Anal. Chem. 1999, 71, 4701-4710
Liquid Chromatography-Tandem Mass Spectrometry of Some Anabolic Steroids P. E. Joos*,‡ and M. Van Ryckeghem
SGS Depauw & Stokoe, Department IAC, Keetberglaan 4, B-9120-Melsele
In this paper a procedure is described for the analysis of 36 anabolic steroids, regularly found in kidney fat matrixes. After preparative HPLC is carried out, six fractions, containing the different steroids, were obtained. These fractions were then separated and on-line mass-analyzed, using APcI LC/MS. When used in tandem mass spectrometry (MRM) mode, it is possible to obtain detection limits below 1 ppb for all steroids and below 0.1 ppb for most of them. As such, it is possible to meet the regulations for detection of these anabolic steroids. To verify the influence of matrixes, real-world samples were analyzed in the same manner. This resulted in almost analog detection limits. According to European regulations, the use of anabolic steroids as growth promoters is prohibited.1,2 By the Belgian Instituut voor Hygie¨ne en Epidemiology, 36 anabolic steroids are regularly checked and are considered as harmful to humans. Some of them, such as estradiol, are present in the human body. However, in elevated concentrations, this compound can cause gynecological cancers. All these steroids are characterized by the presence of a cholestane moiety. Some of the compounds considered here, however, do not have this entity but have an analogous working principle, e.g., diethylstilbestrol (DES). This compound, formerly used in estrogenic hormone therapy, is now held responsible for miscarriage in humans. It is also considered to be a potent carcinogen. These side effects are possible as a result of interaction of DNA3 with hydroxylated compounds. As found by FinkGremmels et al,4 Cytochrome P450 induces hydroxylations of testosterone at the 6β, 2R, 7R and 16R positions. However, other interaction mechanisms are possible, as well. However, compounds which are prohibited are sometimes used illegally. In other cases, drugs are used in too high an amount. When animals in which this has been the case are slaughtered and their meat is consumed, they present a possible threat for human health. The tasks of control organizations are then to trace these animals and eliminate them. When those steroids are injected into animals, they are mostly found back in ‡ Current working address: University of Antwerp, Department of Chemistry, Universiteitsplein 1, B-2610-Wilrijk. * Corresponding author. Tel.: 00-32-3-8202384. Fax: 00-32-3-8202376. Email:
[email protected]. (1) EEC Directive 81/602, No. L222/32, 1981. (2) EEC Directive 88/146, No. L 70/16, 1988. (3) Chiarelli, M. P.; Lay, J. O., Jr. Mass Spectrom. Rev. 1992, 11, 447-493. (4) Fink-Gremmels, J.; van Miert, A. S. Analyst (Cambridge, U.K.) 1994, 119, 2521-2528.
10.1021/ac981073s CCC: $18.00 Published on Web 09/09/1999
© 1999 American Chemical Society
Table 1. Preparative HPLC Elution Procedure, Used During Cleanupa time (min) front cut backflush of guard and precolumn elution
reconditioning stop time a
0 2 2.1 7.1 14-15 15-16 17.1 16.5-17.5 17.5-19 20.5-22.5 22.1 22.5-27.1 27.1 27.1 42.1
flow % % fraction (ml/min) H2O CH3OH collected 3 3
25 0
75 100
3 3 3 3 3 3 3 3 3 3 3 3 3
25 25
75 75
25
75
0
100
0 25 25
100 75 75
1 2 3 3 4 5 5 6
For details see Experimental Section.
fat-rich tissues, like kidney fat. This is quite logical since they are very lipophilic. However, a suitable technique is needed, which provides the necessary detection limits, but which can also generally be applied for the detection of all these steroids. It is also a prerequisite that matrix interference is as low as possible. In this paper, we describe a method for the determination of anabolic steroids in kidney fat with APcI LC-MS/MS (MRM), using preparative HPLC as a precleaning step. EXPERIMENTAL SECTION Chemicals. The different anabolic steroids were purchased from Sigma Aldrich (Bornem, Belgium) and from Pasture, Steraloids (Newport, RI). Solvents (methanol, acetonitrile, and water) were HPLC grade and obtained from Lab-Scan Analytical Sciences (Labscan Ltd., Dublin, Ireland), while trifluoroacetic acid was purchased from Aldrich Chemical Co. Inc. (Milwaukee, WI). A Ranger system (L’Air Liquide, Deurne, Belgium) was used to supply nitrogen gas for LC/MS purposes. Argon gas was also purchased from L’Air Liquide. Procedure. A procedure for the extraction of anabolic steroids out of fat matrixes is described in detail in refs 20-22. The (5) Andre´, F.; Le Bizec, B.; Montrade, M.-P.; Maume, D.; Monteau, F.; Marchand, P. Analyst (Cambridge, U.K.) 1994, 119, 2529-2535. (6) Batjoens, P.; De Brabander, H. F.; Smets, F.; Pottie, G. Analyst (Cambridge, U.K.) 1994, 119, 2607-2610.
