Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

Liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry: a highly sensitive method for the analysis of organic explosives

Gas chromatography/mass spectrometry (GC/MS) is applied to the analysis of volatile and thermally stable compounds, while liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI‐MS) and liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) are preferred for the analysis of compounds with solution acid‐base chemistry. Because organic explosives are compounds with low polarity and some of them are thermally labile, they have not been very well analyzed by GC/MS, LC/APCI‐MS and LC/ESI‐MS. Herein, we demonstrate liquid chromatography/negative ion atmospheric pressure photoionization mass spectrometry (LC/NI‐APPI‐MS) as a novel and highly sensitive method for their analysis. Using LC/NI‐APPI‐MS, limits of quantification (LOQs) of nitroaromatics and nitramines down to the middle pg range have been achieved in full MS scan mode, which are approximately one order to two orders magnitude lower than those previously reported using GC/MS or LC/APCI‐MS. The calibration dynamic ranges achieved by LC/NI‐APPI‐MS are also wider than those using GC/MS and LC/APCI‐MS. The reproducibility of LC/NI‐APPI‐MS is also very reliable, with the intraday and interday variabilities by coefficient of variation (CV) of 0.2–3.4% and 0.6–1.9% for 2,4,6‐trinitrotoluene (2,4,6‐TNT). Copyright © 2008 John Wiley & Sons, Ltd.     

Use of stable isotope labeled probes to facilitate liquid chromatography/mass spectrometry based high‐throughput screening of time‐dependent CYP inhibitors

Inhibition curve shift is a commonly used approach for screening of time‐dependent CYP inhibitors which requires parallel paired incubations to obtain two inhibition curves for comparison. For the control incubation, a test compound is co‐incubated with a probe substrate in human liver microsomes (HLM) fortified with NADPH; for the time‐dependent incubation (TDI), the test compound is pre‐incubated with NADPH‐fortified HLM followed by a secondary incubation with a probe substrate. For both incubations, enzyme activity is measured respectively by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of the CYP‐specific metabolite, and a TDI inhibitor can be readily identified by inhibition curve shifting as a result of CYP inactivation by the test compound during the pre‐incubation. In the present study, we describe an alternative approach to facilitate TDI screening in which stable isotope labeled CYP‐specific probes are used for the TDI, and non‐labeled substrates are included in the control incubation. Because CYP‐specific metabolites produced in the TDI are stable isotope labeled, two sets of incubation samples can be combined and then simultaneously analyzed by LC/MS/MS in the same batch run to reduce the run time. This new method has been extensively validated using both a number of known competitive and TDI inhibitors specific to five most common CYPs such as 1A2, 2C9, 2C19, 2D6, and 3A4. The assay is performed in a 96‐well format and can be fully automated. Compared to the traditional method, this approach in combination with sample pooling and a short LC/MS/MS gradient significantly enhances the throughput of TDI screening and thus can be easily implemented in drug discovery to evaluate a large number of compounds without adding additional resource. Copyright © 2010 John Wiley & Sons, Ltd.     

Improved detection of reactive metabolites with a bromine‐containing glutathione analog using mass defect and isotope pattern matching

Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug‐induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N‐(2‐bromocarbobenzyloxy)‐GSH (GSH‐Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH‐Br for each drug with subsequent analysis by liquid chromatography/high‐resolution mass spectrometry on an electrospray time‐of‐flight (ESI‐TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug‐specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH‐Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH‐Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations. Copyright © 2010 John Wiley & Sons, Ltd.     

High-throughput cytochrome p450 inhibition assays by ultrafast gradient liquid chromatography with tandem mass spectrometry using monolithic columns

A generic method employing ultrafast liquid chromatography with tandem mass spectrometry (LC/MS/MS) was developed and employed for routine screening of drug candidates for inhibition of five major human cytochrome p450 (CYP) isozymes, CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2. The method utilized a monolithic silica rod column to allow fast flow rates to significantly reduce chromatographic run time. The major metabolites of six CYP-specific probe substrates for the five p450 isoforms were monitored and quantified to determine IC(50) values of five drug compounds against each p450 isozyme. Human liver microsomal incubation samples at each test compound concentration were combined and analyzed simultaneously by the LC/MS/MS method. Each pooled sample containing six substrates and an internal standard was separated and detected in only 24 seconds. The combination of ultrafast chromatography and sample pooling techniques has significantly increased sample throughput and shortened assay turnaround time, allowing a large number of compounds to be screened rapidly for potential p450 inhibitory activity, to aid in compound selection and optimization in drug discovery.     

