Chapter 11

Analysis of Steroids by Liquid Chromatography— Atmospheric Pressure Photoionization Mass Spectrometry
Risto Kostiainena,b and Tiina J. Kauppilab

of Pharmacy, Division of Pharmaceutical Chemistry, University of Helsinki, Finland, and bViikki Drug Discovery Technology Center, Faculty of Pharmacy, University of Helsinki, Finland

Electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the most popular ionization techniques used in liquid chromatographymass spectrometry (LC-MS) for qualitative and quantitative analysis. In ESI, the compounds are ionized in the liquid phase and then gas-phase ions are formed by using a high electric field. Since the compounds are ionized without using thermal energy, ESI normally produces little fragmentation. The method is most suitable for ionization of polar and ionic compounds and is capable of ionizing small molecules as well as large biomolecules. In APCI, the compounds are first vaporized in a heated nebulizer, after which the vapor is ionized by a corona discharge needle. This initiates a complex ionization process, which finally leads to the ionization of the analyte. Because the compounds are vaporized by using thermal energy, only relatively small and stable compounds up to about 1,000–1,500 Da can be analyzed. On the other hand, APCI is more suitable for less polar and neutral compounds than ESI. The ionization of nonpolar compounds with ESI or APCI is often impossible, or is achieved with poor ionization efficiency. Atmospheric pressure photoionization (APPI) has recently been introduced as a new ionization method for LC-MS (1–3). The photoionization detector (PID) has long been used as a detection method for gas chromatography (GC) (4–7), but it has also been applied to LC (8–10). Photoionization at atmospheric pressure has been described in connection with ion mobility spectrometry and mass spectrometry (11–13), but before the work of Bruins et al. (1), it had not been used with LCMS. Two distinct APPI apparatuses have been described by Bruins et al. (1) and Syage et al. (2); both share the same operational principles.

The APPI source presented by Bruins et al. was designed to be compatible with a PE/Sciex triple quadrupole mass spectrometer. The system includes a heated nebulizer

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available from Agilent Technologies and ThermoFinnigan (2. which is directly at the end of the heated nebulizer probe. An electric potential of 1. is shown in Figure 11. but in many cases the sensitivity is enhanced if a dopant is used.2. (1). acetonitrile. The source is similar to the Agilent Technologies APCI source. which in turn is usually between 100 and 300 µL/min. but leaves out solvents generally used in LC.and housing identical to the ones used in the Sciex APCI source. by the heated nebulizer. and water (Table 11.1). have described an orthogonal APPI source. Nitrogen is used as both the nebulizing and the auxiliary gas.6 eV). Schematic diagram of the APPI source designed by Bruins et al. A schematic diagram of the Syagen APPI source is presented in Figure 11.3).1. 11. The main difference between Bruins’ and Syagen’s sources is that in Bruins’ source.1. The photoionization lamp is a commercially available krypton discharge lamp emitting 10-eV photons (with a minor fraction at 10. Syage et al. The source was originally designed to be used without a dopant. together with the solvent. with the commercial name of PhotoMate®.5 kV is applied to the mounting bracket of the discharge lamp. except that the corona discharge needle is replaced by a krypton discharge lamp emitting 10eV photons. The source is dependent on the addition of a dopant. The vapor is swept by nitrogen gas flow into the photoionization region. This includes most analytes. Copyright © 2005 AOCS Press . the photoionization takes place inside a closed tube and in Syagen’s source outside. The Ionization Mechanism The ionization in APPI is initiated by the 10-eV photons emitted by the krypton discharge lamp. as well as the Fig. described in more detail by Bruins et al. The APPI source. The photons can ionize molecules that possess ionization energies (IE) below 10 eV. (1). such as methanol.2–1. A typical flow rate of the dopant is about 1/10 of the solvent flow rate. which is added to the auxiliary gas line and vaporized.

