on the composition and flow rate of the make-up solvent relative to the mobile phase [31]. Thus, several tests were performed using a mixture of H2O–MeOH to evaluate the effect of the flow rate (from 0.1 to 0.3 mL min−1 in steps of 0.05), the relative percentages of each solvent (H2O–MeOH 25:75, 50:50, and 75:25), and the addition of acid (0.1 % formic acid) or base (30 mmol L−1 Fig. 3 Chromatograms (TIC) of sulfate and glucuronide-standards separation, obtained on BEH 2EP and BEH columns, respectively. In each chromatogram, isobaric compounds are assigned with the same color. Sulfate separation: 1: Andro 3S; 2: Epiandro 3S; 3: DHEA 3S; 4: EpiT 17S; 5: T 17S; 6: Estrone 3S; 7: 5a-ab 17S; 8: a-E23S; 9: E23S; 10: E217S. Glucuronide separation: 1: Etio 3G; 2: DHEA 3G; 3: Epiandro 3G; 4: EpiT 17G; 5: E1 3G; 6: T 17G; 7: E217G; 8: E23G
ammonium formate). Both flow rate and percentages of each solvent had minor effects on the sensitivity and repeatability (n=5) of the detection (data not shown). Best results were obtained for all compounds with a mixture of H2O–MeOH (50:50, v/v) with 0.1 % formic acid at a constant flow of 0.15 mL min−1. Indeed, the addition of formic acid enhances the ionization efficiency (sensitivity three times higher), as reported in another study [3]. Final conditions Separation of both glucuronide and sulfate steroids was successfully achieved on BEH and BEH 2-EP columns, but with relatively long run time (approximately 40 min; data not shown). Interestingly, the polar BEH phase mainly promoted interactions with the conjugated moiety and therefore provided class separation of conjugates (sulfated first, followed by glucuronated), whereas BEH 2-EP SP mainly interacted with the steroid base regardless of the conjugated form. However, the objective of this study being to provide high-throughput analysis of both types of compound, it seemed more relevant to consider them separately. Additionally, the samplepreparation procedure applied to the urine samples is based on a strong-anion-exchanger SPE method which enables the efficient separation of glucuronated and sulfated steroids and subsequently decreases potential matrix effect. The flow rate of the mobile phase and gradient elution were thus finally optimized on BEH 2-EP and BEH columns for sulfate and glucuronide compounds, respectively. The obtained chromatograms are presented in Fig. 3. Within less than 8 min, sulfate and glucuronide steroids were both efficiently resolved. Steroid isomers for example T 17G, EpiT 17G, and DHEA 3G or T 17S, EpiT 17S, and DHEA 3S were efficiently separated with resolution factor R> 8
6 3 1
Sulfate separation on BEH 2-EP
9
2 4 5 10
7
5 and 6
8
1 23 4
7
Glucuronide separation on BEH
4480 Table 2 Method validation: repeatability (n=6; RSD,%), bias (%), linearity (correlation coefficient R2), and limit of quantification (LOQ) for the different urinary conjugated steroids