LC Determination of Coumarin-3-Acyl Derivatives for Evaluation of the Stability and Monoamine Oxidase Inhibition Mechanism

A simple and rapid LC method has been developed for the determination of coumarin 3-acyl derivatives synthesized and assayed for their inhibitory activity against monoamine oxidases (MAOs). The proposed liquid chromatography method can be used satisfactorily for the purity control of the synthesized compounds and can also be used to study the stability of these potentially reactive compounds. Moreover using this method it has been possible to elucidate the mechanism of MAO inhibition by direct determination of the biochemical reaction mixture.     

Development and validation of a LC‐MS/MS method for the in vitro analysis of 1‐hydroxymidazolam in human liver microsomes: application for determining CYP3A4 inhibition in complex matrix mixtures

Complementary and alternative medicines (CAM) can affect the pharmacokinetics of anticancer drugs by interacting with the metabolizing enzyme cytochrome P450 (CYP) 3A4. To evaluate changes in the activity of CYP3A4 in patients, levels of 1‐hydroxymidazolam in plasma are often determined with liquid chromatography–quadrupole mass spectrometry (LC‐MS/MS). However, validated LC‐MS/MS methods to determine in vitro CYP3A4 inhibition in human liver microsomes are scarce and not optimized for evaluating CYP3A4 inhibition by CAM. The latter is necessary because CAM are often complex mixtures of numerous compounds that can interfere with the selective measurement of 1‐hydroxymidazolam. Therefore, the aim was to validate and optimize an LC‐MS/MS method for the adequate determination of CYP3A4 inhibition by CAM in human liver microsomes. After incubation of human liver microsomes with midazolam, liquid–liquid extraction with tert‐butyl methyl ether was applied and dried samples were reconstituted in 50% methanol. These samples were injected onto a reversed‐phase chromatography consisting of a Zorbax Extend‐C₁₈ column (2.1 × 150 mm, 5.0 µm particle size), connected to a triple quadrupole mass spectrometer with electrospray ionization. The described LC‐MS/MS method was validated over linear range of 1.0–500 nm for 1‐hydroxymidazolam. The results revealed good inter‐assay accuracy (≥85% and ≤115%) and within‐day and between‐day precisions (coefficient of variation ≤ 4.43%). Furthermore, the applicability of this assay for the determination of CYP3A4 inhibition in complex matrix mixtures was successfully demonstrated in an in vitro experiment in which CYP3A4 inhibition by known CAM (β‐carotene, green tea, milk thistle and St. John's wort) was determined. Copyright © 2013 John Wiley & Sons, Ltd.     

Liquid chromatography/multi-stage mass spectrometry of bisphenol A and its halogenated derivatives

We report a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for analyzing bisphenol A (BPA) and its halogenated derivatives. Since only tetrachlorobisphenol A and tetrabromobisphenol A (TBBPA) are commercially available, mono-, di- and trichlorobisphenol A were synthesized and purified in order to be used as analytical standards. This family of compounds was studied using electrospray ionization and an ion trap mass analyzer in order to characterize the new compounds and to propose fragmentation pathways. Multi-stage mass spectrometry was used to confirm the genealogical relationship between the ions. Some product ions were traced from MS/MS to MS(4) and the labelled compounds BPA-d(16) and TBBPA-(13)C(12) were used to assign some product ion structures. In general, the deprotonated molecule [M--H](-) loses a methyl and/or a halogen group during both MS/MS and MS(3), while the neutral loss of CO was also observed in MS(3) spectra. We selected the most intense and characteristic MS/MS transitions for LC/MS/MS analysis. LC separation was performed in a reversed-phase column; methanol/water (no additives) was used as the mobile phase in gradient elution mode; and BPA-d(16) was chosen as the internal standard. Solid-phase extraction (SPE) was used to pre-concentrate and to clean up water samples. The SPE LC/MS/MS method allows BPA and its halogenated derivatives to be detected at a few parts-per-billion (ppb) in surface water.     

