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Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry a
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Sara Odoardi , Erika Castrignanò , Simona Martello , Marcello Chiarotti & Sabina Stranoa
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Institute of Public Health, Forensic Toxicology Laboratory, Università Cattolica del S. Cuore, Rome, Italy Accepted author version posted online: 26 Feb 2015.Published online: 04 Mar 2015.
Click for updates To cite this article: Sara Odoardi, Erika Castrignanò, Simona Martello, Marcello Chiarotti & Sabina Strano-Rossi (2015): Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1014868 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1014868
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1014868
Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry Sara Odoardi, Erika Castrignanò, Simona Martello, Marcello Chiarotti and Sabina Strano-Rossi* Institute of Public Health, Forensic Toxicology Laboratory, Università Cattolica del S. Cuore, Rome, Italy
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(Received 19 November 2014; accepted 30 January 2015) A sensitive method for the identification and quantification of anabolic steroids and clenbuterol at trace levels in dietary supplements by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in atmospheric pressure ionisation (APCI) mode using a single-stage Orbitrap analyser operating at a resolution power of 100 000 full width at half maximum (FWHM) was developed and validated. A total of 1 g of dietary supplement was added with testosterone-d3 as internal standard, dissolved in methanol, evaporated to dryness, diluted in sodium hydroxide solution and extracted with a mixture of pentane/ethyl ether 9:1. The extract was directly injected into the LC-HRMS system. The method was fully validated. Limits of detection (LODs) obtained for anabolic androgenic steroids (AASs) varied from 1 to 25 ng g−1 and the limit of quantitation (LOQ) was 50 ng g−1 for all analytes. The calibration was linear for all compounds in the range from the LOQ to 2000 ng g−1, with correlation coefficients always higher than 0.99. Accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Good values of matrix effect and recovery were achieved. The ease of the sample preparation together with a fast run time of only 16 min permitted rapid identification of the analytes. The method was applied to the analysis of 30 dietary supplements in order to check for the presence of anabolic agents not labelled as being present in these supplements. Many AASs were often detected in the same sample: indeed, androstenedione was detected in nine supplements, 5-androsten-3β-ol-17-one (DHEA) in 12, methandienone in three, stanozolol in one, testosterone in seven and testosterone esters in four of them. A retrospective analysis of suspected compounds not included at the beginning of the method development was also possible by means of the full acquisition spectra obtained with the HRMS technique. Keywords: dietary supplements analysis; anabolic agents; drug testing; LC-HRMS
Introduction Dietary supplements are products intended to supplement nutrients, theoretically present in a normal and balanced diet, such as vitamins, minerals, amino acids and herbs extracts. They are generally in the form of tablets, capsules, powders, energy bars or as oily pearls for oral consumption. All sectors of the population, especially sportsmen at all levels, from amateurs to professional athletes, are often tempted to take dietary supplements in order to improve their performance and/or to gain mass and increase muscle growth. Thus, the spread of supplements, also coming from the black market, has increased dramatically in the last decades (Geyer et al. 2004, 2008; Baume et al. 2006; Parr et al. 2008). The easy availability of supplements through websites, supplements shops or in gyms, where it is possible to buy these products without any prescriptions and clinical approval, has contributed to their spread. In recent years it has been reported that products marketed as dietary supplements may contain non-labelled substances, like clenbuterol (Parr et al. 2008) and anabolic androgenic steroids (AASs) (Kamber et al. 2001; Delbeke et al. 2002; Geyer et al. 2004, 2008; Baume et al. 2006). This discrepancy between the label and the content of preparation might be the result of poor manufacturing practice, but in most cases it is deliberate
adulteration of products by the manufacturers in order to obtain more marked effects. These substances, which are originally produced and sold for the clinical treatment of various diseases, can expose healthy consumers to risks and the consequences of their misuse might be more dangerous, especially for adolescents. Their intake might also lead to an inadvertent doping offence, as the use of anabolic agents is severely banned by the World Anti-Doping Agency (WADA). Therefore, the development of methods able to screen for a high number of possible anabolic agents in supplements with adequate sensitivity is of great interest. The analysis of food supplements requires sample pretreatment in order to have them in the right physical state for analysis, to reduce interferences present in the matrix and to detect low concentrations of analytes. The techniques used till now for these purposes involved a dissolution in a solvent (Van Poucke et al. 2007; Doué et al. 2014; Krug et al. 2014) followed sometimes by liquid– liquid extraction (LLE) (Geyer et al. 2004; Baume et al. 2006; Martello et al. 2007; Strano-Rossi et al. Forthcoming), otherwise by SPE (Becue et al. 2011) or a filtration (Cho et al. 2014). The subsequent detection step of AASs is mostly performed by GC-MS (Kamber et al. 2001; Delbeke et al. 2002; Geyer et al. 2004; Baume et al.
*Corresponding author. Emails: [email protected] and [email protected] © 2015 Taylor & Francis
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2006; Parr et al. 2008) or LC-MS/MS in ESI (Van Poucke et al. 2007; Cho et al. 2014) and atmospheric pressure ionisation (APCI) mode (Martello et al. 2007; Doué et al. 2014). The dissolution of solid matrix in sodium hydroxide and successive extraction by pentane/diethyl ether before the derivatisation for obtaining trimethylsilyl (TMS) derivatives to analyse by GC-MS was described by Delbeke et al. (2002) and Van Thuyne and Delbeke (2004). Parr et al. (2004) added a mixture of diisopropylamino-n-alkanes (DIPAs) to the final extract before the GC-MS analysis of TMS derivative. A recent study on 337 products from the German black market was performed screening using either a target LC-MS/MS method, a LC-QTOF-HRMS method or a GC-QTOFHRMS method after derivatisation (Krug et al. 2014). Recently, Kaufmann (2012) reported on the increasing role of high resolution mass spectrometry (HRMS) in food analysis, specifically for pesticides and veterinary drugs. The main mentioned advantages were the acquisition of full-scan spectra, which allows the measurements of compounds without previous compound-specific tuning, the possibility of retrospective data analysis, and the capability of performing structural elucidations of unknown or suspected compounds. For these reasons, we developed a sensitive method by LC-HRMS in APCI mode for the screening of AASs and clenbuterol (a β-agonist with anabolic activity) in dietary supplements. The method was validated for some specific anabolic agents, allowing their forensic identification and quantification.
Materials and methods Chemicals and reagents Formic acid, sodium hydroxide, ultrapure water and methanol were purchased from Sigma-Aldrich (Milan, Italy). All solvents used were LC-MS grade. 1,4-Androstadien-17β-ol-3-one (boldenone), boldenone propionate, boldenone undecylenate, 4,9,11-estratrien17β-ol-3-one (trenbolone), 5-estren-3-β,17β-diol, 4-androsten-17β-ol-3-one (testosterone) decanoate, testosterone cypionate, testosterone undecanoate, testosterone phenylpropionate, 4-estren-17β-ol-3-one (nandrolone) laurate, 4-estren3,17-dione (norandrostenedione), 1,4-androstadien-17αmethyl-17β-ol-3-one (methandienone), 5α-androstan-1αmethyl-17β-ol-3-one (mesterolone), 1(5α)-androsten-17methyl-17β-ol-3-one (methenolone), methenolone enanthate, methenolone acetate, 5α-androstan-17αmethyl-17β-ol (stanozolol), 1,4-androstadien-3,17-dione (boldione), 5-androsten3β-ol-17-one (DHEA), 5α-androstan-17β-ol-3-one (DHT), 4-androsten-3β,17β-diolo (4-androstendiol), 4-androsten3α,17β-diol, 4-androsten-3β,17α-diol, 5α-androstan3α,17β-diol (3αAdiol), 5α-androstan-3β,17β-diol (3βAdiol), 5-androsten-3β-ol-7,17-dione (7-keto-DHEA), 5-androstene3,17-dione, 5(10)-estren-3β,17β-diol, 5-androsten-
3β,17β-diol, 4-androsten-3,17-dione (androstenedione) were from Steraloids (Newport, RI, USA). Clenbuterol, 4-androsten-17α-ol-3-one (epitestosterone), testosterone enanthate, nandrolone decanoate and testosterone-d3 were from Sigma-Aldrich (St. Louis, MO, USA). Testosterone, testosterone propionate, testosterone acetate and nandrolone were from Fluka (Sigma-Aldrich, Milan, Italy). Individual methanolic solutions of each standard were prepared from the pure powders at a concentration of 1 mg ml−1 and stored at −20°C according to the manufacturers’ specifications. A working solution mixture, at a concentration of 1 μg ml−1 in methanol, was then prepared by diluting stock solutions in methanol and stored in a freezer at −20°C.
Sample preparation Aliquots of nutritional supplements in powders, oily capsules, energy bars and tablets were collected in glass tubes and kept refrigerated at 4°C in the dark in order to avoid photo-degradation. A total of 1 g of the sample was weighted, the internal standard (IS) testosterone-d3 was added to the samples at a concentration of 100 ng g−1 and 5 ml of methanol were added. The specimen was then vortexed for 20 s and centrifuged for 10 min at 4000 rpm. The methanolic phase was evaporated to dryness under nitrogen flow at 40°C and reconstituted in 2 ml of a solution of sodium hydroxide 1 M. An LLE was performed through the addition of 5 ml of a mixture of pentane/ethyl ether 9:1, under agitation for 1 h. After centrifugation, the organic phase was evaporated to dryness under a nitrogen stream at RT and reconstituted with methanol/water/formic acid 6:4:0.03 v/v (100 μl) in a glass vial. A total of 10 μl was then directly injected in the LC-HRMS system.
LC-HRMS Equipment The LC-HRMS system was composed of a Thermo ULTIMATE 3000 system equipped with an analytical column Thermo Acclaim RSLC 120 C18 (2.1 mm × 100 mm, 2.2 μm particle size), coupled to a Thermo single-stage Orbitrap (Exactive) MS system. The whole equipment and the column were provided by Thermo Fisher Scientific (Milan, Italy).
LC-HRMS conditions Mobile phase A was a mixture of ultrapure water/methanol 90/10 v/v with 0.1% formic acid; mobile phase B was a solution of methanol with 0.1% formic acid. The analytical column was maintained at 40°C and sample injection volume was 10 μl. The flow rate was set at 400 μl min–1. Mobile phase gradient was as follows: 60% A for 1 min, linear gradient to 100% B in 7 min, held for 5.0 min,
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Food Additives & Contaminants: Part A column re-equilibration was performed with linear gradient to 60% A in 3.0 min, held for 3.0 min. Ionisation parameters were optimised with direct infusion of testosterone-d3 standard by using external syringe. The optimised tune file was then used to develop the screening method and to analyse the samples. The APCI source was heated at 400°C. Source current was 6 µA, sheath gas and auxiliary gas (either nitrogen) flow rates were 35 and 15 arbitrary units, respectively; capillary temperature was 290°C. During the method setup an HESI-II Ion Max source was also tested in order to choose the better ionisation conditions. Data were acquired in full-scan mode over a mass range of 110–800 m/z. The instrument operated in positive ion mode with a resolving power of 100 000 full width at half maximum (FWHM). In case of confirmation of screening positive results, a further set of experiments was performed with in source collision-induced dissociation (CID) with voltage set at 40 V, acquiring ions from 70 to 500 m/z, with a resolving power of 50 000 FWHM, obtaining the accurate masses of both precursor and fragment ions. Experiments for the determination of the better conditions for in-source CID fragmentation were performed at different voltage settings (20, 30 and 40 V). A 40 V CID showed optimal fragmentations for all the steroids, giving higher signals for the characteristic fragments. Mass calibration was performed according to the guidelines provided by the instrument supplier. The recommended calibration solution is made of MRFA (L-methionyl-arginyl-phenylalanylalanine acetate), caffeine and Ultramark® 139 1621 dissolved in methanol/ water (1:1). For calibration purposes, the automatic calibration feature of the Exactive tune software was used. The mass scale was calibrated every 2 days over the mass range m/z 50–2000. Lock-mass (diisooctyl phtalate ionic species, 391.2843 m/z) was utilised during the analysis of samples in order to compensate any possible mass axis drifts. Data analysis For qualitative screening, data were processed using a homemade database containing the protonated exact monoisotopic masses list and built on the extracted ion chromatograms (EIC) of the expected [M + H]+ ions of each compound. Table 1 reports the raw formulas, exact and accurate masses with respective errors and retention times of the analytes investigated. In the case of DHEA, 4-androsten-3α,17β-diol, 4androsten-3β,17α-diol, 5(10)-estren-3β,17β-diol, and 5androsten-3β,17β-diol, the ionic species detectable were those obtained by the loss of one or two molecules of water, as reported in Table 1. A database of analytes was created by analysing pure standard solutions.
