Rosa Ventura

Derivatization procedures for gas chromatographic–mass spectrometric determination of xenobiotics in biological samples, with special attention to drugs of abuse and doping agents

The development of low cost MS detectors in recent years has promoted an important increase in the applicability of GC-MS system to analyze for the presence of foreign substances in the human body. Drugs and toxic agents are in vivo metabolized in such a way that more polar compounds are usually formed. Derivatization of these metabolites is often an unavoidable requirement for gas chromatographic analysis. Application of derivatization methods in recent years has been relevant, especially for silylation, acylation, alkylation and the formation of cyclic or diastereomeric derivatives. Given the relevance of drug of abuse testing in modern toxicology, main derivatization procedures for opiates, cocaine, cannabis, amphetamines, benzodiazepines and LSD have been reviewed. Papers describing the analyses of drugs of abuse in matrixes other than blood, such as hair or sweat, have received special attention. Advances in derivatization for sports drug testing have been particularly relevant for anabolic steroids, diuretics and corticosteroids. Among the several methodologies applied, the formation of trimethylsilyl, perfluoroacyl or methylated derivatives have proved to be both versatile and extensively used. Further advances in derivatization for GC-MS applications in clinical and forensic toxicology will depend on the one hand on the degree of further use of GC-MS for routine applications and, on the other hand, on the alternative progress made for developments in LC-MS or CE-MS. Last but not least, the appearance of comprehensive libraries in which reference spectra for different derivatives of many drugs and their metabolites are collected will have an important impact on the expansion of derivatization in GC-MS for toxicological applications.

Derivatization procedures for the detection of β2-agonists by gas chromatographic/mass spectrometric analysis

An evaluation of derivatization procedures for the detection of beta(2)-agonists is presented. The study was performed with the beta(2)-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol and terbutaline. Different derivatizating agents were employed, aiming to obtain derivatives with high selectivity to be used in the gas chromatographic/mass spectrometric analysis of beta(2)-agonists in biological samples. Trimethylsilylation was compared with different agents and the role of some catalysts was evaluated. Acylation, combined trimethylsilylation and acylation, and the formation of cyclic methylboronates were also studied. Sterical hindrance caused by different substituents at the nitrogen atom of the beta-ethanolamine lateral chain of beta(2)-agonist molecules is mainly responsible for differences in the abundances of the derivatives obtained. The use of catalysts produces an increase in the derivatization yield, especially for compounds with low steric hindrance (substituents with primary and secondary carbon atoms). The formation of trimethylsilyl (TMS) ethers is not influenced by structural molecular differences when only hydroxy groups are involved in derivatization. Combined trimethylsilylation and acylation showed that compounds with a secondary carbon atom linked to the nitrogen atom form mainly N-TFA-O-TMS derivatives, with a small amount of N-TMS-O-TMS derivatives. Compounds with tert-butyl substituents at the amino group (bambuterol, salbutamol and terbutaline) formed O-TMS derivatives as the main products, although a limited amount of trifluoroacylation at the nitrogen atom also occurred. Cyclic methylboronates were formed with bambuterol, clenbuterol, formoterol, salbutamol and salmeterol. Owing to hydroxy substituents in unsuitable positions for ring formation, this procedure was not effective for fenoterol and terbutaline. Mass spectra of different derivatives and tentative fragmentation profiles are also shown. For screening purpose (e.g. sports drug testing), derivatization with MSTFA or BSTFA alone is recommended as a comprehensive derivatization technique for beta(2)-agonists owing to minimal by-product formation; formation of cyclic methylboronates can be useful for confirmation purposes. Detection limits were obtained for the TMS and cyclic methylboronate derivatives using the derivatizing reagents MSTFA and trimethylboroxine, respectively. For most of the compounds, lower detection limits were found for the TMS derivatives.