M. Doué et al.
Abbreviation
Spiked level (ng mL−1)
Glucuronide steroids Etio 3G 0.5 5 50 DHEA 3G 0.5 5 50 Epiandro 3G 0.5 5 50 EpiT 17G 0.5 5 50 E1 3G 0.5 5 50 T 17G 0.5 5 50 E2 17G 0.5 5 50 E2 3G 0.5 5 50 Sulfate steroids Andro 3S 0.1 5 50 Epiandro 3S 0.1 5 50 DHEA 3S 0.1 5 50 EpiT 17S 0.1 5 50 T 17S 0.1 5 50 E1 3S 0.1 5 50 5a-ab 17S 0.1 5 50 α-E2 3S 0.1 5 50 E2 3S 0.1 5 50 E2 17S 0.1 5 50
Repeatability (RSD,%)
Bias (%)
13.9 4.8 4.2 25.6 15.4 5.8 10.4 9.8 5.1 11.7 9.3 3.2 1.8 6.4 2.2 26.8 5.8 9.3 6.4 2.9 8.3 2.2 11.7 8.9
19.5 17.4 2.3 29.7 18.0 3.6 27.6 13.6 3.1 14.4 11.9 1.6 22.6 19.6 3.8 28.0 12.9 2.3 19.4 8.1 4.7 22.5 17.2 4.9
20.2 8.4 8.6 6.7 9.6 3.0 16.4 8.3 13.4 14.2 1.7 3.4 22.9 0.7 3.3 6.4 7.1 19.9 10.4 11.2 12.9 8.9 6.4 15.9 0.6 12.4 16.8 4.0 6.1 12.9
27.7 7.2 1.1 −20.6 5.0 0.8 11.4 −2.0 −0.6 7.8 −8.3 −1.0 25.3 −2.8 −0.3 20.3 −3.2 0.2 26.0 4.6 1.2 20.6 −7.8 2.6 −24.7 1.3 1.0 22.3 −4.7 0.5
Linearity (correlation coefficient R2)
LOQ (ng mL−1)
0.997
0.5
0.9988
0.5
0.9972
0.5
0.9998
0.5
0.9957
0.5
0.9992
0.5
0.9997
0.5
0.9996
0.5
0.9998
0.1
0.9999
0.1
0.9999
0.1
0.9999
0.1
0.9995
0.1
0.9998
0.1
0.9997
0.1
0.9996
0.1
0.9999
0.1
0.9999
0.1
Conjugated steroids analysis by UHPSFC-MS/MS
4481
1.25. Few co-elutions were observed and they involved compounds of different molecular weight, for example E13G and T 17G, which is not a problem when mass spectrometry is used as the detection method, especially in SRM mode (Table 1), as was the case in this study. Method performance The performance of the method was evaluated using urine samples collected from a young calf (see BStandards and urine samples^ section). Mass-spectrometry acquisition was performed in SRM mode using the transitions indicated in Table 1. Results in terms of repeatability, bias, linearity, and limit of quantification are indicated in Table 2. Fig. 4 Comparison between UHPSFC–MS–MS and UHPLC– MS–MS analyses for selected compounds, namely Etio 3G, Epiandro 3G, T 17S, EpiT 17S, and DHEA 3S, spiked at 1 ng mL−1
Repeatability (n = 6) was determined at 0.5, 5, and 50 ng mL−1 and at 0.1, 5, and 50 ng mL−1 for all glucuronide and sulfate compounds, respectively. Relative standard deviations (RSD) were below 20 % for all compounds except DHEA 3G, T 17G, and T 17S, which had RSD values of 25.6 %, 26.8 %, and 22.9 %, respectively, at the lowest levels. All conjugated steroids had a bias rate lower than 20 % at 5 and 50 ng mL−1. Bias rates were more important at the lowest levels, but remained below 30 % and were therefore still regarded as satisfactory. Calibration curves (three replicates) were constructed using the spike levels 0.5, 2.5, 5.0, 25, and 50 ng mL−1 for glucuronide steroids and 0.1, 2.5, 5.0, 25, and 50 ng mL−1 for sulfate steroids, analyzed using the developed analytical method.