Analytical Chemistry, Vol. 71, No. 20, October 15, 1999 4701
Figure 1. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 1. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) bZ; (b) E3.
extraction procedure for the fecal and urine blanks is essentially the same (for details: see ref 23). Kidney fat (50 g) was cut into fine pieces and put into a flask. Sodium acetate buffer (70 mL; 0.04 M; pH 5.2) was added, and the mixture was heated to 80 °C (7) Hubbard, W.; Bickel, C.; Schleimer, R. P. Anal. Biochem. 1994, 221, 109117. (8) Schoene, C.; Nedderman, A. N. R.; Houghton, E. Analyst (Cambridge, U.K.) 1994, 119, 2537-2542. (9) Bagnati, R.; Fanelli, R. J. Chromatogr. 1991, 547, 325-334. (10) De Brabander, H. F.; Smets, F.; Pottie, G. J. Planar Chromatogr.sMod. TLC 1988, 1, 369. (11) Shackleton,C. H. L. J. Steroid Biochem. Mol. Biol. 1993, 45, 127-140. (12) Shackleton, C. H. L. J. Chromatogr., Biomed. Appl. 1986, 379, 91-156. (13) Liberato, D. J.; Yergey, A. L.; Esteban, N.; Gomez-Sanchez, C. E.; Shackleton, C. H. L. J. Steroid. Biochem. 1987, 27, 61-70. (14) Esteban, N. V.; Yergey, A. L. Steroids 1990, 55, 152-158. (15) Paulson, J.; Lindberg, C. J. Chromatogr. 1991, 554, 149-154. (16) Poon, G. K.; Jarman, M.; McCague, R.; Davies, J. H.; Heeremans, C. E. M.; Hoeven, R. A. M.; Niessen, W. M. A.; Greef, J. J. Chromatogr. 1992, 576, 235-244. (17) Kobayashi, Y.; Saiki, K.; Watanabe, F. Biol. Pharm. Bull. 1993, 16, 11751178. (18) Ma, Y.-C.; Kim, H.-Y. J. Am. Soc. Mass Spectrom. 1997, 8, 1010-1020. (19) Joos, P.; Van Ryckeghem, M. University of Antwerp (UIA), Antwerp, Belgium; SGS Depauw & Stokoe, Melselc, Belgium. Unpublished work, 1998. (20) Smets, F.; Vandewalle, M. Z. Lebensm.-Unters.-Forsch. 1984, 178, 38-40. (21) Verbeke, R. J. Chromatogr. 1979, 177, 69-84. (22) Smets, F. Benelux Economische Unie, SP/LAB/h (88) 33.
4702 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 2. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids, by MRM (details, see text); fraction 1. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) bZ; (b) E3.
for 10 min. After mixing to homogenity, the residue was extracted with 180 mL of methanol. This methanol fraction was centrifuged (5000 g) for 20 min, and the supernatant was filtrated into a flask of 300 mL. Two-fifths of this extract (i.e., corresponding to 20 g of fat) was put into a separating funnel and washed with 2 × 20 mL of hexane. To this residue, 40 mL of water was added, and the mixture was extracted a second time with 2 × 100 mL of diethyl ether. The collected ether fractions were washed with 10 mL of a sodium carbonate buffer (pH < 10.3) and 2 × 15 mL of water. The ether fraction was evaporated to dryness (rotavapor, temperature should not exceed 40 °C) and dissolved in methanol (3 × 1 mL). This solution was then chromatographed on a preparative HPLC system with an automatic injector, an automatic switching valve, a UV-vis detector, and a fraction collector. The separation was done using a semipreparative C18 column (Ultrasphere ODS (5 µ); 10 mm i.d. × 25 cm) preceded by a guard column (pellicular ODS (C18); 37-53 µm; 4.6 mm i.d. × 3 cm) and a precolumn (MCH-10 cartridge C18; Varian; 10 µ, 4.6 mm i.d. × 3 cm). A slightly altered elution procedure was used and is summarized in Table 1. The different fractions, collected by the fraction collector (steroids, present in the different fractions, are (23) Hendriks, L.; Courteyn, D.; Gielen, B.; Leysens, L.; Raus, J. Proceedings of the EuroResidue II, Conference on Residues of Veterinary Drugs in Food, Veldhoven, The Netherlands, May 3-5, 1993; pp 367-371.
