Screening of free 17-alkyl-substituted anabolic steroids in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry

A qualitative liquid chromatography-electrospray ionization tandem mass spectrometry method was developed for screening of the abuse of 4-chlorodehydromethyltestosterone, danazol, fluoxymesterone, formebolone, metandienone, oxandrolone, and stanozolol. The introduced method measures simultaneously nine different 17-alkyl-substituted anabolic androgenic steroids or their unconjugated metabolites in human urine, using methyltestosterone as an internal standard. Sample preparation involved one-step liquid extraction. Liquid chromatographic separation was achieved on a reversed-phase column with methanol-water gradient containing 5 mmol/l ammonium acetate and 0.01% (v/v) acetic acid. Compounds were ionized in the positive mode and detected by multiple reaction monitoring. All steroids within the study could be selectively detected in urine with detection limits of 0.1-2.0 ng/ml. The method showed good linearity up to 250 ng/ml with correlation coefficients higher than 0.9947. With simple and fast sample preparation, low limits of detection, and high selectivity and precision, the developed method provides advantages over the present testing methods and has the potential for routine qualitative screening method of unconjugated 17-alkyl-substituted anabolic steroids in human urine.     

Doping control for methenolone using hair analysis by gas chromatography-tandem mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of methenolone in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 100 mg) was solubilized in 1 ml 1 M NaOH, 15 min at 95 degrees C, in presence of 1 ng testosterone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18 eluted with methanol) and a liquid-liquid (pentane) extraction. The residue was derivatized by adding 50 microl MSTFA-NH4I-2-mercaptoethanol (1000:2:5, v/v/v), then incubated for 20 ml at 60 degrees C. A 1.5-microl aliquot of the derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm I.D., 0.25 microm film thickness) of a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Methenolone was detected by its parent ion at m/z 446 and daughter ions at m/z 208 and 195 through a Finnigan TSQ 700 MS-MS system. The assay was capable of detecting 1 pg/mg of methenolone when approximately 100 mg hair material was processed. Linearity was observed for methenolone concentrations ranging from 2 to 100 pg/mg with a correlation coefficients of 0.965-0.981. Intra-day and between-day precisions at 2, 10 and 25 pg/mg were 10.9-14.1% and 13.7-16.8%, respectively, with an extraction recovery of 97.6%. The analysis of a strand of hair obtained from two bodybuilders, revealed the presence of methenolone at the concentrations of 7.3 and 8.8 pg/mg.     

Characterization and quantification of karlotoxins by liquid chromatography–mass spectrometry

Karlodinium veneficum is a cosmopolitan dinoflagellate with a worldwide distribution in mesohaline temperate waters. The toxins from K. veneficum, or karlotoxins (KmTxs), which have been implicated in fish kill events, have been purified from monoalgal cultures, and shown to possess hemolytic, cytotoxic and ichthyotoxic activities. Three karlotoxins (KmTx 1–1, KmTx 1–3 and KmTx 2) have been isolated from two different North American strains of K. veneficum and characterized using liquid chromatography–mass spectrometry (LC–MS). KmTx 1 karlotoxins have a UV absorption maximum (λ_max 225 nm) at lower wavelengths than KmTx 2 karlotoxins (λ_max 235 nm). The exact masses and predicted empirical formulae for the karlotoxins (KmTx 1–1, 1308.8210, C₆₇H₁₂₀O₂₄; KmTx 1–3, 1322.8637, and C₆₉H₁₂₆O₂₃; KmTx 2, 1344.7938, C₆₇H₁₂₁ClO₂₄) were determined using high resolution mass spectrometry. Although the individual toxins produce a single peak in reverse phase high performance liquid chromatography (HPLC), MS revealed congeners co-eluting within each peak. These congeners could be separated under normal phase chromatography and revealed a single hydroxylation being responsible for the mass differences. Multistage MS (MSⁿ) showed that the three KmTxs and their congeners share a large portion of their structures including an identical 907 amu core fragment.