Etio 3G
Epiandro 3G
UHPSFC-MS/MS
UHPLC-MS/MS
Epiandro 3G Etio 3G
UHPSFC-MS/MS
DHEA 3S EpiT 17S T 17S
DHEA 3S
UHPLC-MS/MS EpiT 17S T 17S
4.74 1.25 12.20 1.54 2.38 2.01
RESEARCH PAPER
Analysis of glucuronide and sulfate steroids in urine by ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry Mickael Doué & Gaud Dervilly-Pinel & Karinne Pouponneau & Fabrice Monteau & Bruno Le Bizec
Received: 3 November 2014 / Revised: 12 February 2015 / Accepted: 17 February 2015 / Published online: 4 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Profiling conjugated urinary steroids to detect anabolic-steroid misuse is recognized as an efficient analytical strategy in both chemical-food-safety and anti-doping fields. The relevance and robustness of such profiling rely on the analysis of glucuronide and sulfate steroids, which is expected to have properties including accuracy, specificity, sensitivity, and, if possible, rapidity. In this context, the ability of ultrahigh-performance supercritical-fluid chromatography (UHPSFC) hyphenated tandem mass spectrometry (MS– MS) to provide reliable and accurate phase II analysis of steroids was assessed. Four stationary phases with sub-2 μm particles (BEH, BEH 2-ethyl-pyridine, HSS C18 SB, and CSH fluorophenyl) were screened for their capacity to separate several conjugated steroid isomers. Analytical conditions including stationary phase, modifier composition and percentage, back pressure, column temperature, and composition and flow rate of make-up solvent were investigated to improve the separation and/or the sensitivity. Thus, an analytical procedure enabling the analysis of eight glucuronide and 12 sulfate steroids by two different methods in 12 and 15 min, respectively, was optimized. The two procedures were evaluated, and UHPSFC–MS–MS analysis revealed its ability to provide sensitive (limits of quantification: 0.1 ng mL −1 and 0.5 ng mL−1 for sulfate and glucuronide steroids, respectively)
Published in the topical collection on Hormone and Veterinary Drug Residue Analysis with guest editors Siska Croubels, Els Daeseleire, Sarah De Saeger, Peter Van Eenoo, and Lynn Vanhaecke. M. Doué : G. Dervilly-Pinel (*) : K. Pouponneau : F. Monteau : B. Le Bizec École Nationale Vétérinaire, Agroalimentaire et de l’alimentation Nantes-Atlantique, Laboratoire d’Etude des Résidus et Contaminants dans les Aliments (LABERCA), LUNAM Université, Oniris, Atlanpole-La Chantrerie, CS 50707, 44307 Nantes, France e-mail: [email protected]
and reliable quantitative performance (R2 >0.995, RSD < 20 %, and bias97.0 367.2>97.0 370.2>98.0 367.2>97.0 370.2>98.0 367.2>97.0 349.2>269.2 371.2>97.0 351.2>271.2 351.2>271.2 351.2>97.0
369.2>97.0
465.3>85.0 463.3>85.0 465.3>85.0 466.3>113.0 463.3>85.0 445.3>269.2 466.3>113.0 463.3>85.0 447.3>85.0 447.3>271.2
Transition quantification
48 45 55 45 55 45 50 55 47 47 55
48
50 50 50 45 50 35 45 50 50 40
Cone energy (V)
35 30 37 30 37 30 30 40 35 35 35
35
30 30 30 25 30 40 25 30 25 45
Collision energy (eV)
97.0>80.0 97.0>80.0 – 97.0>80.0 – 97.0>80.0 269.2>145.1 97.0>80.0 351.2>145.1 271.2>183.0 97.0>80.0
97.0>80.0
465.3>113.0 463.3>113.0 465.3>113.0 – 463.3>113.0 445.3>113.0 – 463.3>113.0 447.3>113.0 447.3>113.0
Transition identification
95 95 – 95 – 95 80 95 45 90 93
95
50 50 50 – 50 35 – 50 45 45
Cone energy (V)
18 18 – 18 – 18 35 18 55 40 15
18
30 30 30 – 30 20 – 30 23 23
Collision energy (eV)
EpiT 17S d3 EpiT 17S d3 – EpiT 17S d3 – T 17S d3 T 17S d3 EpiT 17S d3 T 17S d3 T 17S d3 T 17S d3
EpiT 17S d3
EpiT 17G d3 EpiT 17G d3 EpiT 17G d3 – EpiT 17G d3 T 17G d3 – T 17G d3 T 17G d3 T 17G d3
Internal standard
Table 1 Names, abbreviations, retention times, identification and quantification transitions, and corresponding internal standards for glucuronide and sulfate steroids analyzed in this study. Glucuronidesteroid separation on Acquity UPC2 BEH column, and sulfate separation on Acquity UPC2 2-EP column
Conjugated steroids analysis by UHPSFC-MS/MS 4475
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M. Doué et al.