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Identification of compounds was based on the following criteria: exact mass of monoisotopic ion M + 0 (mass Þðaccurate massÞ 106 , accuracy, Δm, defined as ðexaxt mass ðaccurate massÞ less than 2 ppm), comparison of experimental and calculated isotopic pattern (relative isotopic abundance – RIA (M + 1/M + 0) and RIA (M + 2/M + 0) errors less than 15%), retention time window (±0.2 min). Due to the possibility of analysing analytes in a nontargeted way, we added in the database the protonated exact monoisotopic masses of other anabolic steroids that could in principle be present as adulterants not included in the phase of development and validation of the present analytical method. The list is reported in Table 2. The full-scan data file of each supplement was further screened for these AASs for which a full validation was not performed. In these cases only a ‘suspect positive’ could be assumed, as there was no comparison with an analytical standard, necessary for a certain identification Xcalibur 2.1 software (Thermo Fisher Scientific Inc., San Jose, CA, USA) was used to analyse and process all data for quantitative analysis.
Method validation The following parameters were taken into account for method validation: specificity and selectivity, LOD, LOQ, linearity, repeatability, accuracy, matrix effect and carryover. Specificity of the method was studied by analysing 10 nutritional supplement samples free of steroids in order to assess potential interferences from other substances. There were different kinds of nutritional supplements: energy bars, tablets and powders (in some cases vanilla and cocoa flavoured) and oily solutions. In case of oily solutions, we studied only some validation parameters (i.e. specificity and LOD), so in case of analyses of oily supplements we could only give qualitative results. Selectivity of the method was studied by analysing samples spiked with various classes of drugs such as stimulants, opiates, antidepressants, benzodiazepines and cannabinoids. The LOD was determined by analysing scalar dilutions (1–5–10–25 ng g−1) of fortified samples on five different typologies of supplements and it was figured out at a concentration value giving an S/N > 3 for the extracted ionic trace. The LOQ was figured out as the lower level of calibration curve (50 ng g−1, that should give an S/N > 10 and an acceptable precision (%CV < 20%) and accuracy (%E < 20%). Linearity was studied in the range from the LOQ of each substance (50 ng g−1) to 2000 ng g−1 on three different types of supplements (powder, energy bars and tablets). Calibration curves were built by weighted linear
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Table 1.
Raw formulas, exact and accurate masses with respective errors and retention times of the studied anabolic agents.
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Compound 4-Androstendiol 4-Androsten -3α,17β-diol 4-Androsten-3β,17α-diol 5(10)-Estren-3β,17β-diol 5-Androsten-3β,17β-diol 3α-Adiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
MH+
Exact mass m/z
Accurate mass (mean m/z)
Δm (ppm)
Rt (min)
C19H31O2 − H2O C19H31O2 − H2O C19H31O2 − H2O C18H29O2 C19H31O2 − H2O C19H33O2 C19H33O2 C19H27O3 C19H27O2 C19H27O2 C22H31O3 C30H45O3 C19H25O2 C12H19Cl2N2O C19H29O2 − 2H2O C19H31O3 C19H29O2 C20H33O2 C20H29O2 C20H31O2 C22H33O3 C27H43O3 C18H27O2 C28H45O3 C30H49O3 C18H25O2 C21H33N2O C19H29O2 C21H31O3 C27H41O3 C29H47O3 C26H41O3 C28H37O3 C22H33O3 C30H49O3 C18H23O2
273.2212 273.2212 273.2212 259.2056 273.2212 275.2369 275.2369 303.1955 287.2006 287.2006 343.2268 453.3363 285.1849 277.0869 253.1951 291.2319 289.2162 305.2475 301.2162 303.2319 345.2424 415.3207 275.2006 429.3363 457.3676 273.1849 329.2587 289.2162 331.2268 413.3050 443.3520 401.3050 421.2737 345.2424 457.3676 271.1693
273.2211 273.2211 273.2211 259.2054 273.2211 275.2367 275.2368 303.1953 287.2003 287.2003 343.2266 453.3366 285.1848 277.0868 253.1949 291.2318 289.2161 305.2474 301.2160 303.2316 345.2423 415.3207 275.2005 429.3367 457.3678 273.1848 329.2585 289.2161 331.2268 413.3052 443.3522 401.3048 421.2739 345.2422 457.3679 271.1692
−0.4 −0.4 −0.4 −0.6 −0.4 −0.7 −0.2 −0.7 −0.9 −0.9 −0.6 0.6 −0.5 −0.3 −0.9 −0.5 −0.2 −0.4 −0.7 −1.0 −0.3 0.01 −0.2 0.5 0.5 −0.4 −0.6 −0.3 −0.5 0.4 0.4 −0.4 0.4 −0.5 0.6 −0.6
7.7 8.5 8.4 7.5 7.8 8.7 8.1 5.5 7.3 7.1 9.3 11.4 6.6 1.1 8.0 8.4 8.2 8.6 7.5 8.2 9.6 11.3 7.3 11.8 12.5 6.9 8.4 7.7 9.3 11.2 12.0 11.0 10.5 9.8 12.4 7.0
regression, considering the area ratio between the analyte and the internal standard, and were prepared in triplicate by adding the proper amount of working mixture to the aliquots of 1 g of nutritional supplement in order to obtain the following concentrations: 50–100–500–1000– 2000 ng g−1. The weighting factor used was 1/x. Repeatability and accuracy, expressed as %CV and %E, respectively, were assessed on control samples prepared at three different concentrations, i.e. 50, 500 and 2000 ng g−1, by quintuplicate analyses on five different supplements (two powders, two tablets and one energy bar) in three different days. Matrix effect and recoveries were investigated for each compound on five blank supplements (two powders, two tablets and one energy bar). According to Matuszewski et al. (2003), absolute recoveries were determined by comparing the signals of quality control (QC) samples (in terms of peak areas), prepared with the proposed method, with those obtained spiking blanks supplements
after extraction at the same concentrations. The matrix effect was studied by comparing the signals of pure standards in acidified water/methanol with those obtained spiking blanks supplements after extraction. Carryover was evaluated through the injections of two blank samples after each calibration level and evaluating the eventual presence of memory effect.
Results The proposed method allows the screening of anabolic agents in nutritional supplements, using a simple and effective sample preparation procedure that guarantees optimal results in terms of sensitivities. LODs obtained varied from less than 1 ng g−1, in the case of 5-androsten3,17-dione, androstenedione, epitestosterone, methenolone, methenolone acetate, nandrolone, testosterone, testosterone acetate, testosterone propionate and trenbolone, to 25 ng g−1 for 3α-Adiol, 3β-Adiol, boldenone
Food Additives & Contaminants: Part A Table 2.
Raw formulas and exact masses of other common anabolic agents.
Compound
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4-Hydroxytestosterone Bolasterone/calusterone/norboletone Boldenone acetate Boldenone benzoate Clostebol Danazol Dehydrochlormethyltestosterone Desoxymethyltestosterone/ethylestrenol Drostanolone enanthate Drostanolone propionate Fluoxymesterone Formebolone Furazabol Gestrinone Methasterone Nandrolone benzoate Nandrolone phenylpropionate Norclostebol Oxabolone Oxabolone cipionate Oxandrolone Oxymesterone Oxymetholone Prostanozol Quinbolone Tetrahydrogestrinone/tibolone Testosterone benzoate Testosterone isobutyrate Testosterone isocaproate Trenbolone acetate Trenbolone enanthate Trenbolone hexahydrobenzylcarbonate Zeranol Zilpaterol
propionate, boldenone undecylenate, DHT, methandienone, nandrolone decanoate, nandrolone laurate, oxandrolone, testosterone decanoate and undecanoate. The APCI source was chosen during method development with respect to the ESI due to the higher sensitivities obtained for some of the steroids. In fact, although signals obtained for pure standards of many of the steroids were comparable with the two ionisation techniques and better for clenbuterol with ESI, the signals of 5-estren-3-β, 17βdiol and mesterolone had a dramatic decrease with ESI, rendering these substances undetectable with this technique. Furthermore, the matrix effect on spiked samples was higher for all the analytes when ESI was used as ionisation source, giving an ion suppression ranging from 87% to 33% at 500 ng g−1. Table 3 reports LODs, LOQs and linearity (mean slopes, intercepts and correlation coefficients obtained on three different supplements) for each substance included in the present study. All the investigated compounds were correctly quantified at 50 ng g−1 level. The method showed good linearity in the range from the LOQ of
MH+
Exact mass m/z
C19H29O3 C21H33O2 C21H29O3 C26H31O3 C19H28ClO2 C22H28NO2 C20H28ClO2 C20H33O C27H45O3 C23H37O3 C20H29FO3 C21H29O4 C20H31N2O2 C21H25O2 C21H35O2 C25H31O3 C27H35O3 C18H26ClO2 C18H27O3 C16H39O4 C19H31O3 C20H31O3 C21H33O3 C25H39N2O2 C24H33O2 C21H29O2 C26H33O3 C23H35O3 C26H42NO3 C20H25O3 C25H35O3 C26H35O4 C18H27O5 C14H20N3O2
305.2111 317.2475 329.2111 391.2268 323.1772 338.2115 335.1772 289.2526 417.3363 361.2737 337.2174 345.2060 331.2380 309.1849 319.2632 379.2268 407.2581 309.1616 291.1955 295.2843 307.2268 319.2268 333.2424 399.3006 353.2475 313.2162 393.2424 359.2581 416.3159 313.1798 383.2581 411.2529 323.1853 262.1550
each substance to 2000 ng g−1 of nutritional supplement. We used a weighted least squares linear regression, with 1/ x as a weighting factor. The appropriateness of the linear regression model was assessed by examination of the residual plots and by evaluation of percentage relative errors versus concentrations (Almeida et al. 2002). In case of supplements containing substances at concentration above the quantitation range, a lesser amount of supplement (50 mg instead of 1 g) was submitted to the analytical protocol. Also accuracy, intended as mean relative error and inter- and intra-day repeatability, was acceptable (always lower than 15%). Data on mean inter-day repeatability and accuracy (n = 15) are shown in Table 4. No interferences from the matrix were encountered at the retention times of the analytes. Figure 1 shows the layout of a mixture of blank nutritional supplements sample spiked with the analytes of interest at a concentration of 500 ng g−1. Other sample preparation procedures, such as ‘dilute and shoot’ with methanol and acidified methanol/water, have been tried in order to optimise the method, but the step
4-Androstendiol 4-Androsten -3α,17β-diol 4-Androsten -3β,17α-diol 5(10)-Estren -3β,17β-diol 5-Androsten -3β,17β-diol 3α-Adiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
Compound 5 10 10 10 10 25 25 5 1 10 25 25 10 5 5 25 1 5 25 1 1 5 1 25 25 5 25 5 1 10 25 10 5 1 25 1
LOD (ng g–1) 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
LOQ (ng g–1) 0.0005 ± 0.0001 0.0005 ± 0.0002 0.0003 ± 0.0001 0.0045 ± 0.0005 0.0138 ± 0.0034 0.0002 ± 0.0001 0.0004 ± 0.0001 0.0024 ± 0.0005 0.0127 ± 0.0029 0.0018 ± 0.0008 0.0005 ± 0.0001 0.0013 ± 0.0002 0.0049 ± 0.0006 0.0032 ± 0.0006 0.0142 ± 0.0024 0.0033 ± 0.0003 0.0082 ± 0.0008 0.0019 ± 0.0004 0.0015 ± 0.0001 0.0075 ± 0.0015 0.0049 ± 0.0001 0.0011 ± 0.0001 0.0066 ± 0.0011 0.0005 ± 0.0001 0.0005 ± 0.0001 0.0073 ± 0.0018 0.0621 ± 0.0048 0.0038 ± 0.0008 0.0065 ± 0.0003 0.0017 ± 0.0001 0.0006 ± 0.0001 0.0016 ± 0.0002 0.0035 ± 0.0006 0.0066 ± 0.0002 0.0004 ± 0.0001 0.0068 ± 0.0006
Slope ± SD
Table 3. LODs, LOQs and linearity (mean slopes, intercepts and correlation coefficients with RSDs, n = 5).