These data were used to develop a quantitative LC–MS assay for karlotoxins from cultures and environmental samples. The sensitivity afforded by MS detection compared to UV absorbance allowed toxin quantification at 0.2 ng when injected on column. Aqueous solutions of karlotoxins were found to quantitatively adsorb to PTFE and nylon membrane filters. Aliquots from whole cultures or environmental samples could be concentrated and desalted by adsorption to PTFE syringe filters and karlotoxins eluted with methanol for analysis by LC–MS. This simplified solid phase cleanup afforded new data indicating that each karlotoxin may also exist as sulfated derivatives and also provided a rapid detection method for karlotoxin from environmental samples and whole cultures.     

Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry

We report an ultrahigh-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS) method to quantify all-trans-retinal in biological samples of limited size (15–35 mg), which is especially advantageous for use with adipose. To facilitate recovery, retinal and the internal standard 3,4-didehydroretinal were derivatized in situ into their O-ethyloximes. UHPLC resolution combined with high sensitivity and specificity of MS/MS allowed quantification of retinal-O-ethyloximes with a 5-fmol lower limit of detection and a linear range from 5 fmol to 1 pmol. This assay revealed that extraocular concentrations of retinal range from approximately 2 to 40 pmol/g in multiple tissues—the same range as all-trans-retinoic acid. All-trans-retinoic acid has high affinity (k_d ? 0.4 nM) for its nuclear receptors (RARα, -β, and -γ), whereas retinal has low (if any) affinity for these receptors, making it unlikely that these retinal concentrations would activate RAR. We also show that the copious amount of vitamin A used in chow diets increases retinal in adipose depots 2- to 5-fold relative to levels in adipose of mice fed a vitamin A-sufficient diet, as recommended for laboratory rodents. This assay also is proficient for quantifying conversion of retinol into retinal in vitro and, therefore, provides an efficient method to study metabolism of retinol in vivo and in vitro.     

Miniaturized proteomics and peptidomics using capillary liquid separation and high resolution mass spectrometry

Knowledge of the protein and peptide content in a tissue or a body fluid is vital in many areas of medical and biomedical sciences. Information from proteomic and peptidomic studies may reveal alterations in expression due to, e.g., a disease and facilitate the understanding of the pathophysiology and the identification of biological markers. In this minireview, we discuss miniaturized proteomic and peptidomic approaches that have been applied in our laboratory in order to investigate the protein and peptide contents of body fluids (such as plasma, cerebrospinal and amniotic fluid), as well as extracted tissues. The methods involve miniaturized liquid separation, i.e., capillary liquid chromatography and capillary electrophoresis, combined with high resolution mass spectrometry (MS), i.e., Fourier transform ion cyclotron resonance MS. These approaches provide the opportunity to analyze samples of small volumes with high throughput, high sensitivity, good dynamic range and minimal sample handling. Also, the experiments are relatively easy to automate.     

Proteomic analysis of estrogen response of premalignant human breast cells using a 2-D liquid separation/mass mapping technique

A 2-D liquid-phase separation method based on chromatofocusing and nonporous silica RP-HPLC followed by ESI-TOF-MS was used to analyze proteins in whole cell lysates from estrogen-treated and untreated premalignant, estrogen-responsive cell line MCF10AT1 cells. 2-D mass maps in the pH range 4.6-6.0 were generated with good correlation to theoretical M(r) values for intact proteins. Proteins were identified based on intact M(r), pI and PMF, or MS/MS sequencing. About 300 unique proteins were identified and 120 proteins in mass range 5-75 kDa were quantified upon treatment of estrogen. Around 40 proteins were found to be more highly expressed (>four-fold) and 17 were down-regulated (>four-fold) in treated cells. In our study, we found that many altered proteins have characteristics consistent with the development of a malignant phenotype. Some of them have a role in the ras pathway or play an important role in signal pathways. These changed proteins might be essential in the estrogen regulation mechanism. Our study highlights the use of the MCF10AT1 cell line to examine estrogen-induced changes in premalignant breast cells and the ability of the 2-D mass mapping technique to quantitatively study protein expression changes on a proteomic scale.     