Use of LC-MS/MS for the Open Detection of Steroid Metabolites Conjugated with Glucuronic Acid

In humans, conjugation with glucuronic acid is the most important phase II metabolic reaction of steroidal compounds. Glucuronoconjugated metabolites have been conventionally studied by using β-glucuronidase enzymes to release the phase I metabolites. It is well-known that hydrolysis with β-glucuronidase presents some limitations that may result in the underestimation of some conjugates. The aim of the present work was to develop and to evaluate liquid chromatography-tandem mass spectrometry (LC-MS/MS) scan methods for the open detection of steroid glucuronides in urine samples. The mass spectrometric behavior of thirteen representative steroid glucuronides, used as model compounds, was studied. Characteristic ionization and collision induced dissociation behaviors were observed depending on the steroid glucuronide structure. Neutral loss (NL of 176, 194, 211, and 229 Da) and precursor ion (PI of m/z 141, 159, and 177, in positive mode and m/z 75, 85, and 113, in negative mode) scan methods were evaluated. The NL scan method was chosen for the open detection of glucuronoconjugated steroids due to its sensitivity and the structural information provided by this method. The application of the NL scan method to urine samples collected after testosterone (T) undecanoate administration revealed the presence of two T metabolites which remain conjugated as glucuronides after an enzymatic hydrolysis of the urine. 3α,6β-Dihydroxy-5α-androstan-17-one (6β-hydroxyandrosterone) glucuronide and 3α,6β-dihydroxy-5β-androstan-17-one (6β-hydroxyetiocholanolone) glucuronide were established as the structures for these metabolites, by comparing the structure of the steroids released after chemical hydrolysis with reference materials. An increase of 50-300-fold of these metabolites after oral administration of T undecanoate was observed, proving that their determination can be useful in the doping control field. Moreover, these results exemplify that significant information might be missed, unless direct methods for the determination of steroid glucuronides are employed.

High-throughput and sensitive screening by ultra-performance liquid chromatography tandem mass spectrometry of diuretics and other doping agents.

The reliability of ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for high throughput screening in anti-doping control has been tested. A method to screen for the presence of diuretics and other doping agents in urine has been optimised and validated. The extraction procedure consisted of an alkaline extraction (pH 9.5) with ethyl acetate and salting-out effect (sodium chloride). The extracts were analysed by UPLC-MS/MS. Analysis of 34 forbidden drugs and metabolites was achieved in a total run time of 5 min, using a C18 column (100 mm × 2.1 mm i.d., 1.7 μm particle size) and a mobile phase containing deionised water and acetonitrile with formic acid, with gradient elution at a flow-rate of 0.6 mL min−1. Identification of the compounds was performed by multiple reaction monitoring, using electrospray ionisation in positive- or negative-ion mode. Precursor and product ions were studied for each compound and cone voltage and collision energy were optimised. Due to the different chemical structure of the compounds under study, extraction recoveries varied from less than 10% to 100% depending on the analyte. The limits of detection ranged from 50 ng mL−1 to 200 ng mL−1, and all the compounds comply with the requirements of quality established by the World Anti-doping Agency. Intra-assay precision was evaluated at two concentrations for each compound and, in most cases, a relative standard deviation of the signal ratio lower than 20% was obtained. The method has demonstrated to be reliable when analysing routine samples and the short analysis time resulting from a simple sample preparation and a rapid instrumental analysis allow a fast turn-around time and makes it of great interest for routine anti-doping control purposes.

Validation of qualitative chromatographic methods: strategy in antidoping control laboratories.

An experimental approach for the validation of chromatographic qualitative methods and its application in an antidoping control laboratory is described. The proposed strategy for validation of qualitative methods consists of the verification of selectivity/specificity, limit of detection (LOD), extraction recovery and repeatability (intra-assay precision). A one-day assay protocol, based on the analysis of five blank samples obtained from different sources and four replicates of control samples at two different concentrations of the analytes, has been defined to evaluate the validation parameters. The following evaluation criteria have been applied: absence of interfering substances at the retention time of the analytes in the blank samples to check the selectivity/specificity of the method, the LOD recommended by international sports authorities has to be attained, and for repeatability, the relative standard deviation should be <25% for the low concentration control sample and <15% for the high concentration control sample. Qualitative screening procedures are able to detect a great number of analytes so that extraction and analysis conditions are always a compromise for the different analytes. For this reason, no minimum acceptance criteria have been defined for data of extraction recoveries. The proposed protocol has been used for the validation of the screening and confirmation qualitative methods included in the scope of the accreditation of an antidoping control laboratory according to ISO quality standards.

Progress in the removal of di-[2-ethylhexyl]-phthalate as plasticizer in blood bags.