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0.0019 ± 0.0050 0.0000 ± 0.0044 −0.0017 ± 0.0041 0.0007 ± 0.0017 −0.0559 ± 0.0433 0.0007 ± 0.0024 −0.0017 ± 0.0023 −0.0838 ± 0.0545 −0.0512 ± 0.0728 −0.0041 ± 0.0062 −0.0006 ± 0.0003 0.0008 ± 0.0014 0.0029 ± 0.0339 −0.7505 ± 1.2792 −0.0225 ± 0.0581 −0.1171 ± 0.1931 −0.1081 ± 0.3451 −0.1319 ± 0.2257 −0.0358 ± 0.0504 −0.0171 ± 0.0151 −0.0103 ± 0.0006 0.0619 ± 0.1703 −0.0427 ± 0.2761 0.0084 ± 0.0535 −0.0161 ± 0.0166 −0.1612 ± 0.2970 0.0010 ± 0.0013 −0.0460 ± 0.0318 −0.0005 ± 0.0211 0.1928 ± 0.4410 −0.0213 ± 0.0290 0.0643 ± 0.1776 −0.0579 ± 0.1295 −0.4900 ± 0.6912 −0.0164 ± 0.0372 −0.1395 ± 0.1012
Intercept ± SD
0.9952 ± 0.0039 0.9936 ± 0.0015 0.9943 ± 0.0021 0.9941 ± 0.0004 0.9956 ± 0.0028 0.9929 ± 0.0026 0.9933 ± 0.0028 0.9921 ± 0.0017 0.9964 ± 0.0029 0.9945 ± 0.0024 0.9940 ± 0.0004 0.9923 ± 0.0027 0.9928 ± 0.0023 0.9940 ± 0.0013 0.9931 ± 0.0028 0.9918 ± 0.0013 0.9928 ± 0.0024 0.9939 ± 0.0033 0.9935 ± 0.0026 0.9933 ± 0.0015 0.9918 ± 0.0009 0.9922 ± 0.0008 0.9930 ± 0.0024 0.9914 ± 0.0007 0.9992 ± 0.0025 0.9932 ± 0.0031 0.9951 ± 0.0056 0.9949 ± 0.0026 0.9913 ± 0.0017 0.9925 ± 0.0016 0.9908 ± 0.0009 0.9913 ± 0.0007 0.9916 ± 0.0016 0.9915 ± 0.0001 0.9916 ± 0.0014 0.9940 ± 0.0006
r2 ± SD
6 S. Odoardi et al.
4-Androstendiol 4-Androsten-3α,17β-diol 4-Androsten-3β,17α-diol 5(10)-Estren-3β,17β-diol 5-Androsten-3β,17β-diol 3αAdiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
Compound
−10 10 11
12 13 12 13 11 6 13 12 3 13 −9 2 14 8 9 2 3 −0.1 14 10 14 12 6 14 10 −8 10 14 −5 7 9 8 −0.1
%E (n = 15)
4 13 5
5 9 12 9 11 12 10 5 9 10 12 10 5 8 8 12 14 11 11 10 11 3 7 4 14 12 12 12 11 7 3 6 12
%CV (n = 15)
80 80 67
70 86 81 48 68 78 88 79 56 53 76 64 74 76 80 79 64 81 68 72 84 87 50 87 75 67 65 81 70 88 79 79 82
%ME (n = 5)
50 ng g–1
69 69 64
64 64 75 72 71 73 84 76 73 65 76 71 81 83 88 79 80 86 60 81 85 62 66 65 67 57 63 68 57 61 59 54 68
%R (n = 5)
Table 4. Inter-day repeatability, accuracy, matrix effect and recovery.
5 7 1
8 1 9 3 1 13 5 14 6 3 3 10 1 4 1 9 3 8 11 3 14 14 10 1 14 9 3 7 3 6 1 7 4
−2 −11 −10 −9 −6 −7 −13 −13 −0.4 −9 4 −6 −9 −12 −11 −3 3 −3 −11 −10 10 −3 1 10 3 2 −7 −14 9 6 13 8 13 7 8 −12
%CV (n = 15)
%E (n = 15)
70 69 63
98 78 95 68 53 68 79 75 58 77 57 67 63 87 74 73 61 87 64 86 65 62 40 85 84 77 70 69 62 69 72 68 69
%ME (n = 5)
500 ng g–1
67 59 72
63 64 86 70 67 66 70 79 73 66 75 71 69 70 72 67 70 69 63 76 63 65 63 63 69 58 63 78 54 51 55 50 60
%R (n = 5)
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−8 −6 1
−0.1 1 1 3 1 1 0.4 0.03 1 2 −6 −1 2 1 2 1 −0.4 −0.2 1 −1 −8 −3 1 −3 −3 0.4 −0.01 2 −7 −1 −6 −0.01 −6
%E (n = 15)
11 8 3
3 0.5 2 3 0.2 1 1 4 1 3 10 2 2 4 5 3 1 3 3 1 11 2 2 6 7 1 2 2 10 7 9 2 12
%CV (n = 15)
83 83 62
77 54 84 59 55 65 80 81 61 61 58 60 60 74 76 77 64 78 63 87 75 62 47 89 90 81 80 70 85 83 83 83 83
%ME (n = 5)
2000 ng g–1
57 68 57
60 65 70 61 58 65 66 70 71 64 63 66 63 77 66 76 77 65 71 65 60 63 63 66 78 51 58 62 51 56 67 57 61
%R (n = 5)
Food Additives & Contaminants: Part A 7
8
S. Odoardi et al. 12.37
100
Testosterone undecanoate (Rt = 12.4) Nandrolone laurate (Rt = 12.5)
50 0 100
NL: 2.29E5 m/z = 457.3630-457.3722 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
12.52
NL: 2.47E5 m/z = 443.3476-443.3564 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
11.99
Testosterone decanoate
50 10.19
0 100
11.76
Nandrolone decanoate
NL: 2.64E5 m/z = 429.3320-429.3406 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Relative Abundance
50 0 100
11.42
NL: 1.31E4 m/z = 453.3318-453.3408 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Boldenone undecylenate
50
9.06 9.24
0.50
0 100
10.72
8.85
12.02
12.31
15.28 NL: 5.52E5 m/z = 415.3165-415.3249 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
11.26
Methenolone enanthate 9.94 10.93 11.15
0 100
NL: 9.94E5 m/z = 413.3009-413.3091 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone cypionate 50 0 100
11.02
NL: 4.88E5 m/z = 401.3010-401.3090 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone enathate
50
10.66 10.48
0 100
NL: 1.25E6 m/z = 421.2695-421.2779 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone phenylpropionate
50 0 100
9.75
Methenolone acetate (Rt = 9.6) Testosterone propionate (Rt = 9.8)
50 0 100
NL: 2.68E6 m/z = 345.2389-345.2459 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
9.58
Testosterone acetate
9.26
NL: 2.56E6 m/z = 331.2235-331.2301 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
9.26
NL: 2.03E5 m/z = 343.2234-343.2302 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
50 Relative Abundance
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50
0 100
Boldenone propionate
50 9.60 0 100
10.65 10.93
Mesterolone (Rt = 10.9)
50
NL: 1.70E6 m/z = 305.2444-305.2506 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.62 9.96
0 100
11.35
8.44
Stanozolol (Rt = 8.4)
NL: 7.98E5 m/z = 329.2554-329.2620 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
50 10.26 9.88
0 100
12.09 12.80
NL: 2.52E6 m/z = 291.2290-291.2348 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
DHT (Rt = 9.9)
50 8.38
9.55
0 0
1
2
3
4
5
6
7
8
9
10.16 10.69 11.75 10
11
12
13
14
15
16
Time (min)
Figure 1. (colour online) Extracted ionic chromatogram of a blank nutritional supplement spiked with the analytes of interest at a concentration of 500 ng g−1.
Food Additives & Contaminants: Part A 8.17
100
NL: 2.62E6 m/z = 303.2289-303.2349 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Methenolone (Rt = 8.2) 50 9.68
0 100
10.64
8.70
10.65 11.75
9.49 0 100
Relative Abundance
7.68
NL: 4.91E5 m/z = 273.2185-273.2239 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.38 8.53
11.61 NL: 4.92E5 m/z = 253.1926-253.1976 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.96
DHEA
50 7.68 0 100
8.53 8.17 7.71
Testosterone (Rt = 7.7) Epitestosterone (R = 8.2)
50 0 100
NL: 3.59E6 m/z = 289.2133-289.2191 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1 NL: 2.33E5 m/z = 259.2030-259.2082 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.54
5 (10)-estren-3β, 17β-diolo (Rt = 7.5)
50 9.88 10.36 11.00 11.48 7.83 8.96 9.60 7.45
0 100
14.07 14.24 NL: 4.77E5 m/z = 301.2132-301.2192 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Menthandienone (Rt = 7.4) 50 8.16 0 100
9.37 9.77 10.41 10.69
7.33
Boldenone (Rt = 7.1) Androstenedione (Rt = 7.3)
50
NL: 4.65E6 m/z = 287.1977-287.2035 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.09 0 100
NL: 2.21E6 m/z = 275.1978-275.2034 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.32
Nandrolone
50 0 100
NL: 1.63E6 m/z = 271.1666-271.1720 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
6.94
Trenbolone (Rt = 6.9)
50 Relative Abundance
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0 100
14.12 14.58 15.43
7.78
4-androstendiol (Rt = 7.7) 5-androsten-3β, 17β-diol (Rt = 7.8) 4-androstene-3β, 17α-diol (Rt = 8.4) 4-androstene-3α, 17β-diol (Rt = 8.5)
50
NL: 1.03E5 m/z = 275.2341-275.2397 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.10
3βAdiol (Rt = 8.1) 3αAdiol (Rt = 8.7)
50
9.99
6.61 6.87
0 100
9
NL: 2.43E6 m/z = 273.1822-273.1876 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Norandrostenedione
50 7.32
0 100
NL: 1.13E6 m/z = 285.1820-285.1878 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
6.58
Boldione (Rt = 6.6)
50 5.48 0 100
7.08
8.05
10.15 11.01 NL: 4.67E5 m/z = 303.1925-303.1985 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
5.47
7 keto-DHEA
50 0 100
NL: 4.00E5 m/z = 277.0841-277.0897 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
1.07
Clenbuterol
50 10.69 0 0
Figure 1.
1
2
Continued.