A high-performance liquid chromatography-tandem mass spectrometry assay of the androgenic neurosteroid 3alpha-androstanediol (5alpha-androstane-3alpha,17beta-diol) in plasma

The testosterone metabolite 3alpha-androstanediol (5alpha-androstane-3alpha,17-diol) is a potential GABA(A) receptor-modulating neurosteroid with anticonvulsant properties and hence could act as a key neuromodulator in the central nervous system. However, there is no specific and sensitive assay for quantitative determination of the androgenic neurosteroid 3alpha-androstanediol in biological samples. We have established a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay to measure 3alpha-androstanediol in rat plasma. Standard 3alpha-androstanediol added to rat plasma has been successfully analysed with excellent linearity, specificity, sensitivity, and reproducibility. The sensitivity of the method was < 10 ng/ml with a detection limit of 2 ng/ml (6.8 nmol/l) and a linear range of 10-2000 ng/ml. The method was used for the analysis of testosterone-induced increase in plasma 3alpha-androstanediol levels in rats. Testosterone produced a dose-dependent elevation in plasma 3alpha-androstanediol, which was almost completely prevented by pretreatment with the 5alpha-reductase inhibitor finasteride, indicating that 3alpha-androstanediol is synthesized from testosterone via a 5alpha-reductase pathway. This LC-MS-MS method allows accurate, high-throughput analysis of 3alpha-androstanediol in small amounts (200 microl) of plasma and possibly other biological samples.     

Analysis of the stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde using high-performance liquid chromatography and mass spectrometry

The stability of amino acids derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) was investigated using a combination of high-performance liquid chromatography, solid-phase extraction, photodiode array spectrophotometric detection, and mass spectrometric (MS) characterization. The degradation of amino acid derivatives, generated using beta-mercaptoethanol as a nucleophile, was characterized under a variety of environmental influences, with a focus on understanding the degradation kinetics and identifying the degradation products. The predominant degradation product observed under most reaction conditions was the nonfluorescent lactam form of the originally fluorescent isoindole derivative. First, the time-dependent degradation of the isoindole derivative L-serine-NDA-beta-mercaptoethanol was found to follow pseudo-first order kinetics with a half-life of 2.0 min at pH 9.2 and room temperature. The isoindole derivative was observed to react further with methanol to form a more stable fluorescent methoxy-isoindole, shedding new light on the basis for enhanced stability of these derivatives in methanol. Tandem mass spectrometry (MS/MS) experiments were used to demonstrate unimolecular degradation of the protonated isoindole in the absence of solvent or atmosphere, suggesting an intramolecular reaction mechanism involving the hydroxyethylthio group. Finally, in photobleaching studies, NDA derivatives rapidly degraded into a variety of products within the first 2 min of photobleaching versus timed controls, with the predominant product being the lactam. These results suggest that the degradation pathway for NDA derivatives is similar to the previously reported pathway for o-phthalaldehyde derivatives and clearly identifies the reaction and degradation products under a variety of conditions.     

Metabolism and excretion of anabolic steroids in doping control--new steroids and new insights

The use of anabolic steroids in sports is prohibited by the World Anti-Doping Agency. Until the 1990s, anabolic steroids were solely manufactured by pharmaceutical companies, albeit sometimes on demand from national sports agencies as part of their doping program. Recently the list of prohibited anabolic steroids in sports has grown due to the addition of numerous steroids that have been introduced on the market by non-pharmaceutical companies. Moreover, several designer steroids, specifically developed to circumvent doping control, have also been detected. Because anabolic steroids are most often intensively subjected to phase I metabolism and seldom excreted unchanged, excretion studies need to be performed in order to detect their misuse.

This review attempts to summarise the results of excretion studies of recent additions to the list of prohibited steroids in sports. Additionally an update and insight on new aspects for “older” steroids with respect to doping control is given.     

Concurrent quantification of multiple biomarkers indicative of oxidative stress status using liquid chromatography-tandem mass spectrometry

