LC-MS analysis of steroids

Mass spectrometry of steroids and steroid conjugates 2.1 History 

The analysis of steroids is a challenging task. GC-MS has frequently been applied, but requires analyte derivatization [1]. Therefore, over the years most LC-MS interfaces have been tested or applied in the analysis of steroids. As early as 1981, Henion [2] demonstrated the analysis of dexamethasone and cortisone with micro-LC coupled to a capillary-inlet interface. Van der Greef et al. [3] described the quantitation of progesterone in serum using isotope dilution MS in anunonium Cl mode using the moving-belt interface. Henion and coworkers [4] described 

Nowadays, electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI) are the methods-of-choice in steroid analysis by LC-MS, while the potential of atmospheric-pressure photoionization (APPI) has been evaluated as well. The analysis of steroids by LC-MS is challenging, because of their relatively low polarity and proton affinity. 

While there were already some reports on the LC-MS analysis of steroids [12-18], Ma and Kim [19] reported a comparative study of the performance of APCI and ESI in the analysis of neutral steroids. Both electrospray and APCI were performed at 0.4 ml/min and a 2.1-mm-ID column. Based on mass spectral data and sensitivity, the steroids investigated were classified into three major groups  

B Steroids containing at least one ketone group without conjugation, e.g., estrone and androsterone. 

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The LC-MS analysis of steroids is discussed from a more general perspective in Ch. 13. In environmental analysis, SPE on C,8- or carbon-materials are generally applied for analyte extraction and preconcentration. Gradient elution using 20-100% acetonitrile in water on a Cig-colunm is used in combination with either negative-ion ESI or positive-ion APCI. 

General aspects of the LC-MS analysis of steroids are discussed in detail in Ch. 13. In this section, attention is paid to veterinary residue analysis of steroids. LC-MS has been used at regulatory level in tracing the use of illegal hormones via the analysis of body fluids, e.g., urine, serum, or plasma, and animal tissue, e.g., meat, kidney, liver. 

Dne to their estrogenic activity, steroids have been inclnded in preliminary lists of EDCs. These chemicals are even more diffnsely fonnd in waters, also dne to sensitivities nowadays achieved in advanced LC/MS and LC/MS/MS instrumentations. The interfaces most widely nsed for the LC/ MS determination of steroids, drngs, surfactants, and organic pollntants in an aqnatic environment are ESI, which is particnlarly well suited for the analysis of polar componnds, and APCI, that is more effective in the analysis of medium- and low-polarity snbstances. LC/MS and LC/MS/MS have been mostly applied in the SIM mode and in the MRM mode. 

The applicability of the APCI interface is restricted to the analysis of compounds with lower polarity and lower molecular mass compared with ESP and ISP. An early demonstration of the potential of the APCI interface is the LC-APCI-MS-MS analysis of phenylbutazone and two of its metabolites in plasma and urine (128). Other applications include the LC-APCI-MS analysis of steroids in equine and human urine and plasma (129-131), the determination of six sulfonamides in milk samples after a simple solid-phase extraction and LC separation (132), of tetracyclines in muscle at the 100 ppb level (133), of fenbendazole, oxfendazole, and the sulfone metabolite in muscle at the 10 ppb level, and of five thyreostats in thyroid tissue at the 1 ppm level (134). 

The group of Kostiainen [72-74] reported a number of comparative studies into the performance of APPI relative to APCI and ESI, e.g., in the LC-MS analysis of flavonoids [72], anabolic steroids [73], and naphthalenes [74], A variety of solvents were compared for the toluene-doped APPI of naphthalenes,... 

Preparative LC fractionation of the kidney fat, faeces, and urine extracts into six fractions prior to the LC-MS analysis of 36 anabolic steroids [67],... 

In the only publication on the LC-MS analysis of synthetic steroids or animal samples that is unrelated to any steroid misuse, Magnusson and Sandstrom (2004) reported on a quantitative analysis of eight T metabolites in rat intestine mucosa. 

Steroid profile analysis (simultaneous measurement of various types of steroids) plays an important role in the clinical evaluation of a number of common endocrine disorders in humans and animal models. LC-MS profiles of steroids in bovine adrenal cells, the rat brain, and human serum have been reported during the past five years (Table 7). 

Higashi, T. Ninomiya, Y. IwaM, N. Yamauchi, A. Takayama, N. Shimada, K. Studies on neurosteroids XVin. LC-MS analysis of changes in rat brain and serum testosterone levels induced by immobilization stress and ethanol administration. Steroids 2006, 71 (7), 609-617. 

Abdel-Hamid, M.E. LC-MS analysis of selected sulfur-containing non-steroid antiinflammatory agents Applications to pharmaceutical products, J.Liq.Chromatogr.Rel.Technol., 2000, 23, 3095-8107. [rofecoxib sulindac celecoxib piroxicam tenoxicam]... 

Extensive reviews have been published on the analysis of steroids (e.g. Shimada et al., 2001 Andrew, 2001). GC is still used to measure levels of some volatile steroids, but LC-MS (Shimada et al., 2001) and GC-MS (Kim et al., 2000 Vallee et al., 2000 Purdy et al., 2004 and chapter in this volume) are now used for most analyses because of versatility and selectivity. 

Many methods have been used to quantify steroidal compounds. These include RIA, gas chromatogra-phy-mass spectrometry (GC/MS), high-performance liquid chromatography (HPLC), and liquid chroma-tography-mass spectrometry (LC/MS). Although these techniques are successful in the analysis of steroids, it has been difficult to achieve quantitative analysis of small samples of neurosteroids because of their low concentrations in nervous tissues. Highly specific analytical methods are required to analyze small quantities of neurosteroids and their sulfates. Only with extremely sensitive methods of analysis is it possible to discover whether neurosteroids are synthesized in nervous tissues in quantities sufficient to affect neuronal activity, and whether these neurosteroids are distributed uniformly in brain. 