Fig. 1 Conjugated steroids of the study; isobaric compounds are presented in the same square
CH3
O
CH3
CH3
OR
CH3
CH3
OR
Glucuronide M = 463.3 g mol-1
CH3
Sulfate M = 367.2 g mol-1 RO
O
O
EpiT 17S EpiT 17G
T 17S T 17G
DHEA 3S DHEA 3G CH3
O
CH3
CH3
O
CH3
RO
Sulfate M = 369.2 g mol-1
RO H
H
Epiandro 3S Epiandro 3G
Etio 3G CH3
O
Glucuronide M = 465.3 g mol-1
CH3
RO H
O
CH3
Andro 3S
Glucuronide M = 445.3 g mol-1
CH3
OR
CH3
Sulfate M = 349.2 g mol-1
Sulfate M = 349.2 g mol-1
RO
HO H
E1 3S E1 3G
5a-ab 17S CH3
OR
CH3
OH
Glucuronide M = 447.3 g mol-1 Sulfate M = 351.2 g mol-1
RO
COOH O OH OH
E2 3S / E2 17S E2 3G / E2 17G
two days (D2), and three days (D3) after treatment. All urine samples were stored at −20 °C until analysis. The animal experiment was conducted in agreement with the animalwelfare rules currently in force in Oniris, France. Sample preparation All urine samples were unfrozen at room temperature, homogenized, and centrifuged (4000 rpm, 5 °C for 20 min). The urinary specific gravity (SG) was measured by refractometry and recorded. Solution (50 μL) containing four internal standards (IS), i.e. EpiT 17G d3, T 17G d3, EpiT 17S d3, and T 17S d3, at 10 ng μL−1 in ethanol was evaporated to dryness under a gentle stream of nitrogen and 2 mL urine sample was added. A liquid–liquid extraction with ethyl acetate (1:1, v/v) was then performed. Glucuronide and sulfate steroids present in the aqueous phase were separated by the strong-anionexchanger SPE method (SAX cartridges) described by Anizan et al. [3]. The two collected fractions were evaporated, and reconstituted in 200 μL MeOH.
-E2 3S
HO
OR = G: CH3 Glucuronide
OH O
RO
O
S
CH3
OR = S: Sulfate
O
O
Ultra-high-performance supercritical-fluid chromatography hyphenated tandem mass spectrometry Target compounds were analyzed using an Acquity ultraperformance convergence chromatography (UPC2®) system from Waters (Waters, Milford, MA, USA). The UPC2 system was equipped with a binary solvent delivery pump, an autosampler, a column oven, and a back-pressure regulator (BPR). Four stationary phases were screened for their ability to separate phase II steroids, namely: 1. Acquity UPC2 BEH (3.0×100 mm, 1.7 μm); 2. Acquity UPC2 BEH 2-ethyl-pyridine (BEH 2-EP, 3.0× 100 mm, 1.7 μm); 3. Acquity UPC2 HSS C18 SB (3.0×100 mm, 1.8 μm); and 4. Acquity UPC 2 CSH Fluorophenyl (3.0 × 100 mm, 1.7 μm). Two different analytical conditions were used to separate sulfate and glucuronide steroids.