A novel liquid chromatography/tandem mass spectrometry based depletion method for measuring red blood cell partitioning of pharmaceutical compounds in drug discovery

A novel liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based depletion method for measuring compound partitioning between human plasma and red blood cells (RBC) in a drug discovery environment is presented. Conventionally, RBC partitioning is determined by separate measurements of drug concentrations in equilibrating plasma and whole blood or RBC using separate standards prepared in their respective matrices, i.e., in plasma and whole blood or RBC lysates. The process is very tedious, labor-intensive, and difficult to automate. In addition, interferences from the heme and other highly abundant cellular composites make the measurement of the drug concentration in whole blood or RBC inevitably variable even with a highly specific LC/MS/MS method. Therefore, there is an imminent need to develop a straightforward and fast method to assess the partitioning of drug-like compounds in RBC.This work describes an LC/MS/MS-based depletion assay that measures the compound concentration in plasma that has been equilibrating with RBC. Compounds were spiked into fresh human whole blood and plasma respectively to a final concentration of 500 nM. Both the spiked whole blood and plasma control were incubated at 37 degrees C for up to 60 min. During the time course, aliquots of plasma and whole blood from both incubation mixtures were sampled at 10 and 60 min. The whole blood samples were centrifuged to yield the plasma. The plasma samples from both incubations were extracted using a protein precipitation method, and analyzed using LC/MS/MS under the multiple-reaction monitoring (MRM) mode. The RBC partitioning ratio was calculated using the analyte peak area responses of the plasma samples through an equation deduced in this work.The method was first tested using two commercial compounds, phenoprobamate and acetazolamide, to determine the optimal incubation conditions and the concentration dependency of the assay. The assay reproducibility was also assessed by three inter-day assays for phenoprobamate. This method was further evaluated using 20 commercial compounds of different classes with a wide range of RBC partitioning coefficients and the results were compared with those reported in the literature. Excellent correlation (R2=0.9396) was found between the measured and literature values. In addition, several proprietary compounds were assayed using both the new and traditional methods and the measured partitioning ratios from the two methods are equivalent. The experiments in this work demonstrate that the LC/MS/MS-based depletion method can provide direct and accurate measurement of RBC partitioning for compounds in drug discovery.     

蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶抑制剂

基于酶与底物间的相互作用,建立了蛋白-脂质体复合物毛细管电泳筛选单胺氧化酶(MAO)抑制剂的新方法.分别将不同浓度的4种N-炔丙基胺类化合物添加至含有蛋白-脂质体复合物的毛细管电泳缓冲液中,抑制MAO活性.考察MAO底物犬尿胺(Kyn)在含有不同浓度N-炔丙基胺类化合物缓冲液中的迁移时间比率(RMTR).结果表明,化合物N-炔丙基-N-甲基-R-2-庚胺(R-2-HMP)和N-炔丙基-R-2-庚胺(R-2-HPA)能够明显抑制MAO活性,导致MAO与Kyn的相互作用减弱,Kyn的RMTR值随着R-2-HMP和R-2-HPA的增加呈现出明显增加的趋势.化合物N,N-二炔丙基-R-2-己胺和N,N-二炔丙基-R-2-辛胺对MAO活性抑制不明显,Kyn的RMTR随这两种浓度增加变化不大.此结果与柱外孵育测定化合物活性结果一致.与传统MAO筛选剂筛选方法相比,本方法快速,成本低,酶消耗量少且不受分离电压等于扰因素的影响.     

Cardiac Muscle/Cell Membrane Chromatography-Offline-Liquid Chromatography/Mass Spectrometry Method to Identify Bioactive Components from Traditional Chinese Medicines

An offline analytical method combining cardiac muscle/cell membrane chromatography (CM/CMC) with liquid chromatography/mass spectrometry (LC/MS) to identify bioactive components from traditional Chinese medicines (TCMs) such as Schisandrae Chinensis Fructus (SCF) and Schisandrae Sphenantherae Fructus (SSF) was developed. The stationary phase of the CM/CMC system employed rat cardiac muscle cell membranes. Fractions retained by the CM/CMC column were collected and analyzed by LC/MS under optimum offline conditions. After evaluating the suitability and reliability of the CM/CMC-offline-LC/MS method using nifedipine and nitrendipine as standards, this method was applied for screening and identification of bioactive components from the extracts of SCF and SSF. The main compounds in both species retained by CM/CMC were deoxyschizandrin (DSD) and schisantherin A (STA) as identified by LC/MS. In vivo pharmacological experiments suggested that DSD and STA could significantly decrease the myocardial-infarct size in rats injured by myocardial ischemia–reperfusion. Our CM/CMC-offline-LC/MS method could be used as an effective alternative for screening multiple receptor-binding bioactive components in TCMs such as FSC and FSS used to remedy cardiac diseases and may be useful for drug discovery using medicinal herbs as lead compounds.     