but the ionization efficiency in photoionization techniques can be enhanced by the use of a readily ionizable substance. or otherwise present in the atmospheric pressure ion source. that is. Schematic diagram of the APPI source designed by Syage et al.15) and Copyright © 2005 AOCS Press . In direct photoionization. as the formation of radical cations of the analytes is dependent on the ionization energy of the compound. the use of a dopant had been reported with photoionization-ion mobility spectrometry (PI-IMS) (13. and therefore difficult to ionize using ESI or APCI (14). the analyte forms a radical cation. or abstraction of hydrogen from a protic solvent (Table 11. reaction 3) (2. (3). reaction 2). However. M+! (Table 11.2. which usually are nonpolar molecules. gases used in the nebulization. Charge exchange to another species weakens the signal of the analyte. without dopant.2. a dopant. Possible reactions include charge exchange to species of lower ionization energy (Table 11. reaction 1). which can further react with other gasphase species. Before the development of APPI. Compounds that form protonated molecules via proton transfer usually possess high proton affinities (PA) and could therefore also be ionized using ESI or APCI. The photons typically have a short lifetime. 11.Fig. whereas the formation of a protonated molecule via hydrogen abstraction only transforms the analyte to another ion species and thus does not affect the overall ionization efficiency.2.2. it makes possible the ionization of low-PA compounds.3).

Toluene. Gases.1 Energetics of Typical Solvents. + M D+. N2 Acetonitrile Methanol Water n-Hexane Chloroform Ammonia Acetic acid Formic acid Trifluoroacetic acid aIE.6 783. + D. acetone. the energy of the photons. b PA.37 10. + S (solvent) D (dopant) + 10-eV photons D+.912 — — 1451 12.912 "Gacidd (kJ/mol) 8.13 11. d"G acid . Another possibility is a proton transfer from the dopant radical cation to solvents or analytes that possess proton TABLE 11. benzene.6 10. + e–. cEA.TABLE 11. the dopant radical cation can then react with the analyte via charge exchange (Table 11. and a radical cation (D+!) of the dopant is formed (Table 11.07 10.17).2 9. Copyright © 2005 AOCS Press . PAb (kJ/mol) 784.4 884. gas-phase acidity. + MH+. proton affinity. if IEa (M) < 10 eV if IE (X) < IE (M) if PAb (M+. # # # # # # # # M+. APCI (16). and anisole have been reported as APPI dopants (1.0 — EAc (eV) 0.2 Reactions in Positive-Ion APPI M (analyte) + 10-eV photons M+. + n S D+. electron affinity. MH+ + [S–H]. + M S n H+ + M aIE.0 831. In cases where the IE of the analyte is below that of the dopant.2. + X M+. D+. the photons ionize the dopant first. X+. reaction 5). proton affinity. + M. bPA.0 839.84 12.2 754.4 812.) > PA ([S–H.6 750. all possessing IE below 10 eV. and Modifiers Used in APPI IEa (eV) Toluene Benzyl radical Acetone Anisole Benzene Phenyl radical O2 HO2.243 — 0.2.0 — — 853.622 — — — — 1607 — 1464 — 1429 1415 1328 ionization energy. reaction 4).65 779. + SnH+.2 9. + e–.) if PA (M) > PA (Sn) (1) (2) (3) (4) (5) (6) (7) (8) ionization energy.83 7. MH+ + n S.703 8.2 10. [D–H].3 691.) if PA (M) > PA ([D–H]. In dopant-assisted APPI. M+.7 — — 0. [D–H].]) if IE (M) < IE (D) if PA (Sn) > PA ([D–H].