3
4
5
6
7
8 Time (min)
9
10
11.01 11.44 11
12
13
14
15
16
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10
S. Odoardi et al.
with sodium hydroxide and the following extraction were fundamental for the method in order to have a cleaner extract and therefore to lower the ion suppression, allowing to obtain satisfactory sensitivities and to reduce matrix effect. The results of the mean matrix effect at three different concentrations are listed in Table 4, with a value of 100% indicating no matrix effect, < 100% indicating signal suppression and > 100% indicating signal enhancement. As expected due to the complexity of a multi-component matrix, a matrix effect was observed for some analytes. The highest signal suppression was observed for nandrolone at 60%. Nevertheless, all the substances could be detected at ng mg–1 levels also for those analytes showing higher matrix effect. Recoveries were satisfactory, ranging from 51% to 86% for all the analytes. No significant degradation of analytes or hydrolysis of esters was observed during sample preparation. Only oxandrolone gave very variable results, probably due to its degradation, with unacceptable validation results. Carryover was observed only in the first blank sample after the injection of the higher calibration level (2000 ng g−1) for testosterone undecanoate, decanoate, phenylpropionate and nandrolone laurate, in an amount under the LOQ and therefore not quantifiable. In these cases three blank samples were injected afterwards, and a complete decontamination of the system was ensured by the complete negativity of the third blank injection. The fragmentation pattern of the analytes was used further to confirm positive cases by the exact masses of the characteristic fragments in addition to their retention times, accurate masses by evaluating Δm, and isotopic cluster, by the comparison between experimental and calculated RIA (M + 1/M + 0) and (M + 2/M + 0) errors. This was obtained by performing a further in source CID experiment and by identifying the compound of interest according to the relative abundance of the generated fragments and their accurate masses with respect to those obtained by a reference standard. This method was used for the analysis of 30 nutritional supplements sold in fitness centres and in websites, collected from police officers in Italy in order to check the eventual presence of unlabelled substances. As reported on the labels, they contained proteins, vitamins and fruit, chocolate or vanilla flavours, DHEA in one supplement and androstenedione in another one. Many unlabelled AASs were detected, often in combination: androstenedione was detected in nine supplements from 190 to 8170 ng g−1 (only in one case it was declared on the label, and was at a concentration far above the linearity range); DHEA in 12 samples (from 115 to 32 000 ng g−1), declared on the label in only one of them, methandienone (from 330 to 8850 ng g−1) in three samples, stanozolol in one supplement (at 25 300 ng g−1), epitestosterone (at 3700 ng g−1), testosterone in seven samples (from a concentration below the LOQ to
40 000 ng g−1), and testosterone esters in four of them (testosterone decanoate at 25 ng g−1, testosterone enanthate at 87 and 170 ng g−1, testosterone phenylpropionate at 10 and 92 ng g−1). These results are summarised in Table 5. The mass spectra obtained at a resolution of 100 000 FWHM from the analysis of a nutritional supplement sample containing DHEA at 32 400 ng g−1, testosterone phenylpropionate at 92 ng g−1 and testosterone enanthate at 170 ng g−1 are shown in Figure 2, along with the comparison with calculated theoretical values. Data files were further reprocessed also for the screening of other anabolic steroids that could be present as adulterant in food supplements and for which we did not perform a validation (e.g. steroids listed in Table 1) by the screening of their exact protonated masses. In one of them we suspected the presence of oxymetholone, in another of testosterone isocaproate, along with other testosterone esters, and in another one of tibolone. A comparison with certified analytical standards would be necessary for their determination. Discussion The issue of contaminated/adulterated nutritional supplements is of great concern, both for public health and for possible doping implications. In fact it may happen that elite athletes, submitted to anti-doping controls, have positive results due to an involuntary intake of prohibited substances present in uncontrolled supplements, an event that is anyway considered a doping offence, according to the article 2.1 of WADA’s Anti-Doping Code (2015). The analysis of supplements for undeclared doping agents is therefore necessary. High sensitivities are key factors in analytical methods in order to detect substances at low amounts that can lead to a doping offence, but mainly to a health hazard for unaware consumers. The present method allows an efficient and rapid determination of about 40 anabolic agents in dietary supplements. The isolation of analytes from complex mixtures such as oily or energy bars after an alkaline treatment with sodium hydroxide and subsequent extraction allows an effective clean-up of the samples leading to a low matrix effect and high sensitivities for all the studies compounds. The use of HRMS, due to its high resolving power, permits an excellent selectivity and sensitivity for the target analytes. The use of the accurate mass and the correspondence of isotopic cluster guarantees the identification of the empirical formula, allowing a reliable screening for a large number of anabolic substances. The efficient chromatography obtained with the present method, with a total run time of 16 min, provides a rapid and effective separation of analytes, guaranteeing also the identification of positional isomers or diastereoisomers. Hence, in the case of isobaric compounds, such as for isomeric androsten-diols, the good chromatographic
Fat burner, energy enhancer
Fat burner, energy enhancer
Expectorant, bronchodilator
Fat burner, energy enhancer
Fat burner, energy enhancer
Fat burner, energy enhancer
Fat burner
DHEA nutritional supplement
Fat burner
Muscular growth enhancer Diuretic Ephedrine nutritional supplement Androstenedione nutritional supplement
Fat burner, energy enhancer
Unknown
Unknown
1
2
3
4
5
6
7
8
9
10 11 12 13
14
15
16
Declared type of supplement
Tablets
Oily solution
Tablets
Capsules Capsules Tablets Capsules
Capsules
Capsules
Capsules
Capsules
Tablets
Capsules
Tablets
Tablets
Capsules
Formulation
Table 5. Substances determined in dietary supplements. AASs found
Ephedrine and caffeine alkaloids, amino acids
Androstenedione 1250 ng g−1 DHEA 310 ng g−1 Epitestosterone 3700 ng g−1 Natural products and extracts, amino acids, DHEA 115 ng g−1 Methandienone 8850 ng g−1 minerals Stanozolol 25 300 ng g−1 Testosterone 2600 ng g−1 Testosterone decanoate 25 ng g−1 Ephedrine, guaifenesin Androstenedione 3080 ng g−1 DHEA 4740 ng g−1 Testosterone < LOQ Ephedrine and caffeine alkaloids, natural Androstenedione 577 ng g−1 DHEA 170 ng g−1 products, minerals Testosterone < LOQ Testosterone phenylpropionate 10 ng g−1 Natural products and extracts, amino acids, Androstenedione 770 ng g−1 DHEA 120 ng g−1 minerals Caffeine alkaloids, natural extracts, amino acids, Androstenedione 190 ng g−1 minerals DHEA 410 ng g−1 Methandienone 640 ng g−1 Natural products, amino acids, vitamins Androstenedione 225 ng g−1 DHEA 117 ng g−1 Methandienone 130 ng g−1 Testosterone < LOQ Testosterone enanthate 87 ng g−1 DHEA Androstenedione 8170 ng g−1 DHEA 32,000 ng g−1 Testosterone < LOQ Testosterone enanthate 170 ng g−1 Testosterone phenylpropionate 92 ng g−1 Ephedrine and caffeine alkaloids, natural products Androstenedione 330 ng g−1 and extracts, amino acids, minerals DHEA 330 ng g−1 Amino acids DHEA 8170 ng g−1 Natural extracts, vitamins DHEA 3650 ng g−1 Ephedrine Androstenedione 3275 ng g−1 Androstenedione Androstenedione >> 30 000 ng g−1 DHEA 4100 ng g−1 Testosterone 40 000 ng g−1 Natural products and extracts, amino acids, Androstenedione 250 ng g−1 minerals Testosterone < LOQ Tibolone Unknown Testosterone propionate Testosterone phenylpropionate Testosterone decanoate Testosterone isocaproate Unknown Oxymetholone
Main compounds declared on the label
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Caffeine Ephedrine, caffeine
Caffeine
Ephedrine, caffeine
Caffeine
Ephedrine, pseudophedrine, caffeine
Ephedrine, caffeine, guaifenesin
Ephedrine, caffeine
Alkaloids found
Food Additives & Contaminants: Part A 11
Relative Abundance
12
S. Odoardi et al. 253.1950
100 80 60 40 20 0 100 80 60 40 20 0
DHEA - 2H2O (C19H25) 254.1983 255.2015 253.1951
Theoretical 254.1984 255.2018 252.6
252.8
253.0
253.2
253.4
253.6
253.8
254.0
254.2
254.4
254.6
254.8
255.0
255.2
255.4
255.6
255.8
256.0
Relative Abundance
421.2737
100 80 60 40 20 0 100 80 60 40 20 0
Testosterone phenylpropionate (C28H37O3) 422.2771 421.3526 421.2737
423.2806
Theoretical 422.2771 423.2804 421.0
Relative Abundance
Downloaded by [Northeastern University] at 03:45 11 March 2015
m/z
421.2
421.4
421.6
421.8
422.0
422.2 m/z
422.4
422.6
422.8
423.0
423.2
423.4
423.6
401.3049
100 80 60 40 20 0 100 80 60 40 20 0
Testosterone enanthate (C26H41O3) 402.3083 403.3115 401.3050
Theoretical 402.3084 403.3117 401.2
401.4
401.6
401.8
402.0
402.2 m/z
402.4
402.6
402.8
403.0
403.2
Figure 2. HR mass spectra obtained from the analysis of a nutritional supplement containing DHEA, testosterone phenylpropionate and testosterone enanthate: experimental (upper) and calculated (lower) isotopic patterns of DHEA-2H2O (C19H25), testosterone phenylpropionate (C28H37O3) and testosterone enanthate (C26H41O3).
separation permits identification of different analytes through their retention times. A further confirmatory analysis can be achieved by fragmenting the compound. The exact masses and the relative abundances of ion fragments, in combination with the accurate mass of the parent compound and with retention time, give a high degree of certainty as to the identity of the detected compound. Furthermore the method allows quantification of analytes with good accuracy and repeatability. The high sensitivity of the method allowed for the identification of undeclared AASs in supplements also in low concentrations (testosterone esters in concentration > 100 ng g−1, androstenedione at 190 ng g−1 or DHEA at 115 ng g−1), that could not have a biological activity. These amounts, however, can have health hazard implications, especially if supplements are taken in high amounts and continually, and can result in a doping offence, due to the high sensitivities required by the WADA for determination of AASs in athletes’ urine samples.
Finally, full scan acquisition enables a future retrospective reanalysis of an acquired data file at any time in case of a further substance to be detected. As an example, during the analysis of seized supplements, compounds not included in the phase of development and validation of the present method were also detected at a later stage. Conclusions Due to the high sensitivity, resolving power and specificity of LC-HRMS, a fast and efficient method was developed for the analysis of anabolic steroids and clenbuterol in nutritional supplements by the use of Orbitrap® technology. The effective sample preparation, efficient chromatography and high resolving power of the HRMS instrument, leading to high specificity, allowed good sensitivities and reliable quantitative results to be obtained for a large number of compounds. The application of the method to real supplements demonstrated its suitability
Food Additives & Contaminants: Part A for the identification of anabolic agents not indicated on the labels. Finally, the great potential of this technique is related to the possibility of reprocessing of previously acquired data files when a further banned/dangerous substance needs to be identified.
Funding This work was performed under the framework of the project ‘Smart-Stop’, funded by the Italian Antidrug Policies Department
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References Almeida AM, Castel-Branco MM, Falcao AC. 2002. Linear regression for calibration lines revisited: weighting schemes for bioanalytical methods. J Chromatogr B Analyt Technol Biomed Life Sci. 774:215–222. Baume N, Mahler N, Kamber M, Mangin P, Saugy M. 2006. Research of stimulants and anabolic steroids in dietary supplements. Scand J Med Sci Sports. 16:41–48. Becue I, Van Poucke C, Van Peteghem C. 2011. An LC-MS screening method with library identification for the detection of steroids in dietary supplements. J Mass Spectrom. 46:327–335. Cho S-H, Park HJ, Lee JH, Kim HJ, Cho S, Yoon C-Y, Kim WS. 2014. Monitoring of 35 illegally added steroid compounds in foods and dietary supplements. Food Addit Contam: Part A. 31:1470–1475. Delbeke FT, Van Eenoo P, Van Thuyne W, Desmet N. 2002. Prohormones and sport. J Steroid Biochem Mol Biol. 83:245–251. Doué M, Dervilly-Pinel G, Gicquiau A, Pouponneau K, Monteau F, Le BB. 2014. High throughput identification and quantification of anabolic steroid esters by atmospheric solids analysis probe mass spectrometry for efficient screening of drug preparations. Anal Chem. 86:5649–5655. Geyer H, Parr MK, Koehler K, Mareck U, Schanzer W, Thevis M. 2008. Nutritional supplements cross-contaminated and faked with doping substances. J Mass Spectrom. 43:892–902. Geyer H, Parr MK, Mareck U, Reinhart U, Schrader Y, Schanzer W. 2004. Analysis of non-hormonal nutritional supplements
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for anabolic-androgenic steroids – results of an international study. Int J Sports Med. 25:124–129. Kamber M, Baume N, Saugy M, Rivier L. 2001. Nutritional supplements as a source for positive doping cases? Int J Sport Nutr Exerc Metab. 11:258–263. Kaufmann A. 2012. The current role of high-resolution mass spectrometry in food analysis. Anal Bioanal Chem. 403:1233–1249. Krug O, Thomas A, Walpurgis K, Piper T, Sigmund G, Schänzer W, Laussmann T, Thevis M. 2014. Identification of black market products and potential doping agents in Germany 2010–2013. Eur J Clin Pharmacol. 70:1–9. Martello S, Felli M, Chiarotti M. 2007. Survey of nutritional supplements for selected illegal anabolic steroids and ephedrine using LC-MS/MS and GC-MS methods, respectively. Food Addit Contam: Part A. 24:258–265. Matuszewski BK, Constanzer ML, Chavez-Eng CM. 2003. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC−MS/MS. Anal Chem. 75:3019–3030. Parr MK, Geyer H, Reinhart U, Schänzer W. 2004. Analytical strategies for the detection of non-labelled anabolic androgenic steroids in nutritional supplements. Food Addit Contam: Part A. 21:632–640. Parr MK, Koehler K, Geyer H, Guddat S, Schänzer W. 2008. Clenbuterol marketed as dietary supplement. Biomedl Chromatogr. 22:298–300. Strano-Rossi S, Odoardi S, Castrignanò E, Serpelloni G, Chiarotti M. Forthcoming 2014. Liquid chromatographyhigh resolution mass spectrometry (LC-HRMS) determination of stimulants, anorectic drugs and phosphodiesterase 5 inhibitors (PDE5I) in food supplements. J Pharm Biomed Anal. doi:10.1016/j.jpba.2014.06.011 Van Poucke C, Detavernier C, Van Cauwenberghe R, Van Peteghem C. 2007. Determination of anabolic steroids in dietary supplements by liquid chromatography–tandem mass spectrometry. Anal Chim Acta. 586:35–42. Van Thuyne W, Delbeke FT. 2004. Validation of a GC-MS screening method for anabolizing agents in solid nutritional supplements. Biomed Chromatogr. 18:155–159. Wada Anti-Doping Code. 2015. https://wada-main-prod.s3. amazonaws.com/resources/files/wada-2015-world-anti dopingcode.pdf
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry a
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Sara Odoardi , Erika Castrignanò , Simona Martello , Marcello Chiarotti & Sabina Stranoa
Rossi a
Institute of Public Health, Forensic Toxicology Laboratory, Università Cattolica del S. Cuore, Rome, Italy Accepted author version posted online: 26 Feb 2015.Published online: 04 Mar 2015.