Figure 3. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 2. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) Hex, (b) E2, (c) DE, (d) DES, (e) EE2, (f) E1, (g) Z, (h) MeBol, (i) bNT, (j) FMT, (k) Bol, (l) TB.
Figure 4. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids by MRM (details, see text); fraction 2. For the abbreviations of the names of the anabolic steroids, we refer to Table 2;; (a) Hex, (b) E2, (c) DE, (d) DES, (e) EE2, (f) E1, (g) Z, (h) MeBol, (i) bNT, (j) FMT, (k) Bol, (l) TB.
given in Table 2), were then evaporated to dryness with a Vortex evaporator and redissolved in 50 µL of methanol, following which, another 50 µL of water was added. These six solutions were then used for the analytical HPLC procedure. The obtained recoveries were quite good (>70%). Equipment. A HPLC system HP1100 was equipped with a degasser, quaternary pump, autosampler, and UV/Diode Array Detector, the latter of which was not used during the analysis. For the chromatography part a flow rate of 1 mL/min was used on an Alltima C18 (250 mm × 4.6 mm i.d.; 5 µ, Alltech Europe, Laarne, B). The solvents used were methanol (described in the text as solvent A) and water, acidified with 0.1% trifluoroacetic acid (solvent B). To protect the analytical column, a guard column, having the same stationary phase (7.5 mm × 4.6 mm i.d.; 5 µ, All-Guard Alltech Europe, Laarne, Belgium) was put in front of the analytical column. The exact composition of the mobile phase was dependent on the compound and is given in Table 2. All connections were made using PEEK tubing. Injection volumes for the real-world samples (kidney fat) were 4 µL for standards and 60 µL for the samples. We used 2.0-mL screw top vials Teflon septa, purchased from Varian (Palo Alto, CA) for the standards and 1.1-mL Crimp Top Vials (Varian) for the samples. The LC/
MS apparatus used was a VG Quattro II mass spectrometer (Micromass, Altrincham, UK) with a triple quadrupole analyzer. The Atmospheric Pressure Chemical Ionization (APcI) interface was used in order to insert the eluate from the HPLC into the analyzer. Probe temperature was 600 °C in all cases. The source temperature was set to 150 °C. The other different mass spectrometric parameters (cone voltage, collision energy) are also compound-dependent and are summarized in Table 2. The pressure in the collision cell was always 2.5 × 10-3 mBar. For the MRM parameters an interchannel delay of 0.02 s was used, while the span was set to 0.01 m/z in all experiments. For data processing, the software program MassLynx for Windows NT (Micromass, Altrincham, UK) was used. RESULTS AND DISCUSSION In the past, anabolic steroids were analyzed using GC/MS.5-9 But these procedures are very tedious, since they require sample derivatization. This gives, however, long sample preparation times, and, moreover, not all steroids (e.g., stanozolol) can be derivatized or some can be derivatized but only with or low recoveries. The most routinely used technique is HPTLC (see, e.g., ref 10), but this technique is also very tedious and for some steroids (e.g., Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Table 2. LC/MS/MS (MRM) Conditions of the Different Anabolic Steroids Analyzed steroid
tR (min)/k′ value
taleranol (bZ)
9.4 (k′ ) 6.2)
estriol (E3)
5.6 (k′ ) 3.3)
trenbolone (TB)
12.8 (k′ ) 8.8)
boldenone (Bol)
13.0 (k′ ) 9.0)
fluoxymesterone (FMT)
14.1 (k′ ) 9.8)
β-nortestosterone (bNT)
15.6 (k′ ) 11.0)
methylboldenone (MeBol)
16.2 (k′ ) 11.5)
zeranol (Z)
16.0 (k′ ) 11.3)
estrone (E1)
16.6 (k′ ) 11.8)
ethinylestradiol (EE2)