Profiling of grape monoterpene glycosides (aroma precursors) by ultra‐high performance‐liquid chromatography‐high resolution mass spectrometry (UHPLC/QTOF)

A ‘suspect screening analysis’ method for grape metabolomics by ultra‐high performance‐liquid chromatography (UHPLC) and high‐resolution quadrupole‐time of flight (QTOF) mass spectrometry was recently developed. This method was applied to study grape monoterpene glycosides, the main grape aroma precursors. Since standard compounds were not available, they were tentatively identified by overlapping various analytical approaches, in agreement with the indications recommended in mass spectrometry (MS)‐based metabolomics. Accurate mass and isotopic pattern, MS/MS fragmentation, correlation between fragments observed and putative structures and between liquid chromatography coupled with mass spectrometry (LC/MS) and gas chromatography/mass spectrometry signals were studied. Seventeen monoterpene glycosides were identified without performing the hydrolytic artifacts commonly used to study these compounds which may affect sample profile. This is the first time that a detailed study of these aroma precursors has been carried out by direct LC/MS analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Derivatization in Liquid Chromatography/Mass Spectrometric Analysis of Neurosteroids

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液相色谱-毛细管气相色谱/质谱离线联用分析烟草中的挥发性及半挥发性成分

建立了一种用于烟草样品中挥发性、半挥发性成分分析的液相色谱-毛细管气相色谱/质谱(LC-CGC/MS)离线联用方法。研究了LC-CGC/MS的分离机理。LC分析选用氨基分析柱(250 mm×2.0 mm, 5 μm)作为分析柱,正己烷-二氯甲烷-乙腈(90:6.6:3.4, v/v/v)作为流动相,对挥发性、半挥发性成分进行分离,收集得到5个馏分,并存放在5个氮吹瓶中。多次进样并收集相同时间段的馏分,氮吹浓缩至1 mL,然后分别进行CGC/MS分析,所用的CGC柱为DB-5MS(60 m×0.25 mm×0.25 μm)。结果显示,与直接采用CGC/MS分析相比,采用LC-CGC/MS分析复杂样本的效果更好,定性的可靠性更高。     

In vivo microdialysis and reverse phase ion pair liquid chromatography/tandem mass spectrometry for the determination and identification of acetylcholine and related compounds in rat brain

A method using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. A microdialysis probe was surgically implanted into the striatum of the rats and Ringer's solution was used as the perfusion medium at a flow rate of 2 microL per minute. The samples were then analyzed off-line by LC/MS/MS experiments. The separation of ACh and choline (Ch) was carried out using reverse phase ion pair liquid chromatography with heptafluorobutyric acid as a volatile ion pairing reagent. Analytes were detected by electrospray ionization tandem mass spectrometry in the positive ion mode. The detection limit for ACh was 1.4 fmol on column, which is at least three times lower than previously reported. Three quaternary ammonium compounds in the rat brain microdialysate were also identified by tandem mass spectrometry experiments in which the unknown mass spectra were compared with standard reference compounds. These compounds were identified as carnitine, acetylcarnitine and (3-carboxypropyl)trimethylammonium. This is the first known report of the compound (3-carboxypropyl)trimethylammonium being found in rat brain.     

Derivatization in Liquid Chromatograhy/Mass Spectrometric Analysis of Neurosteroids

 Liquid chromatography/mass spectrometry (LC/MS) is now considered to be the most promising analytical method for the determination of biological substances, especially nonvolatile or highly polar substances However, some compounds do not show enough sensitivity in LC/MS and soft ionization methods commonly used in LC/MS, such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI), sometimes do not give satisfactory structural information This report presents an overview     

Ultra-performance liquid chromatography/tandem mass spectrometric quantification of structurally diverse drug mixtures using an ESI-APCI multimode ionization source