Click for updates To cite this article: Sara Odoardi, Erika Castrignanò, Simona Martello, Marcello Chiarotti & Sabina Strano-Rossi (2015): Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1014868 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1014868
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1014868
Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry Sara Odoardi, Erika Castrignanò, Simona Martello, Marcello Chiarotti and Sabina Strano-Rossi* Institute of Public Health, Forensic Toxicology Laboratory, Università Cattolica del S. Cuore, Rome, Italy
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(Received 19 November 2014; accepted 30 January 2015) A sensitive method for the identification and quantification of anabolic steroids and clenbuterol at trace levels in dietary supplements by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in atmospheric pressure ionisation (APCI) mode using a single-stage Orbitrap analyser operating at a resolution power of 100 000 full width at half maximum (FWHM) was developed and validated. A total of 1 g of dietary supplement was added with testosterone-d3 as internal standard, dissolved in methanol, evaporated to dryness, diluted in sodium hydroxide solution and extracted with a mixture of pentane/ethyl ether 9:1. The extract was directly injected into the LC-HRMS system. The method was fully validated. Limits of detection (LODs) obtained for anabolic androgenic steroids (AASs) varied from 1 to 25 ng g−1 and the limit of quantitation (LOQ) was 50 ng g−1 for all analytes. The calibration was linear for all compounds in the range from the LOQ to 2000 ng g−1, with correlation coefficients always higher than 0.99. Accuracy (intended as %E) and repeatability (%CV) were always lower than 15%. Good values of matrix effect and recovery were achieved. The ease of the sample preparation together with a fast run time of only 16 min permitted rapid identification of the analytes. The method was applied to the analysis of 30 dietary supplements in order to check for the presence of anabolic agents not labelled as being present in these supplements. Many AASs were often detected in the same sample: indeed, androstenedione was detected in nine supplements, 5-androsten-3β-ol-17-one (DHEA) in 12, methandienone in three, stanozolol in one, testosterone in seven and testosterone esters in four of them. A retrospective analysis of suspected compounds not included at the beginning of the method development was also possible by means of the full acquisition spectra obtained with the HRMS technique. Keywords: dietary supplements analysis; anabolic agents; drug testing; LC-HRMS
Introduction Dietary supplements are products intended to supplement nutrients, theoretically present in a normal and balanced diet, such as vitamins, minerals, amino acids and herbs extracts. They are generally in the form of tablets, capsules, powders, energy bars or as oily pearls for oral consumption. All sectors of the population, especially sportsmen at all levels, from amateurs to professional athletes, are often tempted to take dietary supplements in order to improve their performance and/or to gain mass and increase muscle growth. Thus, the spread of supplements, also coming from the black market, has increased dramatically in the last decades (Geyer et al. 2004, 2008; Baume et al. 2006; Parr et al. 2008). The easy availability of supplements through websites, supplements shops or in gyms, where it is possible to buy these products without any prescriptions and clinical approval, has contributed to their spread. In recent years it has been reported that products marketed as dietary supplements may contain non-labelled substances, like clenbuterol (Parr et al. 2008) and anabolic androgenic steroids (AASs) (Kamber et al. 2001; Delbeke et al. 2002; Geyer et al. 2004, 2008; Baume et al. 2006). This discrepancy between the label and the content of preparation might be the result of poor manufacturing practice, but in most cases it is deliberate
adulteration of products by the manufacturers in order to obtain more marked effects. These substances, which are originally produced and sold for the clinical treatment of various diseases, can expose healthy consumers to risks and the consequences of their misuse might be more dangerous, especially for adolescents. Their intake might also lead to an inadvertent doping offence, as the use of anabolic agents is severely banned by the World Anti-Doping Agency (WADA). Therefore, the development of methods able to screen for a high number of possible anabolic agents in supplements with adequate sensitivity is of great interest. The analysis of food supplements requires sample pretreatment in order to have them in the right physical state for analysis, to reduce interferences present in the matrix and to detect low concentrations of analytes. The techniques used till now for these purposes involved a dissolution in a solvent (Van Poucke et al. 2007; Doué et al. 2014; Krug et al. 2014) followed sometimes by liquid– liquid extraction (LLE) (Geyer et al. 2004; Baume et al. 2006; Martello et al. 2007; Strano-Rossi et al. Forthcoming), otherwise by SPE (Becue et al. 2011) or a filtration (Cho et al. 2014). The subsequent detection step of AASs is mostly performed by GC-MS (Kamber et al. 2001; Delbeke et al. 2002; Geyer et al. 2004; Baume et al.
*Corresponding author. Emails: [email protected] and [email protected] © 2015 Taylor & Francis
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2006; Parr et al. 2008) or LC-MS/MS in ESI (Van Poucke et al. 2007; Cho et al. 2014) and atmospheric pressure ionisation (APCI) mode (Martello et al. 2007; Doué et al. 2014). The dissolution of solid matrix in sodium hydroxide and successive extraction by pentane/diethyl ether before the derivatisation for obtaining trimethylsilyl (TMS) derivatives to analyse by GC-MS was described by Delbeke et al. (2002) and Van Thuyne and Delbeke (2004). Parr et al. (2004) added a mixture of diisopropylamino-n-alkanes (DIPAs) to the final extract before the GC-MS analysis of TMS derivative. A recent study on 337 products from the German black market was performed screening using either a target LC-MS/MS method, a LC-QTOF-HRMS method or a GC-QTOFHRMS method after derivatisation (Krug et al. 2014). Recently, Kaufmann (2012) reported on the increasing role of high resolution mass spectrometry (HRMS) in food analysis, specifically for pesticides and veterinary drugs. The main mentioned advantages were the acquisition of full-scan spectra, which allows the measurements of compounds without previous compound-specific tuning, the possibility of retrospective data analysis, and the capability of performing structural elucidations of unknown or suspected compounds. For these reasons, we developed a sensitive method by LC-HRMS in APCI mode for the screening of AASs and clenbuterol (a β-agonist with anabolic activity) in dietary supplements. The method was validated for some specific anabolic agents, allowing their forensic identification and quantification.
Materials and methods Chemicals and reagents Formic acid, sodium hydroxide, ultrapure water and methanol were purchased from Sigma-Aldrich (Milan, Italy). All solvents used were LC-MS grade. 1,4-Androstadien-17β-ol-3-one (boldenone), boldenone propionate, boldenone undecylenate, 4,9,11-estratrien17β-ol-3-one (trenbolone), 5-estren-3-β,17β-diol, 4-androsten-17β-ol-3-one (testosterone) decanoate, testosterone cypionate, testosterone undecanoate, testosterone phenylpropionate, 4-estren-17β-ol-3-one (nandrolone) laurate, 4-estren3,17-dione (norandrostenedione), 1,4-androstadien-17αmethyl-17β-ol-3-one (methandienone), 5α-androstan-1αmethyl-17β-ol-3-one (mesterolone), 1(5α)-androsten-17methyl-17β-ol-3-one (methenolone), methenolone enanthate, methenolone acetate, 5α-androstan-17αmethyl-17β-ol (stanozolol), 1,4-androstadien-3,17-dione (boldione), 5-androsten3β-ol-17-one (DHEA), 5α-androstan-17β-ol-3-one (DHT), 4-androsten-3β,17β-diolo (4-androstendiol), 4-androsten3α,17β-diol, 4-androsten-3β,17α-diol, 5α-androstan3α,17β-diol (3αAdiol), 5α-androstan-3β,17β-diol (3βAdiol), 5-androsten-3β-ol-7,17-dione (7-keto-DHEA), 5-androstene3,17-dione, 5(10)-estren-3β,17β-diol, 5-androsten-
3β,17β-diol, 4-androsten-3,17-dione (androstenedione) were from Steraloids (Newport, RI, USA). Clenbuterol, 4-androsten-17α-ol-3-one (epitestosterone), testosterone enanthate, nandrolone decanoate and testosterone-d3 were from Sigma-Aldrich (St. Louis, MO, USA). Testosterone, testosterone propionate, testosterone acetate and nandrolone were from Fluka (Sigma-Aldrich, Milan, Italy). Individual methanolic solutions of each standard were prepared from the pure powders at a concentration of 1 mg ml−1 and stored at −20°C according to the manufacturers’ specifications. A working solution mixture, at a concentration of 1 μg ml−1 in methanol, was then prepared by diluting stock solutions in methanol and stored in a freezer at −20°C.
Sample preparation Aliquots of nutritional supplements in powders, oily capsules, energy bars and tablets were collected in glass tubes and kept refrigerated at 4°C in the dark in order to avoid photo-degradation. A total of 1 g of the sample was weighted, the internal standard (IS) testosterone-d3 was added to the samples at a concentration of 100 ng g−1 and 5 ml of methanol were added. The specimen was then vortexed for 20 s and centrifuged for 10 min at 4000 rpm. The methanolic phase was evaporated to dryness under nitrogen flow at 40°C and reconstituted in 2 ml of a solution of sodium hydroxide 1 M. An LLE was performed through the addition of 5 ml of a mixture of pentane/ethyl ether 9:1, under agitation for 1 h. After centrifugation, the organic phase was evaporated to dryness under a nitrogen stream at RT and reconstituted with methanol/water/formic acid 6:4:0.03 v/v (100 μl) in a glass vial. A total of 10 μl was then directly injected in the LC-HRMS system.
LC-HRMS Equipment The LC-HRMS system was composed of a Thermo ULTIMATE 3000 system equipped with an analytical column Thermo Acclaim RSLC 120 C18 (2.1 mm × 100 mm, 2.2 μm particle size), coupled to a Thermo single-stage Orbitrap (Exactive) MS system. The whole equipment and the column were provided by Thermo Fisher Scientific (Milan, Italy).