Conjugated steroids analysis by UHPSFC-MS/MS
Sulfate-steroid separation was achieved on an Acquity UPC2 BEH 2-EP. A mixture of MeOH–H2O–ammonium formate (95:5:30, v/v/mmol L−1) (B) was used as co-solvent with CO2 (A), in gradient mode (A:B): 95:5 during 0–1 min, 82:18 at 4 min, 80:20 at 6 min, 78:22 at 7 min, 77:23 at 8.5 min, 70:30 during 10.5–13 min, and 95:5 during 13.1– 15 min. The back pressure was set at 130 bar. The flow was 2 mL min−1, the column temperature was 50 °C, and the injection volume was 2 μL. Glucuronide steroids were separated on an Acquity UPC2 BEH. A mixture of MeOH–H 2 O–ammonium formate (95:5:30, v/v/mmol L−1) (B) was used as co-solvent with CO2 (A), in gradient mode (A:B): 95:5 during 0–1 min, 72:28 during 4–7 min, 70:30 during 7.5–9.5 min, 95:5 during 9.6–12 min. The back pressure was 130 bar. The flow was 2 mL min−1, the column temperature was 40 °C, and the injection volume was 2 μL. To avoid any carry-over, 600 μL MeOH was used as weak wash solvent and 200 μL MeOH–H2O (90:10) was used as strong wash solvent between each injection. The Acquity UPC2 system was hyphenated with tandem mass spectrometry (UHPSFC–MS–MS). The coupling with MS consisted of a double T with PEEK and PEEKsil tubing of fixed dimension, and was placed after the BPR. The double T enabled the addition of make-up solvent to the column effluent to bring the compounds into the ion source of the mass spectrometer and to enhance ionization. The make-up solvent was a mixture of H2O–MeOH (50:50, v/v) with 0.1 % formic acid and was delivered by the 515 HPLC pump (Waters) at a constant flow of 0.15 mL min−1. Data were acquired on a XEVO TQ-S mass spectrometer (Waters) operating in ESI− mode. The optimized settings were: capillary voltage: 2.3 kV; source offset: 40 V; desolvation temperature: 500 °C; source temperature: 150 °C; desolvation flow: 500 L h−1; cone flow: 100 L h−1; and nebulizer: 6 bar. Argon was used as collision gas at 0.15 mL min−1 for selected reaction monitoring (SRM); collision energy and cone voltage were previously optimized for each compound (two transitions: one for quantification and one for identification) [3] and are reported in Table 1.
Ultra-high-performance liquid chromatography hyphenated tandem mass spectrometry (UHPLC–MS–MS) UHPLC–MS–MS analysis was performed on an Acquity UPLC (Waters, Milford, MA, USA) coupled to a Xevo TQS mass spectrometer (Waters) operating in ESI− mode. The liquid-chromatography separation was developed and described elsewhere [3]. Regarding mass-spectrometry acquisition, the optimized conditions were the same as those used for UHPSFC–MS–MS analysis, enabling comparison of the methods.
4477
Data processing MassLynx (Waters) software was used for the data acquisition, data handling, and instrument control, whereas TargetLynx (Waters) was used for the integration and assignment of all chromatographic peaks acquired in SRM mode. Urinary specific gravity (SG) was used as a normalization strategy for quantification purposes. Indeed, using the specific density of the samples, inter-sample variability in the measured concentration caused by differences in density can be corrected. Correction was performed using the average value of 1.033 for the SG of the urine samples, according to the equation: Final concentration ¼
1:033−1 SG sample−1 Sample concentration
ð1Þ
The unambiguous identification of target compounds was on the basis of their retention time and their transition ratios, according to the criteria of 2002/657/EC Decision [24].
Results and discussion Method development The efficiency of UHPSFC for reliable separation of conjugated steroids, including steroid isomers (Fig. 1), was evaluated under different chromatographic conditions. On the basis of a recently published comprehensive tutorial [18], all important conditions of UHPSFC analysis, including stationary phase, modifier composition and percentage, dilution solvent, back pressure, column temperature, and composition and flow of make-up solvent, were investigated. Stationary phase, including modifier composition and percentage SFC is regarded as a unified separation method because it enables the use of both non-polar and polar stationary phases (SP) with the same mobile phase [25]. Thus four SP, namely Waters Acquity UPC2 BEH, BEH 2-EP, HSS C18 SB, and CSH fluorophenyl, with different selectivity properties and therefore suitable for a first screening step [26], were evaluated for their ability to provide good peak shape, resolution, and sensitivity for each conjugated steroid. For all four SP, methanol, ethanol, and acetonitrile were used as co-solvent (B) with CO2 (A), in a gradient from 5 to 30 % B in 20 min. First the development of a unique method for both glucuronide and sulfate forms was attempted. Preliminary results revealed that glucuronide and sulfate steroids were eluted