17.2 (k′ ) 12.2)
diethylstilbestrol (DES)
17.4 (k′ ) 12.4)
hexestrol (Hex)
19.9 (k′ ) 14.3)
estradiol (R/β mixture) (E2)
18.2 (k′ ) 13.0)
dienestrol (DE)
18.0 (k′ ) 12.8)
β-testosterone (bT)
7.1 (k′ ) 4.5)
R-nortestosterone (aNT)
6.6 (k′ ) 4.4)
estranediol (Ed)
8.2 (k′ ) 5.3)
acetoxyprogesterone (AP)
7.2 (k′ ) 4.5)
delmadinone acetate (DMA)
7.4 (k′ ) 4.7)
norgestrel (NG)
7.2 (k′ ) 4.5)
estranediol (Ed)
8.2 (k′ ) 5.3)
R-testosterone (aT)
7.7 (k′ ) 4.9)
methyltestosterone (MT)
8.3 (k′ ) 5.4)
methandriol (MAD)
9.6 (k′ ) 6.4)
MRM transition
collision energy (eV)
relative ratio
20 20 20 15 15 15
20 20 20 15 15 15
0.86 1.00 0.49 0.31 1.00 0.54
Group 2 (A/B 65/35) 271f199 271f253 271f165 287f135 287f121 287f91 337f105 337f131 337f181 275f109 275f145 275f239 301f149 301f121 301f91 305f123 305f189 305f161 157f128 253f157 253f197 279f133 279f159 279f105 269f107 269f135 269f77 135f107 135f77 135f79 255f159 255f133 255f115 267f107 267f121 267f77
20 20 20 15 20 20 25 20 25 20 20 15 10 10 15 20 20 20 25 20 20 15 15 15 10 15 15 10 15 15 18 18 15 20 20 20
20 20 50 10 20 45 50 20 50 20 20 15 15 25 45 20 20 20 25 20 20 15 15 30 40 10 45 15 25 25 15 15 45 20 20 45
1.00 0.56 0.68 0.40 1.00 0.32 1.00 0.72 0.74 1.00 0.33 0.34 0.75 1.00 0.31 0.95 1.00 0.58 0.55 1.00 0.70 1.00 0.81 0.43 1.00 0.99 0.29 1.00 0.43 0.13 1.00 0.34 0.19 1.00 0.85 0.56
Group 3 (A/B 80/20) 289f97 289f109 289f79 275f109 275f145 275f239 243f147 243f91 243f67 313f109 313f79 373f97 403f205 403f181 403f145 313f109 313f91 313f185
20 20 20 20 20 15 20 15 15 20 15 15 15 15 15 20 15 15
20 20 20 20 20 15 20 40 40 20 50 20 20 20 40 20 45 20
1.00 0.84 0.03 1.00 0.43 0.40 1.00 0.77 0.83 1.00 0.93 0.16b 1.00 0.97 0.86 0.99 1.00 0.20
Group 4 (A/B 80/20) 243f147 243f91 243f67 289f97 289f109 289f79 303f97 303f109 303f79 269f159 269f91 269f213
20 15 15 20 20 20 20 20 20 20 20 20
20 40 40 20 20 45 20 20 45 20 45 15
1.00 0.77 0.83 1.00 0.92 0.54 1.00 0.92 0.57 0.65 1.00 0.49
Group 1 (A/B 305f123 305f189 305f161 253f159 253f157 253f197
4704 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
cone voltage (V) 65/35)a
Table 2 (Continued) steroid
tR (min)/k′c value
medroxyprogesterone acetate (MPA)
8.7 (k′ ) 5.7)
progesterone (P)
10.3 (k′ ) 6.9)
trenbolone acetate (TBA)
9.5 (k′ ) 6.3)
megestrol acetate (MegA)
8.6 (k′ ) 5.6)
chlormadinone acetate (CMA)
8.5 (k′ ) 5.5)
melengestrol acetate (MelA)
9.3 (k′ ) 6.2)
norethandrolone (NE)
9.7 (k′ ) 6.5)
chlorotestosterone acetate (ClTA)
7.1 (k′ ) 4.5)
stanozolol (Stan)
4.2 (k′ ) 2.2)
hydroxyprogesterone (HP)
6.4 (k′ ) 3.9)
caproxyprogesterone (CP)
7.9 (k′ ) 5.1)
MRM transition
cone voltage (V)
collision energy (eV)
relative ratio
Group 5 (A/B 80/20) 327f97 327f123 327f91 315f109 315f97 315f79 313f253 313f107 313f91 325f187 325f224 325f175 345f175 345f207 345f43 337f279 337f187 337f294 303f285 303f109 303f79
20 20 15 20 20 15 20 15 20 20 20 20 15 20 15 20 20 20 15 20 20
20 20 45 20 20 45 20 30 50 20 20 20 15 20 30 20 20 15 10 20 45
0.34 1.00 0.25 0.73 1.00 0.38 1.00 0.19 0.26 0.95 0.46/0.97c 1.00 1.00 0.89 0.10 0.47 1.00 0.34 0.42 1.00 0.98
Group 6 (A/B 90/10) 365f305 365f143 365f131 329f95 329f81 329f107 317f109 317f97 317f299 313f109 313f79 313f91
15 20 15 20 20 25 20 20 15 20 20 20
15 20 30 40 35 40 20 20 10 20 45 50
0.29 1.00 0.79 0.42 1.00 0.28 0.82 1.00 0.05 1.00 0.50 0.42
a Solvent A ) CH OH; solvent B ) H O, 0.1 % CF COOH. For further details: see the Experimental Section. b If no norgestrel is present (see 3 2 3 text). c When equal amounts of medroxyprogesterone acetate and melengestrol acetate are present (see text).