LC-HRMS conditions Mobile phase A was a mixture of ultrapure water/methanol 90/10 v/v with 0.1% formic acid; mobile phase B was a solution of methanol with 0.1% formic acid. The analytical column was maintained at 40°C and sample injection volume was 10 μl. The flow rate was set at 400 μl min–1. Mobile phase gradient was as follows: 60% A for 1 min, linear gradient to 100% B in 7 min, held for 5.0 min,
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Food Additives & Contaminants: Part A column re-equilibration was performed with linear gradient to 60% A in 3.0 min, held for 3.0 min. Ionisation parameters were optimised with direct infusion of testosterone-d3 standard by using external syringe. The optimised tune file was then used to develop the screening method and to analyse the samples. The APCI source was heated at 400°C. Source current was 6 µA, sheath gas and auxiliary gas (either nitrogen) flow rates were 35 and 15 arbitrary units, respectively; capillary temperature was 290°C. During the method setup an HESI-II Ion Max source was also tested in order to choose the better ionisation conditions. Data were acquired in full-scan mode over a mass range of 110–800 m/z. The instrument operated in positive ion mode with a resolving power of 100 000 full width at half maximum (FWHM). In case of confirmation of screening positive results, a further set of experiments was performed with in source collision-induced dissociation (CID) with voltage set at 40 V, acquiring ions from 70 to 500 m/z, with a resolving power of 50 000 FWHM, obtaining the accurate masses of both precursor and fragment ions. Experiments for the determination of the better conditions for in-source CID fragmentation were performed at different voltage settings (20, 30 and 40 V). A 40 V CID showed optimal fragmentations for all the steroids, giving higher signals for the characteristic fragments. Mass calibration was performed according to the guidelines provided by the instrument supplier. The recommended calibration solution is made of MRFA (L-methionyl-arginyl-phenylalanylalanine acetate), caffeine and Ultramark® 139 1621 dissolved in methanol/ water (1:1). For calibration purposes, the automatic calibration feature of the Exactive tune software was used. The mass scale was calibrated every 2 days over the mass range m/z 50–2000. Lock-mass (diisooctyl phtalate ionic species, 391.2843 m/z) was utilised during the analysis of samples in order to compensate any possible mass axis drifts. Data analysis For qualitative screening, data were processed using a homemade database containing the protonated exact monoisotopic masses list and built on the extracted ion chromatograms (EIC) of the expected [M + H]+ ions of each compound. Table 1 reports the raw formulas, exact and accurate masses with respective errors and retention times of the analytes investigated. In the case of DHEA, 4-androsten-3α,17β-diol, 4androsten-3β,17α-diol, 5(10)-estren-3β,17β-diol, and 5androsten-3β,17β-diol, the ionic species detectable were those obtained by the loss of one or two molecules of water, as reported in Table 1. A database of analytes was created by analysing pure standard solutions.
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Identification of compounds was based on the following criteria: exact mass of monoisotopic ion M + 0 (mass Þðaccurate massÞ 106 , accuracy, Δm, defined as ðexaxt mass ðaccurate massÞ less than 2 ppm), comparison of experimental and calculated isotopic pattern (relative isotopic abundance – RIA (M + 1/M + 0) and RIA (M + 2/M + 0) errors less than 15%), retention time window (±0.2 min). Due to the possibility of analysing analytes in a nontargeted way, we added in the database the protonated exact monoisotopic masses of other anabolic steroids that could in principle be present as adulterants not included in the phase of development and validation of the present analytical method. The list is reported in Table 2. The full-scan data file of each supplement was further screened for these AASs for which a full validation was not performed. In these cases only a ‘suspect positive’ could be assumed, as there was no comparison with an analytical standard, necessary for a certain identification Xcalibur 2.1 software (Thermo Fisher Scientific Inc., San Jose, CA, USA) was used to analyse and process all data for quantitative analysis.
Method validation The following parameters were taken into account for method validation: specificity and selectivity, LOD, LOQ, linearity, repeatability, accuracy, matrix effect and carryover. Specificity of the method was studied by analysing 10 nutritional supplement samples free of steroids in order to assess potential interferences from other substances. There were different kinds of nutritional supplements: energy bars, tablets and powders (in some cases vanilla and cocoa flavoured) and oily solutions. In case of oily solutions, we studied only some validation parameters (i.e. specificity and LOD), so in case of analyses of oily supplements we could only give qualitative results. Selectivity of the method was studied by analysing samples spiked with various classes of drugs such as stimulants, opiates, antidepressants, benzodiazepines and cannabinoids. The LOD was determined by analysing scalar dilutions (1–5–10–25 ng g−1) of fortified samples on five different typologies of supplements and it was figured out at a concentration value giving an S/N > 3 for the extracted ionic trace. The LOQ was figured out as the lower level of calibration curve (50 ng g−1, that should give an S/N > 10 and an acceptable precision (%CV < 20%) and accuracy (%E < 20%). Linearity was studied in the range from the LOQ of each substance (50 ng g−1) to 2000 ng g−1 on three different types of supplements (powder, energy bars and tablets). Calibration curves were built by weighted linear
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Table 1.
Raw formulas, exact and accurate masses with respective errors and retention times of the studied anabolic agents.
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Compound 4-Androstendiol 4-Androsten -3α,17β-diol 4-Androsten-3β,17α-diol 5(10)-Estren-3β,17β-diol 5-Androsten-3β,17β-diol 3α-Adiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
MH+
Exact mass m/z
Accurate mass (mean m/z)
Δm (ppm)
Rt (min)
C19H31O2 − H2O C19H31O2 − H2O C19H31O2 − H2O C18H29O2 C19H31O2 − H2O C19H33O2 C19H33O2 C19H27O3 C19H27O2 C19H27O2 C22H31O3 C30H45O3 C19H25O2 C12H19Cl2N2O C19H29O2 − 2H2O C19H31O3 C19H29O2 C20H33O2 C20H29O2 C20H31O2 C22H33O3 C27H43O3 C18H27O2 C28H45O3 C30H49O3 C18H25O2 C21H33N2O C19H29O2 C21H31O3 C27H41O3 C29H47O3 C26H41O3 C28H37O3 C22H33O3 C30H49O3 C18H23O2
273.2212 273.2212 273.2212 259.2056 273.2212 275.2369 275.2369 303.1955 287.2006 287.2006 343.2268 453.3363 285.1849 277.0869 253.1951 291.2319 289.2162 305.2475 301.2162 303.2319 345.2424 415.3207 275.2006 429.3363 457.3676 273.1849 329.2587 289.2162 331.2268 413.3050 443.3520 401.3050 421.2737 345.2424 457.3676 271.1693
273.2211 273.2211 273.2211 259.2054 273.2211 275.2367 275.2368 303.1953 287.2003 287.2003 343.2266 453.3366 285.1848 277.0868 253.1949 291.2318 289.2161 305.2474 301.2160 303.2316 345.2423 415.3207 275.2005 429.3367 457.3678 273.1848 329.2585 289.2161 331.2268 413.3052 443.3522 401.3048 421.2739 345.2422 457.3679 271.1692
−0.4 −0.4 −0.4 −0.6 −0.4 −0.7 −0.2 −0.7 −0.9 −0.9 −0.6 0.6 −0.5 −0.3 −0.9 −0.5 −0.2 −0.4 −0.7 −1.0 −0.3 0.01 −0.2 0.5 0.5 −0.4 −0.6 −0.3 −0.5 0.4 0.4 −0.4 0.4 −0.5 0.6 −0.6
7.7 8.5 8.4 7.5 7.8 8.7 8.1 5.5 7.3 7.1 9.3 11.4 6.6 1.1 8.0 8.4 8.2 8.6 7.5 8.2 9.6 11.3 7.3 11.8 12.5 6.9 8.4 7.7 9.3 11.2 12.0 11.0 10.5 9.8 12.4 7.0
regression, considering the area ratio between the analyte and the internal standard, and were prepared in triplicate by adding the proper amount of working mixture to the aliquots of 1 g of nutritional supplement in order to obtain the following concentrations: 50–100–500–1000– 2000 ng g−1. The weighting factor used was 1/x. Repeatability and accuracy, expressed as %CV and %E, respectively, were assessed on control samples prepared at three different concentrations, i.e. 50, 500 and 2000 ng g−1, by quintuplicate analyses on five different supplements (two powders, two tablets and one energy bar) in three different days. Matrix effect and recoveries were investigated for each compound on five blank supplements (two powders, two tablets and one energy bar). According to Matuszewski et al. (2003), absolute recoveries were determined by comparing the signals of quality control (QC) samples (in terms of peak areas), prepared with the proposed method, with those obtained spiking blanks supplements
after extraction at the same concentrations. The matrix effect was studied by comparing the signals of pure standards in acidified water/methanol with those obtained spiking blanks supplements after extraction. Carryover was evaluated through the injections of two blank samples after each calibration level and evaluating the eventual presence of memory effect.
Results The proposed method allows the screening of anabolic agents in nutritional supplements, using a simple and effective sample preparation procedure that guarantees optimal results in terms of sensitivities. LODs obtained varied from less than 1 ng g−1, in the case of 5-androsten3,17-dione, androstenedione, epitestosterone, methenolone, methenolone acetate, nandrolone, testosterone, testosterone acetate, testosterone propionate and trenbolone, to 25 ng g−1 for 3α-Adiol, 3β-Adiol, boldenone
Food Additives & Contaminants: Part A Table 2.
Raw formulas and exact masses of other common anabolic agents.
Compound
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4-Hydroxytestosterone Bolasterone/calusterone/norboletone Boldenone acetate Boldenone benzoate Clostebol Danazol Dehydrochlormethyltestosterone Desoxymethyltestosterone/ethylestrenol Drostanolone enanthate Drostanolone propionate Fluoxymesterone Formebolone Furazabol Gestrinone Methasterone Nandrolone benzoate Nandrolone phenylpropionate Norclostebol Oxabolone Oxabolone cipionate Oxandrolone Oxymesterone Oxymetholone Prostanozol Quinbolone Tetrahydrogestrinone/tibolone Testosterone benzoate Testosterone isobutyrate Testosterone isocaproate Trenbolone acetate Trenbolone enanthate Trenbolone hexahydrobenzylcarbonate Zeranol Zilpaterol
propionate, boldenone undecylenate, DHT, methandienone, nandrolone decanoate, nandrolone laurate, oxandrolone, testosterone decanoate and undecanoate. The APCI source was chosen during method development with respect to the ESI due to the higher sensitivities obtained for some of the steroids. In fact, although signals obtained for pure standards of many of the steroids were comparable with the two ionisation techniques and better for clenbuterol with ESI, the signals of 5-estren-3-β, 17βdiol and mesterolone had a dramatic decrease with ESI, rendering these substances undetectable with this technique. Furthermore, the matrix effect on spiked samples was higher for all the analytes when ESI was used as ionisation source, giving an ion suppression ranging from 87% to 33% at 500 ng g−1. Table 3 reports LODs, LOQs and linearity (mean slopes, intercepts and correlation coefficients obtained on three different supplements) for each substance included in the present study. All the investigated compounds were correctly quantified at 50 ng g−1 level. The method showed good linearity in the range from the LOQ of
MH+
Exact mass m/z
C19H29O3 C21H33O2 C21H29O3 C26H31O3 C19H28ClO2 C22H28NO2 C20H28ClO2 C20H33O C27H45O3 C23H37O3 C20H29FO3 C21H29O4 C20H31N2O2 C21H25O2 C21H35O2 C25H31O3 C27H35O3 C18H26ClO2 C18H27O3 C16H39O4 C19H31O3 C20H31O3 C21H33O3 C25H39N2O2 C24H33O2 C21H29O2 C26H33O3 C23H35O3 C26H42NO3 C20H25O3 C25H35O3 C26H35O4 C18H27O5 C14H20N3O2
305.2111 317.2475 329.2111 391.2268 323.1772 338.2115 335.1772 289.2526 417.3363 361.2737 337.2174 345.2060 331.2380 309.1849 319.2632 379.2268 407.2581 309.1616 291.1955 295.2843 307.2268 319.2268 333.2424 399.3006 353.2475 313.2162 393.2424 359.2581 416.3159 313.1798 383.2581 411.2529 323.1853 262.1550
each substance to 2000 ng g−1 of nutritional supplement. We used a weighted least squares linear regression, with 1/ x as a weighting factor. The appropriateness of the linear regression model was assessed by examination of the residual plots and by evaluation of percentage relative errors versus concentrations (Almeida et al. 2002). In case of supplements containing substances at concentration above the quantitation range, a lesser amount of supplement (50 mg instead of 1 g) was submitted to the analytical protocol. Also accuracy, intended as mean relative error and inter- and intra-day repeatability, was acceptable (always lower than 15%). Data on mean inter-day repeatability and accuracy (n = 15) are shown in Table 4. No interferences from the matrix were encountered at the retention times of the analytes. Figure 1 shows the layout of a mixture of blank nutritional supplements sample spiked with the analytes of interest at a concentration of 500 ng g−1. Other sample preparation procedures, such as ‘dilute and shoot’ with methanol and acidified methanol/water, have been tried in order to optimise the method, but the step
4-Androstendiol 4-Androsten -3α,17β-diol 4-Androsten -3β,17α-diol 5(10)-Estren -3β,17β-diol 5-Androsten -3β,17β-diol 3α-Adiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
Compound 5 10 10 10 10 25 25 5 1 10 25 25 10 5 5 25 1 5 25 1 1 5 1 25 25 5 25 5 1 10 25 10 5 1 25 1
LOD (ng g–1) 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50
LOQ (ng g–1) 0.0005 ± 0.0001 0.0005 ± 0.0002 0.0003 ± 0.0001 0.0045 ± 0.0005 0.0138 ± 0.0034 0.0002 ± 0.0001 0.0004 ± 0.0001 0.0024 ± 0.0005 0.0127 ± 0.0029 0.0018 ± 0.0008 0.0005 ± 0.0001 0.0013 ± 0.0002 0.0049 ± 0.0006 0.0032 ± 0.0006 0.0142 ± 0.0024 0.0033 ± 0.0003 0.0082 ± 0.0008 0.0019 ± 0.0004 0.0015 ± 0.0001 0.0075 ± 0.0015 0.0049 ± 0.0001 0.0011 ± 0.0001 0.0066 ± 0.0011 0.0005 ± 0.0001 0.0005 ± 0.0001 0.0073 ± 0.0018 0.0621 ± 0.0048 0.0038 ± 0.0008 0.0065 ± 0.0003 0.0017 ± 0.0001 0.0006 ± 0.0001 0.0016 ± 0.0002 0.0035 ± 0.0006 0.0066 ± 0.0002 0.0004 ± 0.0001 0.0068 ± 0.0006
Slope ± SD
Table 3. LODs, LOQs and linearity (mean slopes, intercepts and correlation coefficients with RSDs, n = 5).