4478
M. Doué et al.
almost all together over the same, relatively long period of time (more than 4 min), regardless of the SP or co-solvent used (data not shown). A combination of the limited solubility of the conjugated steroids in the mobile phase and the presence of residual silanol groups in the SPs probably results in the broad peaks observed. When the percentages of ethanol and acetonitrile exceeded 25 % (flow of 2.5 mL min−1) it resulted in high pressures which were incompatible with the Acquity UPC2 system (maximum pressure 413 bar). Because no differences between the co-solvents could be observed at this stage, only methanol was subsequently used. The use of low concentrations of additives, usually acids or bases, in the methanol increases the solubility of analytes and provides coverage of the active sites, resulting in a beneficial effect on the peak shape [27]. Several additives compatible with MS detection, including ammonia, formic acid, ammonium acetate, ammonium formate, and water, were therefore tested. Results obtained with BEH 2-EP SP for the eight selected glucuronide compounds are presented in Fig. 2. Data were acquired in scan mode, and the extracted ion chromatograms of m/z455±11 enabled the selective detection of all the glucuronide compounds on the basis of their [M−H]− forms (Table 1). The addition of formic acid decreased the retention time of all target compounds but had no effect on the peak shape and the separation (Fig. 2c). In contrast all bases, namely ammonia (Fig. 2b), ammonium acetate (Fig. 2d), and ammonium formate (Fig. 2e), used with MeOH resulted in improved Fig. 2 Extracted ion chromatograms of m/z455±11 obtained for the separation of the eight selected glucuronide steroids, detected on the basis of their [M−H]− forms, with BEH 2EP SP. Gradient of CO2 (A) and MeOH (B) from 5 to 30 % B in 20 min with different additives: (a) no additive; (b) 0.2 % ammonia (NH4OH); (c) 0.1 % formic acid (FA); (d) 20 mmol L−1 ammonium acetate (CH3COONH4); (e) 20 mmol L−1 ammonium formate (HCOONH4); (f) 5 % H2O; (g) 20 mmol L−1 HCOONH4 +5 % H2O
2a. No additive 2b. 0.2% NH4OH 2c. 0.1% FA 2d. 20 mM CH3COONH4 2e. 20 mM HCOONH4 2f. 5% H2O 2g. 20 mM HCOONH4 + 5% H2O
resolution of the conjugated steroids whilst reducing analysis time. A small amount of water added to MeOH also seemed to increase the efficiency of the separation (Fig. 2f). The reason for this is that the use of water enhanced the solvating power of the mobile phase and possibly introduced HILIC-like analyte partition [18]. On the basis of these results, a combination of water with ammonium formate as additive in MeOH was tested and, as illustrated in Fig. 2g, proved to be the best option. With regard to the stationary phases, the two polar SPs, i.e. BEH 2-EP and BEH, resulted in better resolution and peak shape for both sulfate and glucuronide compounds. These two SPs were therefore used. Additional tests were performed to establish the percentage of each additive; the best results were obtained with 5 % water and 30 mmol L−1 COONH4 in MeOH. Dilution solvent, back pressure, and column temperature The choice of the solvent in which the extract is finally reconstituted is as important in UHPSFC as it is for UHPLC applications because it affects the efficiency of the separation [28]. The sample should be dissolved in a solvent close to (or weaker than) the mobile phase in terms of polarity. Supercritical CO2 is regarded as having a polarity close to that of hexane or heptane. However, glucuronide and sulfate compounds cannot be solvated in such apolar solvents. Thus, several injection solvents enabling the dissolution of conjugated steroids were tested: MeOH; MeOH–H2O (95:5); acetonitrile;
Conjugated steroids analysis by UHPSFC-MS/MS
4479