stanozolol and boldenone) give very low sensitivity. LC/MS can offer simpler sample preparation. However, thermospray LC/ MS11-16 has only a low sensitivity. APcI LC/MS17,18 has only been used recently for steroids, which are found in biological fluids. As is described in another paper,19 it is possible to analyze all steroids, mentioned in Table 2, with APcI LC/MS, quite in contrast with the other prementioned techniques. However, when these anabolic steroids were analyzed in selected ion monitoring mode (SIR), interpretation of the data became very difficult and, hence, gave bad detection limits. Therefore, we suggested the use of tandem mass spectrometry (MRM) in order to reduce matrix interference. The product-ion spectra of the 36 anabolic steroids are presented in another article.19 Flow Injection Analysis. Flow injection analysis of the different anabolic steroids in MRM mode was done by using the most abundant peaks in the product-ion spectra. Optimization of cone voltage and collision energy was carried out for each compound. The optimal conditions are summarized in Table 2. The conditions are very similar, the cone voltage being around 20 V for most compounds. This is quite logical, since the structures of the anabolic steroids are very alike. However, the protonated molecule was not used as the parent ion for all steroids because of the lower abundance of this ion in the mass spectrum. For these compounds, the cone voltages and collision energies used were a little lower. For example, hexestrol did not give a
protonated molecule in the mass spectrum at all,19 and fragmentation had to happen on the ion with m/z 135. As a general rule, these compounds had a lower sensitivity in MRM mode. The same was true for estranediol and estriol. However, estrogens did not have this property, and a good sensitivity was obtained for most of them, when the [MH - H2O]+ signal was used as the precursor ion. A similar remark can be made for the esterified steroids, where [MH - RCOOH]+ (R ) CH3 or C5H11) was the precursor ion. For identification, three MRM transitions were obtained for each compound. The relative ratios between these transitions are also listed in Table 2 and, as will be stated in a later section, these are an important additional proof. Finally, we observed that a better S/N ratio could be obtained when CF3COOH was added in a 0.1% concentration. The results of this research will also be presented in a future article.19 Aside from a little correction in the chromatographic conditions, this gave no serious problem to the chromatographic separation, as will be stated in the next section. Liquid Chromatography. To analyze real-world samples, it was important to set up a separation procedure for the steroids, to minimize matrix effects. As described elsewhere,20-22 the different anabolic steroids were extracted from the fat matrix with methanol, followed by a second extraction with diethyl ether. The diethyl fractions were washed with a sodium carbonate buffer, Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
4705
Figure 5. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 3. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) Ed, (b) DMA, (c) AP, (d) NG, (e) bT, (f) aNT.