We report in this paper an ultra-performance liquid chromatography/tandem mass spectrometric (UPLC(R)/MS/MS) method utilizing an ESI-APCI multimode ionization source to quantify structurally diverse analytes. Eight commercial drugs were used as test compounds. Each LC injection was completed in 1 min using a UPLC system coupled with MS/MS multiple reaction monitoring (MRM) detection. Results from three separate sets of experiments are reported. In the first set of experiments, the eight test compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes (ESI+, ESI-, APCI-, and APCI+) during an LC run. Approximately 8-10 data points were collected across each LC peak. This was insufficient for a quantitative analysis. In the second set of experiments, four compounds were analyzed as a single mixture. The mass spectrometer was switching rapidly among four ionization modes during an LC run. Approximately 15 data points were obtained for each LC peak. Quantification results were obtained with a limit of detection (LOD) as low as 0.01 ng/mL. For the third set of experiments, the eight test compounds were analyzed as a batch. During each LC injection, a single compound was analyzed. The mass spectrometer was detecting at a particular ionization mode during each LC injection. More than 20 data points were obtained for each LC peak. Quantification results were also obtained. This single-compound analytical method was applied to a microsomal stability test. Compared with a typical HPLC method currently used for the microsomal stability test, the injection-to-injection cycle time was reduced to 1.5 min (UPLC method) from 3.5 min (HPLC method). The microsome stability results were comparable with those obtained by traditional HPLC/MS/MS.     

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen

The evaluation of interactions between drug candidates and transporters such as P‐glycoprotein (P‐gp) has gained considerable interest in drug discovery and development. Inhibition of P‐gp can be assessed by performing bi‐directional permeability studies with in vitro P‐gp‐expressing cellular model systems such as Caco‐2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying ³H‐digoxin with liquid scintillation counting (LSC) detection or assaying non‐labeled digoxin with liquid chromatography–tandem mass spectrometric (LC‐MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire?, an on‐line solid‐phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3‐digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF‐MS/MS analysis. The RF‐MS/MS method was more than 16 times faster than the LC‐MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P‐gp inhibition results of multiple validation compounds obtained with this RF‐MS/MS method were in agreement with those generated by both the LC‐MS/MS method and the ³H‐radiolabel assay. This method has been successfully deployed to assess P‐gp inhibition potential as an important early liability screen for drug–transporter interaction. Copyright © 2011 John Wiley & Sons, Ltd.     

Combination of liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry for the detection of 21 anabolic steroid residues in bovine urine

For the detection of anabolic steroid residues in bovine urine, a highly sensitive liquid chromatographic/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed using both positive and negative ionization. For four compounds the ESI mode was not sensitive enough and gas chromatographic/mass spectrometric GC/MS detection was therefore still necessary as a complementary method. The sample clean-up consisted of solid-phase extraction (SPE) on a C(18) column followed by enzymatic hydrolysis and a second solid-phase extraction on a combination of a C(18) and a NH(2) column. After this last SPE clean-up, the eluate was split into two equal aliquots. One aliquot was further purified and after derivatization used for GC/MS analysis. The other aliquot was analyzed with LC/MS/MS in both ESI+ and ESI- modes. The method was validated according to the European Commission Decision 2002/657/EC. Decision limits (CCalpha) were between 0.16 and 1 ng ml(-1) for the compounds detected with the LC/MS/MS method. The developed method is used in routine analysis in our laboratory.     

Mass spectrometry applied to the analysis of estrogens in the environment

Environmental analytical chemistry has recently changed focus from analysis of non-polar, persistent contaminants (e.g. polychlorinated biphenyls and dioxins) to more polar and labile compounds that interfere with biological processes. For example, natural and synthetic estrogens and their metabolites have been detected in sewage treatment plant effluents at nanogram/liter concentrations that are similar to those at which both total sex reversal and intersex (containing both testes and ova) is induced in fish exposed to these compounds in laboratory experiments. The development of techniques for the analysis of natural and synthetic estrogens in biological fluids (i.e. serum and urine) has been a priority in the biomedical field. However, the recent recognition that estrogen hormones are contaminants in the environment that may contribute to endocrine disruption has focused attention on the need for highly sensitive and specific techniques that are applicable for trace analysis in complex environmental matrices. Three optimized mass spectrometric protocols have been developed for the determination and quantitation of steroid hormones in environmental matrices using gas chromatography/tandem mass spectrometry (GC/MS/MS), liquid chromatography/mass spectrometry selected ion monitoring, (LC/MS - SIM) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The advantages and disadvantages of each method are presented.