19). have also been reported to take place in the APPI source. has a PA below that of methanol and acetonitrile solvent clusters and therefore gives up its proton if these solvents are present. the ionization of the analytes via proton transfer is more likely than ionization via charge exchange. and chloroform. Since the reactions depend on the differences in PA and IE of the reacting species. whereas solvents of low proton affinity make possible the ionization of analytes via charge exchange. they can obviously also be affected by the choice of the dopant. Also.2. which may complicate the ionization process.2. but can form solvent clusters that possess PA above the PA of the benzyl radical. In addition to charge exchange and proton transfer reactions. the dopant used most often has been toluene. respectively. Thus. reactions 6 and 7). chloroform. which also has a low IE (8. surfaceinduced reduction processes have been observed to take place in APPI (21). Only in solvents such as hexane. or benzyl radical. The dopant should have an IE below the energy of the photons (10 eV for the krypton discharge lamp). These species have different IE and PA than the original molecules. so that it can produce radical cations. they neutralize the dopant radical cations and charge exchange becomes unlikely (Table 11. Use of solvents having low PA. which complicates the ionization process and can lead to an increase in the proton transfer reaction (20). Recently. Instead. In cases where the proton transfer takes place. does the toluene radical cation stay in the system and make the charge exchange reaction possible (14). Acetonitrile and methanol have PA below that of the benzyl radical (deprotonated toluene radical cation). can react with atmospheric oxygen. acetonitrile has been reported to form a species of lower ionization energy. Therefore. Benzene and toluene. reaction 6) (22).2. or water. this means that with reversed-phase liquid chromatography (RPLC) solvents. reaction 8).affinities above that of the deprotonated dopant radical cation [D–H]. In practice. Solvents of high proton affinity promote ionization of analytes via proton transfer. Because of the nature of APPI. it would be desirable for the solvent to have a PA below that of the dopant in order to ionize nonpolar compounds efficiently by charge exchange. can enhance the charge exchange reaction and increase the efficiency of the ionization of analytes of low PA (14). The deprotonated toluene radical cation. which can then be ionized by the 10-eV photons. the charge exchange reaction is suppressed as the dopant radical cations are depleted and no longer available for charge exchange reactions. forming phenol and cresol. which can be used as dopants. Thus far. (Table 11. the proton transfer to analytes that possess PA above that of the solvent can take place with a high efficiency (Table 11. reaction 8) (14). other reactions.2 eV) and can readily be ionized by Copyright © 2005 AOCS Press . the reactions that take place and the ionization of the analytes can be greatly affected by the solvent. anisole. The resulting species may explain the protonation of the analytes when dopant is not present (18. hexane. in cases where the analyte possesses a PA above that of the solvent (Table 11. such as water. Protonated analyte molecules can also be formed by a proton transfer between the protonated solvent molecules and the analyte. When radiated with photons.2.

) if EA (M) > EA (O2) = 0.451 eV X = H. and an increasing number of competitive reactions at higher flow rate (24).3. Copyright © 2005 AOCS Press . The reactions in negative-ion APPI are far less studied than the reactions in positive-ion APPI.. [M–X+O]– + OX. reaction 1) (14) or from the ion source metal surfaces due to irradiation with photons (25). The situation has been found to be the same with both Agilent and Sciex ion sources. and therefore it does not give up its protons to solvents such as methanol or acetonitrile.. + F– S–. M + O2–. especially when the flow rate is increased above 500 µL/min. + O2. but some phenomena are already known.24).3.the 10-eV photons. which is present in the atmospheric pressure ion source. It has a positive electron affinity and can therefore capture electrons from its surroundings.) if "Gacid (M) < "Gacid (HO2. has been introduced as a new dopant for APPI (17). Cl. such as analytes.. Oxygen. gas-phase acidity. Anisole has a somewhat higher PA than toluene (Table 11. although the Agilent source seems to be less dependent on the flow rate when high-PA analytes are analyzed (24). if EAa (M) > 0 eV 2 [S–H]– + HO2.2. The ionization efficiency in APPI has been observed to be at its best at flow rates in the range 10–300 µL/min (23. + e– M–. NO2 X = H. but instead its radical cation lingers in the system even with these solvents. solvents. nonpolar compounds can be ionized through chargeexchange reaction by using reversed-phase liquid chromatography. O2– ! is TABLE 11. the ionization process is started by a release of an electron by the photoionization of the dopant (Table 11. These low-energy electrons may be captured by species that possess positive electron affinity. M + O2–. loss of photons in collisions with the increased volume of solvent. thereby forming a superoxide ion O2– ! (Table 11. the main reason for the signal drop for low-PA analytes was considered to be the neutralization of the dopant radical cations by solvent or solvent impurities that possess high PA.2). the ionization efficiency decreases. b"G acid. Cl.. NO2 if "Gacid (M) < "Gacid (S) (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) electron affinity. 2 # # # # # # # # # # # D+. With dopant-assisted APPI. M–. Possible reasons for the loss of high-PA analytes include neutralization of ions in recombination with electrons or ions of opposite charge. [M–F]. + O M + [S–H]– aEA. O –. At flow rates above this. In negative-ion APPI.. collisions with source walls.3 The Reactions in Negative-Ion APPI D + h$ M + e– M + e– S + e– O2 + e– S + O2–. or gases (Table 11. has been observed to play a critical role in the APPI source. [M–H]– + HO2. [M–H]– + S. reactions 2–5). [M–X+O]– + OX. M–. M + O2–. reaction 5). if "Gacidb (S) < "Gacid (HO2. The consequence is very practical: With anisole.