and isopropanol. Surprisingly, the effect of the dissolution solvent was barely perceptible (data not shown); no differences in terms of separation efficiency and peak width could be observed. A possible explanation may be the presence of water in the mobile phase which, combined with the use of a polar dilution solvent, would not induce any peak distortions. Thus MeOH was retained as the dilution solvent for the final extract, on the basis of its compatibility with the samplepreparation procedure (see BSample preparation^ section). The back pressure and the column temperature directly affect the elution power of supercritical fluid by changing the density of supercritical CO2 [29]. Nevertheless, because the composition of the mobile phase is the most important factor in controlling SFC separation when a high percentage of co-solvent (5–30 %) is used, the back-pressure and temperature optimizations were used only for fine tuning [18, 30]. In this work, an optimum back pressure of 130 bar was chosen (tested values: from 100 to 150 bar in steps of 10 bar). Regarding the column temperature, better resolution was obtained at 50 °C for the sulfate forms, whereas 40 °C was the best compromise for glucuronides (tested values: from 30 °C to 50 °C in steps of 5 °C). Composition and flow rate of make-up solvent To bring the compounds into the ion source, a make-up solvent was added after the back-pressure regulator. In this case, ionization efficiency depends mainly on the composition and flow rate of the make-up solvent relative to the mobile phase [31]. Thus, several tests were performed using a mixture of H2O–MeOH to evaluate the effect of the flow rate (from 0.1 to 0.3 mL min−1 in steps of 0.05), the relative percentages of each solvent (H2O–MeOH 25:75, 50:50, and 75:25), and the addition of acid (0.1 % formic acid) or base (30 mmol L−1 Fig. 3 Chromatograms (TIC) of sulfate and glucuronide-standards separation, obtained on BEH 2EP and BEH columns, respectively. In each chromatogram, isobaric compounds are assigned with the same color. Sulfate separation: 1: Andro 3S; 2: Epiandro 3S; 3: DHEA 3S; 4: EpiT 17S; 5: T 17S; 6: Estrone 3S; 7: 5a-ab 17S; 8: a-E23S; 9: E23S; 10: E217S. Glucuronide separation: 1: Etio 3G; 2: DHEA 3G; 3: Epiandro 3G; 4: EpiT 17G; 5: E1 3G; 6: T 17G; 7: E217G; 8: E23G
ammonium formate). Both flow rate and percentages of each solvent had minor effects on the sensitivity and repeatability (n=5) of the detection (data not shown). Best results were obtained for all compounds with a mixture of H2O–MeOH (50:50, v/v) with 0.1 % formic acid at a constant flow of 0.15 mL min−1. Indeed, the addition of formic acid enhances the ionization efficiency (sensitivity three times higher), as reported in another study [3]. Final conditions Separation of both glucuronide and sulfate steroids was successfully achieved on BEH and BEH 2-EP columns, but with relatively long run time (approximately 40 min; data not shown). Interestingly, the polar BEH phase mainly promoted interactions with the conjugated moiety and therefore provided class separation of conjugates (sulfated first, followed by glucuronated), whereas BEH 2-EP SP mainly interacted with the steroid base regardless of the conjugated form. However, the objective of this study being to provide high-throughput analysis of both types of compound, it seemed more relevant to consider them separately. Additionally, the samplepreparation procedure applied to the urine samples is based on a strong-anion-exchanger SPE method which enables the efficient separation of glucuronated and sulfated steroids and subsequently decreases potential matrix effect. The flow rate of the mobile phase and gradient elution were thus finally optimized on BEH 2-EP and BEH columns for sulfate and glucuronide compounds, respectively. The obtained chromatograms are presented in Fig. 3. Within less than 8 min, sulfate and glucuronide steroids were both efficiently resolved. Steroid isomers for example T 17G, EpiT 17G, and DHEA 3G or T 17S, EpiT 17S, and DHEA 3S were efficiently separated with resolution factor R> 8
6 3 1
Sulfate separation on BEH 2-EP
9
2 4 5 10
7
5 and 6
8
1 23 4
7
Glucuronide separation on BEH
4480 Table 2 Method validation: repeatability (n=6; RSD,%), bias (%), linearity (correlation coefficient R2), and limit of quantification (LOQ) for the different urinary conjugated steroids