Downloaded by [Northeastern University] at 03:45 11 March 2015
0.0019 ± 0.0050 0.0000 ± 0.0044 −0.0017 ± 0.0041 0.0007 ± 0.0017 −0.0559 ± 0.0433 0.0007 ± 0.0024 −0.0017 ± 0.0023 −0.0838 ± 0.0545 −0.0512 ± 0.0728 −0.0041 ± 0.0062 −0.0006 ± 0.0003 0.0008 ± 0.0014 0.0029 ± 0.0339 −0.7505 ± 1.2792 −0.0225 ± 0.0581 −0.1171 ± 0.1931 −0.1081 ± 0.3451 −0.1319 ± 0.2257 −0.0358 ± 0.0504 −0.0171 ± 0.0151 −0.0103 ± 0.0006 0.0619 ± 0.1703 −0.0427 ± 0.2761 0.0084 ± 0.0535 −0.0161 ± 0.0166 −0.1612 ± 0.2970 0.0010 ± 0.0013 −0.0460 ± 0.0318 −0.0005 ± 0.0211 0.1928 ± 0.4410 −0.0213 ± 0.0290 0.0643 ± 0.1776 −0.0579 ± 0.1295 −0.4900 ± 0.6912 −0.0164 ± 0.0372 −0.1395 ± 0.1012
Intercept ± SD
0.9952 ± 0.0039 0.9936 ± 0.0015 0.9943 ± 0.0021 0.9941 ± 0.0004 0.9956 ± 0.0028 0.9929 ± 0.0026 0.9933 ± 0.0028 0.9921 ± 0.0017 0.9964 ± 0.0029 0.9945 ± 0.0024 0.9940 ± 0.0004 0.9923 ± 0.0027 0.9928 ± 0.0023 0.9940 ± 0.0013 0.9931 ± 0.0028 0.9918 ± 0.0013 0.9928 ± 0.0024 0.9939 ± 0.0033 0.9935 ± 0.0026 0.9933 ± 0.0015 0.9918 ± 0.0009 0.9922 ± 0.0008 0.9930 ± 0.0024 0.9914 ± 0.0007 0.9992 ± 0.0025 0.9932 ± 0.0031 0.9951 ± 0.0056 0.9949 ± 0.0026 0.9913 ± 0.0017 0.9925 ± 0.0016 0.9908 ± 0.0009 0.9913 ± 0.0007 0.9916 ± 0.0016 0.9915 ± 0.0001 0.9916 ± 0.0014 0.9940 ± 0.0006
r2 ± SD
6 S. Odoardi et al.
4-Androstendiol 4-Androsten-3α,17β-diol 4-Androsten-3β,17α-diol 5(10)-Estren-3β,17β-diol 5-Androsten-3β,17β-diol 3αAdiol 3β-Adiol 7-Keto-DHEA Androstenedione Boldenone Boldenone propionate Boldenone undecylenate Boldione Clenbuterol DHEA DHT Epitestosterone Mesterolone Methandienone Methenolone Methenolone acetate Methenolone enanthate Nandrolone Nandrolone decanoate Nandrolone laurate Norandrostenedione Stanozolol Testosterone Testosterone acetate Testosterone cypionate Testosterone decanoate Testosterone enanthate Testosterone phenylpropionate Testosterone propionate Testosterone undecanoate Trenbolone
Compound
−10 10 11
12 13 12 13 11 6 13 12 3 13 −9 2 14 8 9 2 3 −0.1 14 10 14 12 6 14 10 −8 10 14 −5 7 9 8 −0.1
%E (n = 15)
4 13 5
5 9 12 9 11 12 10 5 9 10 12 10 5 8 8 12 14 11 11 10 11 3 7 4 14 12 12 12 11 7 3 6 12
%CV (n = 15)
80 80 67
70 86 81 48 68 78 88 79 56 53 76 64 74 76 80 79 64 81 68 72 84 87 50 87 75 67 65 81 70 88 79 79 82
%ME (n = 5)
50 ng g–1
69 69 64
64 64 75 72 71 73 84 76 73 65 76 71 81 83 88 79 80 86 60 81 85 62 66 65 67 57 63 68 57 61 59 54 68
%R (n = 5)
Table 4. Inter-day repeatability, accuracy, matrix effect and recovery.
5 7 1
8 1 9 3 1 13 5 14 6 3 3 10 1 4 1 9 3 8 11 3 14 14 10 1 14 9 3 7 3 6 1 7 4
−2 −11 −10 −9 −6 −7 −13 −13 −0.4 −9 4 −6 −9 −12 −11 −3 3 −3 −11 −10 10 −3 1 10 3 2 −7 −14 9 6 13 8 13 7 8 −12
%CV (n = 15)
%E (n = 15)
70 69 63
98 78 95 68 53 68 79 75 58 77 57 67 63 87 74 73 61 87 64 86 65 62 40 85 84 77 70 69 62 69 72 68 69
%ME (n = 5)
500 ng g–1
67 59 72
63 64 86 70 67 66 70 79 73 66 75 71 69 70 72 67 70 69 63 76 63 65 63 63 69 58 63 78 54 51 55 50 60
%R (n = 5)
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−8 −6 1
−0.1 1 1 3 1 1 0.4 0.03 1 2 −6 −1 2 1 2 1 −0.4 −0.2 1 −1 −8 −3 1 −3 −3 0.4 −0.01 2 −7 −1 −6 −0.01 −6
%E (n = 15)
11 8 3
3 0.5 2 3 0.2 1 1 4 1 3 10 2 2 4 5 3 1 3 3 1 11 2 2 6 7 1 2 2 10 7 9 2 12
%CV (n = 15)
83 83 62
77 54 84 59 55 65 80 81 61 61 58 60 60 74 76 77 64 78 63 87 75 62 47 89 90 81 80 70 85 83 83 83 83
%ME (n = 5)
2000 ng g–1
57 68 57
60 65 70 61 58 65 66 70 71 64 63 66 63 77 66 76 77 65 71 65 60 63 63 66 78 51 58 62 51 56 67 57 61
%R (n = 5)
Food Additives & Contaminants: Part A 7
8
S. Odoardi et al. 12.37
100
Testosterone undecanoate (Rt = 12.4) Nandrolone laurate (Rt = 12.5)
50 0 100
NL: 2.29E5 m/z = 457.3630-457.3722 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
12.52
NL: 2.47E5 m/z = 443.3476-443.3564 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
11.99
Testosterone decanoate
50 10.19
0 100
11.76
Nandrolone decanoate
NL: 2.64E5 m/z = 429.3320-429.3406 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Relative Abundance
50 0 100
11.42
NL: 1.31E4 m/z = 453.3318-453.3408 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Boldenone undecylenate
50
9.06 9.24
0.50
0 100
10.72
8.85
12.02
12.31
15.28 NL: 5.52E5 m/z = 415.3165-415.3249 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
11.26
Methenolone enanthate 9.94 10.93 11.15
0 100
NL: 9.94E5 m/z = 413.3009-413.3091 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone cypionate 50 0 100
11.02
NL: 4.88E5 m/z = 401.3010-401.3090 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone enathate
50
10.66 10.48
0 100
NL: 1.25E6 m/z = 421.2695-421.2779 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Testosterone phenylpropionate
50 0 100
9.75
Methenolone acetate (Rt = 9.6) Testosterone propionate (Rt = 9.8)
50 0 100
NL: 2.68E6 m/z = 345.2389-345.2459 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
9.58
Testosterone acetate
9.26
NL: 2.56E6 m/z = 331.2235-331.2301 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
9.26
NL: 2.03E5 m/z = 343.2234-343.2302 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
50 Relative Abundance
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50
0 100
Boldenone propionate
50 9.60 0 100
10.65 10.93
Mesterolone (Rt = 10.9)
50
NL: 1.70E6 m/z = 305.2444-305.2506 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.62 9.96
0 100
11.35
8.44
Stanozolol (Rt = 8.4)
NL: 7.98E5 m/z = 329.2554-329.2620 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
50 10.26 9.88
0 100
12.09 12.80
NL: 2.52E6 m/z = 291.2290-291.2348 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
DHT (Rt = 9.9)
50 8.38
9.55
0 0
1
2
3
4
5
6
7
8
9
10.16 10.69 11.75 10
11
12
13
14
15
16
Time (min)
Figure 1. (colour online) Extracted ionic chromatogram of a blank nutritional supplement spiked with the analytes of interest at a concentration of 500 ng g−1.
Food Additives & Contaminants: Part A 8.17
100
NL: 2.62E6 m/z = 303.2289-303.2349 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Methenolone (Rt = 8.2) 50 9.68
0 100
10.64
8.70
10.65 11.75
9.49 0 100
Relative Abundance
7.68
NL: 4.91E5 m/z = 273.2185-273.2239 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.38 8.53
11.61 NL: 4.92E5 m/z = 253.1926-253.1976 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.96
DHEA
50 7.68 0 100
8.53 8.17 7.71
Testosterone (Rt = 7.7) Epitestosterone (R = 8.2)
50 0 100
NL: 3.59E6 m/z = 289.2133-289.2191 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1 NL: 2.33E5 m/z = 259.2030-259.2082 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.54
5 (10)-estren-3β, 17β-diolo (Rt = 7.5)
50 9.88 10.36 11.00 11.48 7.83 8.96 9.60 7.45
0 100
14.07 14.24 NL: 4.77E5 m/z = 301.2132-301.2192 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Menthandienone (Rt = 7.4) 50 8.16 0 100
9.37 9.77 10.41 10.69
7.33
Boldenone (Rt = 7.1) Androstenedione (Rt = 7.3)
50
NL: 4.65E6 m/z = 287.1977-287.2035 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.09 0 100
NL: 2.21E6 m/z = 275.1978-275.2034 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
7.32
Nandrolone
50 0 100
NL: 1.63E6 m/z = 271.1666-271.1720 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
6.94
Trenbolone (Rt = 6.9)
50 Relative Abundance
Downloaded by [Northeastern University] at 03:45 11 March 2015
0 100
14.12 14.58 15.43
7.78
4-androstendiol (Rt = 7.7) 5-androsten-3β, 17β-diol (Rt = 7.8) 4-androstene-3β, 17α-diol (Rt = 8.4) 4-androstene-3α, 17β-diol (Rt = 8.5)
50
NL: 1.03E5 m/z = 275.2341-275.2397 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
8.10
3βAdiol (Rt = 8.1) 3αAdiol (Rt = 8.7)
50
9.99
6.61 6.87
0 100
9
NL: 2.43E6 m/z = 273.1822-273.1876 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
Norandrostenedione
50 7.32
0 100
NL: 1.13E6 m/z = 285.1820-285.1878 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
6.58
Boldione (Rt = 6.6)
50 5.48 0 100
7.08
8.05
10.15 11.01 NL: 4.67E5 m/z = 303.1925-303.1985 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
5.47
7 keto-DHEA
50 0 100
NL: 4.00E5 m/z = 277.0841-277.0897 F: FTMS {1,1} + p APCI corona Full ms [110.00-800.00] MS 121205__500ng_g_smart_stop_1
1.07
Clenbuterol
50 10.69 0 0
Figure 1.