better selectivity for these kinds of analytes may be achieved. reaction 11) (14). O2 and O2– ! have also been observed to react with some analytes via oxidation reactions (Table 11. Solvents that possess high electron affinities (chloroform.and disaccharides have also been analyzed successfully with APPI-MS (45). it can react with solvents or analytes via charge exchange. low-PA ions such as HCOO–.g.46–54).50. quinones (36.55.48. Also. This would also explain why the formation of deprotonated analytes is decreased (26). ESI performed best (40.a relatively strong gas-phase base ("Gacid of HO2 ! = 151 kJ/mol. and 11 in Table 11. APPI gave equal or better sensitivity than APCI and was less susceptible to matrix effects than ESI or APCI (23. Deprotonation of the analytes can also take place in a proton transfer between the analyte and a solvent species of lower gas-phase acidity (Table 11. thus leaving an insufficient amount for the analytes that otherwise would form ions via electron capture. Other analytes of environmental interest. Problematic analytes such as mono. reactions 6 and 7).27–30). fungicides (33).35). trifluoroacetic acid. and hydrophobic peptides (41) have been successfully analyzed using APPI as the method of ionization.3. however. In addition. reactions 9 and 10).. such as mycotoxins (42). Analytes that possess higher gas-phase acidities than the solvents can. CH3COO–. be deprotonated. APPI has been found to be an efficient ionization method for a variety of compounds. which thus cannot react via charge exchange with the high-EA analytes. With nonpolar molecules.3. If solvents that possess high gas-phase acidities (e. APCI. In fact. explosives (34. APPI usually showed a much higher sensitivity than ESI or APCI.37). by the use of acidic solvents. Table 11. The suitability of APPI in drug discovery and drug analysis has been examined by several research groups (23.40). but when the analyte was polar. etc. in case the other species possesses an electron affinity above that of O2 (Table 11.56). This would also prevent the formation of superoxide ions from oxygen. and trifluoroacetic acid) are introduced to the ion source. and thus eliminate the possibility of proton transfer between the analytes/solvents and O2– !. Copyright © 2005 AOCS Press . They are thought to deplete the electrons from the system.57).3.42–44. In most of the comparisons between ESI. organic fluorine compounds (43). formic acid. and CF3COO– resulting from the solvent become dominant in the system and prevent the deprotonation of analytes of low gas-phase acidity via reactions 6. Analytes such as polyaromatic hydrocarbons (PAH) (14.3.3) and can therefore accept protons from other species and take part in the deprotonation of solvents and analytes that have higher gas-phase acidities (Table 11. and APPI. and harmful antibiotics (44) have also been analyzed using APPI. they can protonate the superoxide ion. 7. nonpolar vitamins (38). flavonoids (39.3. Applications Because of its suitability to analysis of both nonpolar and polar analytes.) have also been observed to deteriorate the signal of the analytes in negative ion APPI. reaction 8). Negative-ion APPI is also very dependent on the solvent composition. acetic acid. pesticides (31.32).