8-Hydroxy-2-deoxyguanosine (8-OHdG), 8-nitroguanine (8-NO₂Gua), 8-iso-prostaglandin F_2α (8-IsoPGF_2α), and N-acetyl-S-(tetrahydro-5-hydroxy-2-pentyl-3-furanyl)-L-cysteine (HNE-MA) are well-studied and representative biomarkers for oxidative DNA damage, inflammation, and lipid peroxidation; all of which have been associated with increases in risks of various diseases and cancers. A rapid and highly sensitive isotope-dilution liquid-chromatography tandem mass spectrometry (LC-MS/MS) method was developed to simultaneously quantify the aforementioned biomarkers in urine. Upon validation, this method shows excellent feasibility, sensitivity (0.008–0.03 ng/mL) and satisfactory recoveries (88.7–95.4%); the calibration curves displayed excellent linearity with coefficients of determination (R²) greater than 0.998. Additionally, low variations were observed in the relative standard deviation for intra- and inter-day measurements for the four analytes. The relative matrix effects for all four analytes ranged from 2.04 to 3.27%, which signaled that interferences from endogenous levels of the analytes were deemed statistically insignificant. This study successfully developed an analytical method capable to simultaneously quantify urinary 8-OHdG, 8-NO₂Gua, 8-IsoPGF_2α, and HNE-MA. This analytical protocol can be applied towards conducting epidemiological studies to reveal the mechanisms related to disease development, and thus evaluate the associated risks of diseases.     

Development of a method for determination of the malondialdehyde-deoxyguanosine adduct in urine using liquid chromatography-tandem mass spectrometry

A procedure is described for the quantification of the major malondialdehyde deoxyguanosine adduct, pyrimido[1,2-alpha]purin-10(3H)-one-deoxyribose (M(1)GdR) in urine. M(1)GdR is isolated from urine by a combination of C(18) solid-phase extraction and immunoaffinity chromatography. Sodium borohydride treatment reduces M(1)GdR to the 5,6-dihydro derivative, which is quantified by liquid chromatography-mass spectrometry. Authentic [7,9-15N,8-13C]M(1)GdR is added to urine as an internal standard. A detection limit of 50 fmol M(1)GdR/ml urine is achieved starting with 5 ml of urine. Analysis of urine samples from control rats or rats treated with CCl(4) indicates that the levels of M(1)GdR are below the detection limit of the assay. This method is easily adaptable to the analysis of M(1)GdR in DNA samples or biological fluids.     

Molecular constituents of the postsynaptic density fraction revealed by proteomic analysis using multidimensional liquid chromatography-tandem mass spectrometry

Protein constituents of the postsynaptic density (PSD) fraction were analysed using an integrated liquid chromatography (LC)-based protein identification system, which was constructed by coupling microscale two-dimensional liquid chromatography (2DLC) with electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and an automated data analysis system. The PSD fraction prepared from rat forebrain was solubilized in 6 m guanidium hydrochloride, and the proteins were digested with trypsin after S-carbamoylmethylation under reducing conditions. The tryptic peptide mixture was then analysed with the 2DLC-MS/MS system in a data-dependent mode, and the resultant spectral data were automatically processed to search a genome sequence database for protein identification. In triplicate analyses, the system allowed assignments of 5264 peptides, which could finally be attributed to 492 proteins. The PSD contained various proteins involved in signalling transduction, including receptors, ion channel proteins, protein kinases and phosphatases, G-protein and related proteins, scaffold proteins, and adaptor proteins. Structural proteins, including membrane proteins involved in cell adhesion and cell-cell interaction, proteins involved in endocytosis, motor proteins, and cytoskeletal proteins were also abundant. These results provide basic data on a major protein set associated with the PSD and a basis for future functional studies of this important neural machinery.     

Quantitative analysis of tissue folate using ultra high-performance liquid chromatography tandem mass spectrometry

The tissue distribution of folate in its numerous coenzyme forms may influence the development of disease at different sites. For instance, the susceptibility of human colonic mucosa to localized folate deficiency may predispose to the development of colorectal cancer. We report a sensitive and robust ultra high-performance liquid chromatography (UHPLC) tandem mass spectrometry method for quantifying tissue H₄folate, 5-CH₃-H₄folate, 5-CHO-H₄folate, folic acid, and 5,10-CH⁺-H₄folate concentration. Human colonic mucosa (20–100 mg) was extracted using lipase and conjugase enzyme digestion. Rapid separation of analytes was achieved on a UHPLC 1.9-μm C18 column over 7 min. Accurate quantitation was performed using stable isotopically labeled (¹³C₅) internal standards. The instrument response was linear over physiological concentrations of tissue folate (R² > 0.99). Limits of detection and quantitation were less than 20 and 30 fmol on column, respectively, and within- and between-run imprecision values were 6–16%. In colonic mucosal samples from 73 individuals, the average molar distribution of folate coenzymes was 58% 5-CH₃-H₄folate, 20% H₄folate, 18% formyl-H₄folate (sum of 5-CHO-H₄folate and 5,10-CH⁺-H₄folate), and 4% folic acid. This assay would be useful in characterizing folate distribution in human and animal tissues as well as the role of deregulated folate homeostasis on disease pathogenesis.     