Figure 6. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids by MRM (details, see text); fraction 3. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) Ed, (b) DMA, (c) AP, (d) NG, (e) bT, (f) aNT.
resulting in an extract that could be separated in six fractions by preparative HPLC, on the basis of retention times and the use of a gradient elution (Table 1). In Table 2, the different groups are listed. It should be mentioned that estranediol (Ed) can be present in fractions 3 and 4, since its elution time is very near the cutoff time of fraction 3. Hence, estranediol has to be determined in both fractions. However, loop injections of the different fractions, i.e., without further chromatographic separation, resulted in interference with matrix components still present in the mixture. This was especially true for the last fractions, for which it is expected that matrix interference can occur (see Table 1 for detailed cleanup separation procedure). Therefore, we decided to set up an isocratic HPLC analysis method, since this would decrease the waiting time between two injections. The first and second fraction could be analyzed using CH3OH/H2O (0.1% CF3COOH) 65/35, while for the fractions 3-5 an 80/20 composition of the same solvents was used. Finally, the components in the last fraction were eluted with CH3OH/H2O (0.1% CF3COOH) 90/ 10. Retention times of the different compounds are also listed in Table 2. A problem arose when a test mixture of the last group, containing the four anabolic steroids, was injected. However, only three chromatographic peaks were seen. After a separate injection of the different steroids, the missing compound seemed to be
stanozolol. This is not very surprising, since stanozolol (containing an imidazole moiety) is charged in the pH conditions used (pH ) 2). Consequently, it will interact with the stationary phase, more precisely with the nonderivatized silanol groups, resulting in a large retention time. Therefore, an Alltima column (Alltech Europe) was used. This column has undergone an additional derivatization step for removing all free silanol sites (end-capped). When this column was used, stanozolol eluted as the first peak in the chromatogram, as expected. Another problem existed in the second fraction, where not all of the compounds could fully be separated. As stated in the next section, full separation of all steroids is not necessary under MRM conditions. The epimers R- and β-estradiol even resulted in only one chromatographic peak. However, at the moment, regulations do not require that these two compounds have to be reported separately. Liquid Chromatography/Mass Spectrometry (MRM). Once the chromatographic and mass spectrometric parameters were optimized, coupling was carried out under the same conditions. As a test mixture, six solutions of 5 ng/µL and 500 pg/µL were used, containing those steroids which eluted in each fraction, as mentioned in Table 2. When 2 µL was injected from the six solutions of 5ng/µL (i.e., an injection of 10 ng of each component), all MRM transitions for all the steroids gave a detectable signal
4706 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 7. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 4. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) MAD, (b) MT, (c) aT.
with a S/N ratio of at least 10. However, when the same was done with the 500 pg/µL solution, not all three MRM transitions of each compound gave a signal with a S/N ratio larger than 3. This was especially true for compounds in the second fraction, due to the longer retention times used in that analysis. For example, only two transitions with a S/N ratio higher than 3 could be used for the identification of methyl boldenone and hexestrole. This, however, is not a great problem, since the regulations demand a limit of 2 ppb, i.e., 20 ng in 10 g of kidney fat. Since this amount of fat is available in most cases, detection should not really pose a problem, and these detection limits are highly satisfying. Another problem was the fact that in the third fraction acetoxyprogesterone and norgestrel fell in the same retention area (tR ≈ 7.3 min), and both of them used the same ion, m/z 313, as the parent ion of most transitions. We solved this problem by including transitions, taking the protonated molecule as the parent ion for acetoxyprogesterone. The transition considered here (m/z 373 f m/z 97) is less sensitive, but is still present in a high enough S/N ratio after an injection of 2 ng. The presence of a signal at this transition is a proof of the presence of acetoxyprogesterone in the mixture. Since the regulations only require a semiquantitative measurement for the steroids, determination of the amount of both compounds in the mixture is not required. An additional proof was found in the ratio of the transitions. For example, when the
Figure 8. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids by MRM (details, see text); fraction 4. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) MAD, (b) MT, (c) aT (d) Ed. Ed could also be present in this fraction, because of the small difference between its retention time and the cutoff time of fraction 3.
transition of m/z 373 f m/z 97 is compared to m/z 313 f m/z 109 and equal amounts of both compounds are present, then the ratio of both is 1 to 10. However, when only acetoxyprogesterone is present, this ratio is only 1 to 6. The same is true when a great amount (>500 ng) of the compounds medroxyprogesterone acetate and megestrol acetate was injected. These compounds have nearly the same molecular mass (m/z 327 for medroxyprogesterone and m/z 325 for megestrol acetate) and could not be separated under the chromatographic conditions cited above. When 1 µg of medroxyprogesterone acetate was injected, a clear signal was also obtained from the MRM transitions of megestrol acetate. Raising the resolution for the first quadrupole resulted only in a diminishing of the signal, while a now smaller, but still detectable, signal for megestrol acetate was present. Taking the ratios between the different transitions into account could also solve this problem. When one notes a great amount of medroxyprogesterone acetate (higher than 100 ppb) and, consequently, a signal for megestrol acetate is present, it is advisable to check the ratios, as they have to be in agreement with those mentioned in Table 2. When no megestrol acetate is present in the mixture, the ratio between the transitions m/z 325 f 224 and m/z 325 f Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
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Figure 9. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 5. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) P, (b) NE, (c) TBA, (d) MelA, (e) MPA, (f) CMA, (g) MegA.