Also the combination of APPI with APCI has extended the range of compounds that can be analyzed in a single run and increased the analyte signal levels (56). Recently introduced. causing the expression of certain genes. Vitamin D–derived steroid hormones regulate the uptake and metabolism of Ca2+ and phosphate. APCI and ESI have been the most commonly used ionization techniques for LC-MS analysis of steroids. The analysis of steroids by LC-MS has garnered increased interest during recent years. Corticosteroids affect the metabolism of carbohydrates (glucocorticoids) and regulate the concentrations of electrolytes in the blood (mineralocorticoids).27). and steroids. The most important steroids are the adrenocortical hormones. The common method for the analysis of steroids is GC-MS after a derivatization step. The steroids that are present in the brain and whose concentration is independent of peripheral sources are called neurosteroids. peptides. APPI in the Analysis of Steroids Steroids are a highly important class of compounds in biological systems. Because of the low flow rates used in APPI. including the formation and mobilization of calcium phosphate in bone. and during convalescence after chronic debilitating disease. which have very different characteristics and thus could not be ionized simultaneously using ESI or APPI alone (29).Unlike ESI. Anabolic steroids are used to treat cases of protein and bone wasting and osteoporosis. especially with ESI. ESI (29. since the method allows direct analysis without time-consuming sample pretreatment and derivatization. The newest trend in the development of APPI seems to be combining it with other atmospheric pressure ion sources. proteins. Copyright © 2005 AOCS Press . the sex hormones and the vitamin D–derived hormones.61) and APCI (56). it has also been found to be a suitable interface for capillary electrophoresis-mass spectrometry (CE-MS) (56) and it is more tolerant than ESI toward phosphate buffers. So far. as the use of a nonpolar solvent promotes the charge exchange reaction and thus the ionization of nonpolar analytes (14. The androgens and the estrogens are sex hormones and affect sexual development and sexual behavior. and premenstrual syndrome (64). They affect several types of behavior. The use of NP solvents can even be an advantage when the analytes are nonpolar. Steroids are lipid-soluble and readily pass through plasma membranes into the cytosol of target cells.51. APPI can be used under either reversed-phase or normal-phase (NP) liquid chromatographic conditions (14. which are required for good CE separation (49). the ionization efficiency for less-polar steroids may be poor. highly sensitive and selective analysis methods are required. However.58–60). Hanold et al.28. have successfully combined APPI with ESI and have been able to analyze polyaromatic hydrocarbons. The use of anabolic androgenic steroids (AAS) to improve athletic performance is one of the most widespread problems in sports despite the fact that it was banned in 1976. stress (63). such as anxiety (62). Because steroids can exist in biological matrices at very low concentrations.

since it has been shown that ionization efficiency of APPI is significantly better at low flow rates. 6&-hydroxy-4-chloro-dehydromethyltestosterone (HC). M+! being more abundant with water than with methanol for all of the corticosteroids studied. but at higher flow rates APCI is a bit more efficient than APPI (68).08–0. therefore. The ionization efficiency of APPI without dopant was compared to APCI and ESI using a flow rate of 8 µL/min. being somewhat higher than with ESI (0. compared LC-MS using ESI and APPI in the analysis of cortisol (55). and methylprednisolone) by APPI using toluene as the dopant were in agreement with the results presented by Greig et al. The relative abundances of MH+ and M+! were dependent on the solvent. The detection limit with APPI was 20 pg injected on the column. methylprednisolone at 500 ng/mL whereas corticosterone was not detectable even at 1000 ng/mL (40). (40) in the analysis of three glucorticosteroids (corticosterone. A comparison of the analysis of corticosterone. Eluent composition. prednisolone. The detection limits with LC-MS/MS using APPI and APCI were at the same level (0. All the APPI-MS spectra of the analytes detected without the dopant showed MH+ and/or M+!. All the APPI-MS spectra showed protonated analyte molecules and more extensive fragmentation than did APCI and ESI. obviously due to better desolvation of methanol. Leinonen et al. and fragmentation were optimized for ESI. because prednisolone could be detected only at 100 ng/mL. ion source parameters.5 nmol/mL). However. This was clearly lower than with LC-APCI-MS/MS.APPI (1.9 nmol/mL). Copyright © 2005 AOCS Press . (67). the results cannot be generalized to higher flow rates. In this comparison. after which the methods were compared with respect to specificity and detection limit. but its flow rate within a range of 5–25 µL/min did not affect the results. The results obtained using toluene and acetone as dopants were similar. Toluene as the dopant yielded approximately 20–50% higher sensitivity than acetone. The performance of APPI coupled to Fourier transform ion cyclotron resonance MS was compared to APCI and ESI in the analysis of six corticosteroids (67). and oxandrolone (OX) as model compounds. APPI was found to ionize corticosteroids with much higher efficiency than either ESI or APCI. prednisolone. Quenzer et al. Use of methanol as a solvent provided about five times better sensitivity than water. as well as fragments formed by losses of water and neutral hydrogens. The use of dopant was essential for ionization in APPI. which was 2–3 times lower than with ESI.06–0.3. interest in APPI as a method for analysis of steroids has increased. The results presented by Saba et al. (66) studied the applicability of LC-tandem mass spectrometry (LC-MS/MS) to the detection of free anabolic steroids in a fraction of human urine using 3%-hydroxystanozolol (HS). The detection limits obtained for the glucorticosteroids by analysis with LC-APPI-MS/MS were about 20 ng/mL. and methylprednisolone by APPI and APCI is shown in Figure 11. and APPI. However. the use of dopant resulted in changes in signal that ranged from a tenfold increase to a complete signal loss.3) has been shown to be a very powerful method for the analysis of nonpolar neutral compounds (65). APCI.