1
2
Continued.
3
4
5
6
7
8 Time (min)
9
10
11.01 11.44 11
12
13
14
15
16
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10
S. Odoardi et al.
with sodium hydroxide and the following extraction were fundamental for the method in order to have a cleaner extract and therefore to lower the ion suppression, allowing to obtain satisfactory sensitivities and to reduce matrix effect. The results of the mean matrix effect at three different concentrations are listed in Table 4, with a value of 100% indicating no matrix effect, < 100% indicating signal suppression and > 100% indicating signal enhancement. As expected due to the complexity of a multi-component matrix, a matrix effect was observed for some analytes. The highest signal suppression was observed for nandrolone at 60%. Nevertheless, all the substances could be detected at ng mg–1 levels also for those analytes showing higher matrix effect. Recoveries were satisfactory, ranging from 51% to 86% for all the analytes. No significant degradation of analytes or hydrolysis of esters was observed during sample preparation. Only oxandrolone gave very variable results, probably due to its degradation, with unacceptable validation results. Carryover was observed only in the first blank sample after the injection of the higher calibration level (2000 ng g−1) for testosterone undecanoate, decanoate, phenylpropionate and nandrolone laurate, in an amount under the LOQ and therefore not quantifiable. In these cases three blank samples were injected afterwards, and a complete decontamination of the system was ensured by the complete negativity of the third blank injection. The fragmentation pattern of the analytes was used further to confirm positive cases by the exact masses of the characteristic fragments in addition to their retention times, accurate masses by evaluating Δm, and isotopic cluster, by the comparison between experimental and calculated RIA (M + 1/M + 0) and (M + 2/M + 0) errors. This was obtained by performing a further in source CID experiment and by identifying the compound of interest according to the relative abundance of the generated fragments and their accurate masses with respect to those obtained by a reference standard. This method was used for the analysis of 30 nutritional supplements sold in fitness centres and in websites, collected from police officers in Italy in order to check the eventual presence of unlabelled substances. As reported on the labels, they contained proteins, vitamins and fruit, chocolate or vanilla flavours, DHEA in one supplement and androstenedione in another one. Many unlabelled AASs were detected, often in combination: androstenedione was detected in nine supplements from 190 to 8170 ng g−1 (only in one case it was declared on the label, and was at a concentration far above the linearity range); DHEA in 12 samples (from 115 to 32 000 ng g−1), declared on the label in only one of them, methandienone (from 330 to 8850 ng g−1) in three samples, stanozolol in one supplement (at 25 300 ng g−1), epitestosterone (at 3700 ng g−1), testosterone in seven samples (from a concentration below the LOQ to
40 000 ng g−1), and testosterone esters in four of them (testosterone decanoate at 25 ng g−1, testosterone enanthate at 87 and 170 ng g−1, testosterone phenylpropionate at 10 and 92 ng g−1). These results are summarised in Table 5. The mass spectra obtained at a resolution of 100 000 FWHM from the analysis of a nutritional supplement sample containing DHEA at 32 400 ng g−1, testosterone phenylpropionate at 92 ng g−1 and testosterone enanthate at 170 ng g−1 are shown in Figure 2, along with the comparison with calculated theoretical values. Data files were further reprocessed also for the screening of other anabolic steroids that could be present as adulterant in food supplements and for which we did not perform a validation (e.g. steroids listed in Table 1) by the screening of their exact protonated masses. In one of them we suspected the presence of oxymetholone, in another of testosterone isocaproate, along with other testosterone esters, and in another one of tibolone. A comparison with certified analytical standards would be necessary for their determination. Discussion The issue of contaminated/adulterated nutritional supplements is of great concern, both for public health and for possible doping implications. In fact it may happen that elite athletes, submitted to anti-doping controls, have positive results due to an involuntary intake of prohibited substances present in uncontrolled supplements, an event that is anyway considered a doping offence, according to the article 2.1 of WADA’s Anti-Doping Code (2015). The analysis of supplements for undeclared doping agents is therefore necessary. High sensitivities are key factors in analytical methods in order to detect substances at low amounts that can lead to a doping offence, but mainly to a health hazard for unaware consumers. The present method allows an efficient and rapid determination of about 40 anabolic agents in dietary supplements. The isolation of analytes from complex mixtures such as oily or energy bars after an alkaline treatment with sodium hydroxide and subsequent extraction allows an effective clean-up of the samples leading to a low matrix effect and high sensitivities for all the studies compounds. The use of HRMS, due to its high resolving power, permits an excellent selectivity and sensitivity for the target analytes. The use of the accurate mass and the correspondence of isotopic cluster guarantees the identification of the empirical formula, allowing a reliable screening for a large number of anabolic substances. The efficient chromatography obtained with the present method, with a total run time of 16 min, provides a rapid and effective separation of analytes, guaranteeing also the identification of positional isomers or diastereoisomers. Hence, in the case of isobaric compounds, such as for isomeric androsten-diols, the good chromatographic
Fat burner, energy enhancer
Fat burner, energy enhancer
Expectorant, bronchodilator
Fat burner, energy enhancer
Fat burner, energy enhancer
Fat burner, energy enhancer
Fat burner
DHEA nutritional supplement
Fat burner
Muscular growth enhancer Diuretic Ephedrine nutritional supplement Androstenedione nutritional supplement
Fat burner, energy enhancer
Unknown
Unknown
1
2
3
4
5
6
7
8
9
10 11 12 13
14
15
16
Declared type of supplement
Tablets
Oily solution
Tablets
Capsules Capsules Tablets Capsules
Capsules
Capsules
Capsules
Capsules
Tablets
Capsules
Tablets
Tablets
Capsules
Formulation
Table 5. Substances determined in dietary supplements. AASs found
Ephedrine and caffeine alkaloids, amino acids
Androstenedione 1250 ng g−1 DHEA 310 ng g−1 Epitestosterone 3700 ng g−1 Natural products and extracts, amino acids, DHEA 115 ng g−1 Methandienone 8850 ng g−1 minerals Stanozolol 25 300 ng g−1 Testosterone 2600 ng g−1 Testosterone decanoate 25 ng g−1 Ephedrine, guaifenesin Androstenedione 3080 ng g−1 DHEA 4740 ng g−1 Testosterone < LOQ Ephedrine and caffeine alkaloids, natural Androstenedione 577 ng g−1 DHEA 170 ng g−1 products, minerals Testosterone < LOQ Testosterone phenylpropionate 10 ng g−1 Natural products and extracts, amino acids, Androstenedione 770 ng g−1 DHEA 120 ng g−1 minerals Caffeine alkaloids, natural extracts, amino acids, Androstenedione 190 ng g−1 minerals DHEA 410 ng g−1 Methandienone 640 ng g−1 Natural products, amino acids, vitamins Androstenedione 225 ng g−1 DHEA 117 ng g−1 Methandienone 130 ng g−1 Testosterone < LOQ Testosterone enanthate 87 ng g−1 DHEA Androstenedione 8170 ng g−1 DHEA 32,000 ng g−1 Testosterone < LOQ Testosterone enanthate 170 ng g−1 Testosterone phenylpropionate 92 ng g−1 Ephedrine and caffeine alkaloids, natural products Androstenedione 330 ng g−1 and extracts, amino acids, minerals DHEA 330 ng g−1 Amino acids DHEA 8170 ng g−1 Natural extracts, vitamins DHEA 3650 ng g−1 Ephedrine Androstenedione 3275 ng g−1 Androstenedione Androstenedione >> 30 000 ng g−1 DHEA 4100 ng g−1 Testosterone 40 000 ng g−1 Natural products and extracts, amino acids, Androstenedione 250 ng g−1 minerals Testosterone < LOQ Tibolone Unknown Testosterone propionate Testosterone phenylpropionate Testosterone decanoate Testosterone isocaproate Unknown Oxymetholone
Main compounds declared on the label
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Caffeine Ephedrine, caffeine
Caffeine
Ephedrine, caffeine
Caffeine
Ephedrine, pseudophedrine, caffeine
Ephedrine, caffeine, guaifenesin
Ephedrine, caffeine
Alkaloids found
Food Additives & Contaminants: Part A 11
Relative Abundance
12
S. Odoardi et al. 253.1950
100 80 60 40 20 0 100 80 60 40 20 0
DHEA - 2H2O (C19H25) 254.1983 255.2015 253.1951
Theoretical 254.1984 255.2018 252.6
252.8
253.0
253.2
253.4
253.6
253.8
254.0
254.2
254.4
254.6
254.8
255.0
255.2
255.4
255.6
255.8
256.0
Relative Abundance
421.2737
100 80 60 40 20 0 100 80 60 40 20 0
Testosterone phenylpropionate (C28H37O3) 422.2771 421.3526 421.2737
423.2806
Theoretical 422.2771 423.2804 421.0
Relative Abundance
Downloaded by [Northeastern University] at 03:45 11 March 2015
m/z
421.2
421.4
421.6
421.8
422.0
422.2 m/z
422.4
422.6
422.8
423.0
423.2
423.4
423.6
401.3049
100 80 60 40 20 0 100 80 60 40 20 0
Testosterone enanthate (C26H41O3) 402.3083 403.3115 401.3050
Theoretical 402.3084 403.3117 401.2
401.4
401.6
401.8
402.0
402.2 m/z
402.4
402.6
402.8
403.0
403.2
Figure 2. HR mass spectra obtained from the analysis of a nutritional supplement containing DHEA, testosterone phenylpropionate and testosterone enanthate: experimental (upper) and calculated (lower) isotopic patterns of DHEA-2H2O (C19H25), testosterone phenylpropionate (C28H37O3) and testosterone enanthate (C26H41O3).
separation permits identification of different analytes through their retention times. A further confirmatory analysis can be achieved by fragmenting the compound. The exact masses and the relative abundances of ion fragments, in combination with the accurate mass of the parent compound and with retention time, give a high degree of certainty as to the identity of the detected compound. Furthermore the method allows quantification of analytes with good accuracy and repeatability. The high sensitivity of the method allowed for the identification of undeclared AASs in supplements also in low concentrations (testosterone esters in concentration > 100 ng g−1, androstenedione at 190 ng g−1 or DHEA at 115 ng g−1), that could not have a biological activity. These amounts, however, can have health hazard implications, especially if supplements are taken in high amounts and continually, and can result in a doping offence, due to the high sensitivities required by the WADA for determination of AASs in athletes’ urine samples.
Finally, full scan acquisition enables a future retrospective reanalysis of an acquired data file at any time in case of a further substance to be detected. As an example, during the analysis of seized supplements, compounds not included in the phase of development and validation of the present method were also detected at a later stage. Conclusions Due to the high sensitivity, resolving power and specificity of LC-HRMS, a fast and efficient method was developed for the analysis of anabolic steroids and clenbuterol in nutritional supplements by the use of Orbitrap® technology. The effective sample preparation, efficient chromatography and high resolving power of the HRMS instrument, leading to high specificity, allowed good sensitivities and reliable quantitative results to be obtained for a large number of compounds. The application of the method to real supplements demonstrated its suitability
Food Additives & Contaminants: Part A for the identification of anabolic agents not indicated on the labels. Finally, the great potential of this technique is related to the possibility of reprocessing of previously acquired data files when a further banned/dangerous substance needs to be identified.
Funding This work was performed under the framework of the project ‘Smart-Stop’, funded by the Italian Antidrug Policies Department
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