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.     

Quantitative analysis of adenosine using liquid chromatography/atmospheric pressure chemical ionization-tandem mass spectrometry (LC/APCI-MS/MS)

Adenosine-secreting cellular brain implants constitute a promising therapeutic approach for the treatment of epilepsy. To engineer neural stem cells for therapeutic adenosine delivery, a reliable and fast analytical method is necessary to quantify cell-based adenosine release. Here we describe the development, optimization and validation of adenosine measurement using liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry (LC–APCI-MS/MS). LC–MS/MS in positive ion mode used selected reaction monitoring at m/z of 268.2/136.1 and 302.2/170.0 for adenosine and the internal standard, respectively. The bias was within 15% of the nominal value and evaluation of precision showed a relative standard deviation lower than 15% for all measured concentrations. The lower limit of quantification of adenosine was 15.6 ng/ml. Freeze and thaw stability and processed sample stability also fulfilled the acceptance criteria. Evaluation of the matrix effect showed that the method is not affected by relative matrix effects. The major advantages of this method are the absence of an extraction phase and the combination of the high selectivity and sensitivity characteristic for the LC–MS/MS technique, with a short run time of 4.5 min. These results demonstrate that this method is a useful tool to measure adenosine concentrations in culture medium released from stem cells in vitro.     

A sensitive liquid chromatography-tandem mass spectrometry method for the quantification of mometasone furoate in human plasma

A robust, rapid, selective and sensitive liquid chromatography-negative atmospheric pressure chemical ionization (LC-(APCI(-))-MS-MS) method has been developed for the quantification of mometasone furoate (MF) in human plasma utilizing a solid-phase extraction clean-up step and 13C-fluticasone propionate as internal standard. The intra- and inter-day coefficients of variation were < or = 15% and the lower limit of quantification (LLOQ) was 15 pg/ml. This method is ideally suited for pharmacokinetic investigations of low MF levels following inhalation of MF.     

Doping control for metandienone using hair analyzed by gas chromatography-tandem mass spectrometry

A sensitive, specific and reproducible method for the quantitative determination of the anabolic metandienone in human hair has been developed. The preparation involved a decontamination step with methylene chloride. The hair sample (about 50 mg) was solubilised in 1 ml 1 M NaOH, 10 min at 95 degrees C, in presence of 2 ng of nandrolone-d(3) used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase extraction (Isolute C(18) eluted with methanol) and a liquid-liquid extraction with pentane. The residue was derivatized by adding 5 microl MSTFA/NH(4)I/2-mercaptoethanol (250 microl; 5 mg; 15 microl) and 45 microl MSTFA, then incubated for 20 min at 60 degrees C. A 1 microl aliquot of derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.32 mm i.d., 0.25 microm film thickness) of a Hewlett Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Metandienone was identified using three transitions (its daughter ions at m/z 339 and 206 for the parent 444 and 191 for 206) using a Waters Quattro Micro MS-MS system. The transition m/z 444 to 206 has been used as quantification transition and the others as identification transitions. The assay was capable of detecting 2 pg/mg of metandienone when approximately 50 mg of hair material was processed. Linearity was observed for metandienone concentrations ranging from 2 to 500 pg/mg with a correlation coefficient of 0.9997. Intra-day and between-day precisions at 50 pg/mg were 13.4-16.5% and 22.0%, respectively, with an extraction recovery of 48%. The analysis of hair, cut into four segments, obtained from an athlete, revealed the presence of metandienone at the concentrations of 78, 7, 10 and 108 pg/mg in each segment of hair (0-1, 1-2, 2-3 and 3 cm to the tip).     

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C₁₈ column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2 → 120.2 and 152.1 → 110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1–500 μg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.