and dehydroepiandrosterone) and their diacetyl-pentafluorobenzyl (Ac-PFBO) derivatives in spiked rat brain samples. APPI Fig. APPI 1000 ppb. Comparison between APPI and APCI in the detection of prednisolone (1). 11.4).9994) 5400 3600 1800 0 200 400 600 Concentration (ng/mL) 800 1000 Fig.2 (r = 0. Furthermore.34 ' + 53. and corticosterone (3). APCI 50 ppb. APCI 50 ppb.4. (59) compared APPI and APCI in the analysis of four neurosteroids (testosterone. Alary et al.1000 ppb. Calibration curve obtained by atmospheric pressure photoionization for corticosterone. 11. LC-APPI-MS/MS showed good quantitative linearity for all the three glucorticosteroids between 20 and 1000 ng/mL. From Saba et al. prenelonone. 2002 (40). From Saba et al. methylprednisolone (2). 7200 Corticosterone: y – 7. with a correlation coefficient better than 0. progesterone.. 11..995 (Fig. Copyright © 2005 AOCS Press . 2002 (40).3. The sensitivity was better with APPI for nonderivatized compounds.

B. more comparative studies should be done under carefully optimized conditions for each of the ionization techniques. Atmospheric Pressure Photoionization: An Ionization Method for Liquid Chromatography-Mass Spectrometry. Copyright © 2005 AOCS Press . Both ion sources showed similar dynamic range. in negative-ion mode APCI produced a fivefold more intense signal than APPI (51). Limits of detection (on-column) obtained with APPI for Ac-PFBO–derivatized steroids were 90–190 fg. and accuracy.o n e . 1 7&-diol. precision. 72: 3653–3659 (2000). Fernández-Metzler showed that APPI is about 10 times more sensitive for ethynyl estradiol than APCI.whereas APCI showed better sensitivity for Ac-PFBO derivatives. APPI is a very young technique.3 .R. APCI and is often better than the sensitivity obtained by ESI. The performance of APPI and APCI was compared in an LC-MS/MS analysis of 5(. APPI provides reliable quantitative analysis with a wide linear range. which will be developed further as the understanding of the fundamentals of the APPI ionization process increases. most of the studies have been presented at scientific meetings. Bruins. The limits of detection (on-column) with normalphase and reversed-phase LC-APPI-MS/MS were quite comparable.67). Conclusions APPI seems to be a very powerful and versatile ionization technique that overcomes the difficulties associated with APCI and especially with ESI in the analysis of nonpolar neutral compounds such as steroids. On the other hand. as well as good stability. Covey. However.o l . indicating that LCAPPI-MS/MS provides a potential new method for the analysis of neurosteroids. 50/50%) to obtain an intense signal. or at the same level as. Instead. 5(-androstan-17&. Similarly. The MS spectra measured by APPI and APCI were very similar. With both ion sources. and accuracy. only a couple of publications concerning the analysis of steroids with APPI have been presented in refereed scientific journals (66.1 and 10 pg. References 1. In contrast. The use of a dopant (toluene) in APPI was found to be essential in reversed-phase mode (MeOH/H2O. the mass spectra of Ac-PFBO derivatives showed a significantly more intense protonated molecule and less fragmentation and fewer background ions than the nonderivatized neurosteroids. and A. and ethynyl estradiol (58). Chem. Robb. most of the comparative studies concerning steroid analysis by LC-MS show that the sensitivity achieved with APPI is better than. At this time. 94/6%). showing protonated analyte molecules and extensive fragmentation.. being between 0. precision. D. which was 3–20 times lower than with LC-APCI-MS/MS.3 (. However. Furthermore. Anal.a n d r o s t a n . the photoionization process occurred with very good efficiency without any added dopant under normal-phase conditions (isooctane/isopropanol. testosterone. In order to make a realistic conclusion on the performance of APPI in steroid analysis. T.P.

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