Steroids hormones as environmental pollutants Analysis of steroids as environmental endocrine disrupting compounds sample preparation, e.g., LLE vs. SPE immunoassay vs. GC-MS/ MS and LC-MS/MS analyses sensitivities, e.g., LOD at pg-ng/mL level. [13]... 

One of the major advantages of LC-MS/MS over GC-MS or GC-MS/MS is that steroid hormones may be analyzed directly by LC-MS/MS without derivatization procedures, which are time-consuming and tedious [22,50-53], However, a number of studies demonstrated that the chemically derivatized steroid hormones were significantly more sensitive to LC-MS/MS detection than the underivatized hormones, because the neutral molecules of estrogens and metabolites might not be effectively ionized under electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) modes [4, 21, 25, 54, 55], In order to enhance the steroid hormone molecules sensitivity for LC-MS/MS analysis at pg/mL level, chemical derivatization is an effective technique for analysis of steroid hormones and metabolites. A list of derivatization reagents and application examples for steroid hormone analyses by LC-MS/MS and GC-MS are presented in Table 3. 

An ideal derivatization reagent is able to react with steroid hormones and metabolites selectively and quantitatively under mild conditions within a short time, and those hormone derivatives are stable and easily ionized during LC-MS/MS analysis. Based on their functional groups, the derivatization reagents used for LC-MS/MS analyses of steroid hormones and metabolites may be classified into seven major classes ... 

Huopalahti and Henion " reported anabolic steroid extraction from bovine tissues. SFE was performed with CO2 at 60°C and 405 bar, with analytes collected in precooled methanol and quantitated using LC-MS analysis. Residues of 2-thiouradl, 6-methyl-2-thiouradl, 6-propyl-2-thiouracil, and 6-phenyl-2-thiouracil in bovine muscle tissues were extracted with SC CO2 and analyzed using GC-MS. ... 

A. Leinonen, T. Kuuranne, R. Kostiainen, LC-MS in anabolic steroid analysis -optimization and comparison of three ionization techniques ESI, APCI and APPI-MS, J. Mass Spectrom., 37 (2002) 693. 

Figure 13.3 LC-ESI-MS analysis of 11 steroids (20 pg each injected) by means of a methanol-water gradient on a 2.1-mm-lD coluttm, operated at a flow-rate of 0.4 ml/min. Reprinted from [19] with permission. 1997, American Society for Mass Spectrometry.

In early studies, LC-ESI-MS was applied in the analysis of steroid sulfates in eqnine mine [12], and in the identification of methandrostenolone and stanozolol metabolites in eqnine and human urine [13-15]. 

The sample-pretreatment method applied in the regulatory analysis of steroids to some extent depends on the matrix. Urine is first submitted to an enzymatic hydrolysis step in order to convert the steroid conjugates into free steroids. The next step for all matrices is a liquid extraction or LLE, generally followed by a clean-up of the extract by SPE. The sample is then injected into the LC-MS system. In most studies, an IS is added prior to the sample pretreatment. This IS can be an analogue, but more recently isotopically-labelled IS are used more frequently. 

In veterinary medicine, boldenone, a synthetic anabolic steroid (Figure 2.1), is commercially available, hence it is a concern in the horseracing industry. Pu et al. (2004) used ion-trap LC-MS analysis to detect boldenone conjugates (sulfate and glucuronide) and their 17-epimers in horse mine after intramuscular administration of boldenone undecylenate. Soon afterwards. Ho et al. (2004) reported the occurrence of endogenous boldenone sulfate in the urine of uncastrated male horses, and quantitated it by quadrupole time-of-flight (Q-TOF) LC-MS-MS. 

In anticipation of potential studies involving endogenous steroids, including neurosteroids, or exogenous steroids, or metabolites, Ma and Kim (1997) reported results obtained by LC-MS analysis by APCI or ESI of 29 steroid standards. Twelve of those had an androstane nucleus. They were either 3-one-4-enes or unconjugated ketones or they had only hydroxyl groups. The use of different LC mobile phases for each ionization mode helped to optimize the sensitivity. 

However, the utilization of Qg SPE prior to shotgun negative-ESI-MS, or online Qg LC-ESI-MS, allows the facile analysis of steroid sulphates [38]. Shown in Figure 2.6 is a negative-ion ESI-MS of the steroid fraction from infant plasma (Figure 2.7). 

Even when adopting a targeted approach, the coverage of lipids in a specific class can still be restricted by the dominance of a few very abundant lipids. This may be the case for steroid analysis where cholesterol is often the most abundant steroid by two orders of magnitude. This problem can be minimized by subdivision of the steroid class according to hydropho-bicity where cholesterol and more hydrophobic steroids represent one class, and oxysterols (oxidized forms of cholesterol and its precursors) and less hydrophobic metabolites represent a second class [45]. This is an approach we adopted for the analysis of steroid sulphates and bile acid/ alcohol conjugates by shotgun steroidomics, and in our LC-ESl-MS/MS work on oxysterols and cholestenoic acids in brain [46,47], plasma [48], and cerebrospinal fluid (CSF) [49]. 

Mass spectrometry (MS) in combination with the chromatographic separation techniques of gas chromatography (GC) or liquid chromatography (LC) have become the gold standard methods for the analysis of steroids [1-3], These methods allow the identification of novel steroids and in combination with reference standards quantification. Biological MS stretches back to the 1950s, when steroids was one of the first class of biomolecules analyzed by MS [4-9],... 

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