Figure 10. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids by MRM (details, see text); fraction 5. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) P, (b) NE, (c) TBA, (d) MelA, (e) MPA, (f) CMA, (g) MegA.
175 should be 1 to 2. When, however, equal amounts of both compounds are present, the ratio between the two transitions approximates unity. The analysis of a standard mixture of the different steroids is summarized in Figures 1, 3, 5, 7, 9, and 11 for each of the six fractions, respectively. Analysis of Some Blank Matrixes. Before analyzing some real-world samples, we prepared some blank samples, to verify the influence of the matrix on the analysis. The whole procedure, used for real samples (i.e., using the same workup procedure by preparative HPLC), was applied on different types of blanks (kidney fat, feces, and urine). The results were satisfying, since only a flat baseline was obtained for all samples and this was true for all fractions. Only for the last fraction a slight elevation and an irregular pattern of the baseline was noticed, but it was not of the order that it would offer any difficulties for further analysis. The fact that more apolar components, which are abundant in the matrixes, are present and that for the last fraction a longer collection time is used, can be held responsible for this phenomenon (see Experimental Section and Table 1). Hence, the elevated baseline and the irregularity can be attributed to these apolar compounds, coming off at higher retention times, and higher methanol content. Analysis of Some Real-World Samples. To evaluate the method, as developed above, we analyzed a series of samples,
which were prepared by adding anabolic steroids in differing amounts to 50 g of melted kidney fat, such that a broad concentration area was covered (0.1-50 ppb). The procedure was then repeated, i.e., a preparative workup procedure by HPLC and a final analysis of the samples with LC/MSMS (MRM). As already mentioned above, the baseline of most fractions was still low, but it was noticed that when samples which had undergone repetitive melting (due to, e.g., use of these fat samples for other experiments), were analyzed the baseline was also higher and more irregular. This can influence the sensitivity, and hence, repetitive melting of these samples should be avoided as much as possible. Again, this baseline effect was more pronounced in the case of the last fraction, in which sensitivity of the compounds was not that limiting. In a few cases, even chromatographic peaks were seen but at a different retention time than is normally expected for the component. Although these signals had a S/N ratio smaller than 3, we like to stress that analysis of the samples was unsatisfactory when only loop injections were used. Therefore, it can be stated that a steroid is present in a sample only when a chromatographic peak is seen at the correct retention time in the three ion chromatograms and the peak has a S/N ratio higher than three. As an example, we depict the analysis of such a sample (Figures 2, 4, 6, 8, 10, and 12). Each chromatogram within a
4708 Analytical Chemistry, Vol. 71, No. 20, October 15, 1999
Figure 11. Analysis of a standard of anabolic steroids (each 5 ng/ µL) by MRM; fraction 6. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) CP, (b) ClTA, (c) HP, (d) Stan.
Figure 12. Analysis of a sample of kidney fat, spiked with a series of anabolic steroids by MRM (details, see text); fraction 6. For the abbreviations of the names of the anabolic steroids, we refer to Table 2; (a) CP, (b) ClTA, (c) HP, (d) Stan.
fraction is the sum of three transitions of the same compound. The different steroids that can be expected in the chromatogram are mentioned in the figures. In the first group (Figure 2), no steroids (i.e., estriol or taleranol) were present in the sample. This could be concluded from the fact that no chromatographic peaks were observed at tR 5.6 (Figure 2a) or 9.4 min (Figure 2b) in, respectively, the first and second chromatogram in Figure 2. The second fraction (Figure 4), in which possibly 12 steroids can be found, is thus subdivided into twelve chromatograms. The presence of four steroids could be revealed, since a chromatographic signal was observed for trenbolone (tR 12.9 min; Figure 4l), estrone (tR 17.2 min; Figure 4f), ethinylestradiol (tR 17.2 min; Figure 4e), and estradiol (tR 18.2 min (as an epimeric mixture; well within the 10% range in retention time); Figure 4b). Concentrations of the different compounds were relatively low (