M. Doué et al.
Abbreviation
Spiked level (ng mL−1)
Glucuronide steroids Etio 3G 0.5 5 50 DHEA 3G 0.5 5 50 Epiandro 3G 0.5 5 50 EpiT 17G 0.5 5 50 E1 3G 0.5 5 50 T 17G 0.5 5 50 E2 17G 0.5 5 50 E2 3G 0.5 5 50 Sulfate steroids Andro 3S 0.1 5 50 Epiandro 3S 0.1 5 50 DHEA 3S 0.1 5 50 EpiT 17S 0.1 5 50 T 17S 0.1 5 50 E1 3S 0.1 5 50 5a-ab 17S 0.1 5 50 α-E2 3S 0.1 5 50 E2 3S 0.1 5 50 E2 17S 0.1 5 50
Repeatability (RSD,%)
Bias (%)
13.9 4.8 4.2 25.6 15.4 5.8 10.4 9.8 5.1 11.7 9.3 3.2 1.8 6.4 2.2 26.8 5.8 9.3 6.4 2.9 8.3 2.2 11.7 8.9
19.5 17.4 2.3 29.7 18.0 3.6 27.6 13.6 3.1 14.4 11.9 1.6 22.6 19.6 3.8 28.0 12.9 2.3 19.4 8.1 4.7 22.5 17.2 4.9
20.2 8.4 8.6 6.7 9.6 3.0 16.4 8.3 13.4 14.2 1.7 3.4 22.9 0.7 3.3 6.4 7.1 19.9 10.4 11.2 12.9 8.9 6.4 15.9 0.6 12.4 16.8 4.0 6.1 12.9
27.7 7.2 1.1 −20.6 5.0 0.8 11.4 −2.0 −0.6 7.8 −8.3 −1.0 25.3 −2.8 −0.3 20.3 −3.2 0.2 26.0 4.6 1.2 20.6 −7.8 2.6 −24.7 1.3 1.0 22.3 −4.7 0.5
Linearity (correlation coefficient R2)
LOQ (ng mL−1)
0.997
0.5
0.9988
0.5
0.9972
0.5
0.9998
0.5
0.9957
0.5
0.9992
0.5
0.9997
0.5
0.9996
0.5
0.9998
0.1
0.9999
0.1
0.9999
0.1
0.9999
0.1
0.9995
0.1
0.9998
0.1
0.9997
0.1
0.9996
0.1
0.9999
0.1
0.9999
0.1
Conjugated steroids analysis by UHPSFC-MS/MS
4481
1.25. Few co-elutions were observed and they involved compounds of different molecular weight, for example E13G and T 17G, which is not a problem when mass spectrometry is used as the detection method, especially in SRM mode (Table 1), as was the case in this study. Method performance The performance of the method was evaluated using urine samples collected from a young calf (see BStandards and urine samples^ section). Mass-spectrometry acquisition was performed in SRM mode using the transitions indicated in Table 1. Results in terms of repeatability, bias, linearity, and limit of quantification are indicated in Table 2. Fig. 4 Comparison between UHPSFC–MS–MS and UHPLC– MS–MS analyses for selected compounds, namely Etio 3G, Epiandro 3G, T 17S, EpiT 17S, and DHEA 3S, spiked at 1 ng mL−1
Repeatability (n = 6) was determined at 0.5, 5, and 50 ng mL−1 and at 0.1, 5, and 50 ng mL−1 for all glucuronide and sulfate compounds, respectively. Relative standard deviations (RSD) were below 20 % for all compounds except DHEA 3G, T 17G, and T 17S, which had RSD values of 25.6 %, 26.8 %, and 22.9 %, respectively, at the lowest levels. All conjugated steroids had a bias rate lower than 20 % at 5 and 50 ng mL−1. Bias rates were more important at the lowest levels, but remained below 30 % and were therefore still regarded as satisfactory. Calibration curves (three replicates) were constructed using the spike levels 0.5, 2.5, 5.0, 25, and 50 ng mL−1 for glucuronide steroids and 0.1, 2.5, 5.0, 25, and 50 ng mL−1 for sulfate steroids, analyzed using the developed analytical method.
Etio 3G
Epiandro 3G
UHPSFC-MS/MS
UHPLC-MS/MS
Epiandro 3G Etio 3G
UHPSFC-MS/MS
DHEA 3S EpiT 17S T 17S
DHEA 3S
UHPLC-MS/MS EpiT 17S T 17S
4.74 1.25 12.20 1.54 2.38 2.01