Polyvinylchloride (PVC) is used in blood component containers as well as in many other medical devices because it shows optimal inertness, durability, and resistance to heat and chemicals. However, the polymer itself does not provide good handling characteristics or the necessary properties for red blood cell (RBC) survival. PVC thus needs to be plasticized, and di-(2-ethylhexyl)phthalate (DEHP) has been the most common plasticizer to produce the required flexibility to PVC. However, DEHP has been found to leach out from the containers, causing toxic effects, as demonstrated mainly in rodents. It is considered to be a possible carcinogen and suspected to also produce endocrine effects especially in young males. Although controversial, it is commonly accepted that in vulnerable patients such as newborns, trauma patients, or pregnant women, high exposure to DEHP should be avoided. The replacement of the common PVC + DEHP blood bags poses technical challenges due to the positive influence of the DEHP molecules on the red blood cell (RBC) membrane, and thus it has been shown to affect RBC storage and survival after transfusion. Different approaches are thus being taken to find a suitable alternative to DEHP for blood components bags. Environmentalists even favor the substitution of the PVC to avoid not only the plasticizers but even the other residues contained in the polymer material. Consequently, whereas the simplest solution is the substitution of the DEHP by other plasticizers, alternative plasticizer-free materials are becoming explored. Even modifications of existing materials are being presented by some research groups, ranging from covering of the DEHP-containing materials to alloys or special additive solutions. Different strategies as well as the most promising approaches are presented in this review. In any case, the degree of stabilization of RBCs undergoing prolonged storage will dictate the final acceptance for such alternatives.

New potential markers for the detection of boldenone misuse

Boldenone is one of the most frequently detected anabolic androgenic steroids in doping control analysis. Boldenone misuse is commonly detected by the identification of the active drug and its main metabolite, 5β-androst-1-en-17β-ol-3-one (BM1), by gas chromatography-mass spectrometry (GC-MS), after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. However, some cases of endogenous boldenone and BM1 have been reported. Nowadays, when these compounds are detected in urine at low concentrations, isotope ratio mass spectrometry (IRMS) analysis is needed to confirm their exogenous origin. The aim of the present study was to identify boldenone metabolites conjugated with sulphate and to evaluate their potential to improve the detection of boldenone misuse in sports. Boldenone was administered to a healthy volunteer and urine samples were collected up to 56h after administration. After a liquid-liquid extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using electrospray ionisation in negative mode by monitoring the transition of m/z 365-350, specific for boldenone sulphate. Boldenone sulphate was identified in the excretion study urine samples and, moreover, another peak with the same transition was observed. Based on the MS/MS behaviour the metabolite was identified as epiboldenone sulphate. The identity was confirmed by isolation of the LC peak, solvolysis and comparison of the retention time and MS/MS spectra with an epiboldenone standard. These sulphated metabolites have not been previously reported in humans and although they account for less than 1% of the administered dose, they were still present in urine when the concentrations of the major metabolites, boldenone and BM1, were at the level of endogenous origin. The sulphated metabolites were also detected in 10 urine samples tested positive to boldenone and BM1 by GC-MS. In order to verify the usefulness of these new metabolites to discriminate between endogenous and exogenous origin of boldenone, four samples containing endogenous boldenone and BM1, confirmed by IRMS, were analysed. In 3 of the 4 samples, neither boldenone sulphate nor epiboldenone sulphate were detected, confirming that these metabolites were mainly detected after exogenous administration of boldenone. In contrast, boldenone sulphate and, in some cases, epiboldenone sulphate were present in samples with low concentrations of exogenous boldenone and BM1. Thus, boldenone and epiboldenone sulphates are additional markers for the exogenous origin of boldenone and they can be used to reduce the number of samples to be analysed by IRMS. In samples with boldenone and BM1 at the concentrations suspicion for endogenous origin, only if boldenone and epiboldenone sulphates are present, further analysis by IRMS will be needed to confirm exogenous origin.

Prenatal Phthalate Exposure and Childhood Growth and Blood Pressure: Evidence from the Spanish INMA-Sabadell Birth Cohort Study

Background Human evidence on the effects of early life phthalate exposure on obesity and cardiovascular disease risks, reported by experimental studies, is limited to a few cross-sectional studies. Objectives We evaluated the associations between prenatal phthalate exposure and childhood growth and blood pressure in a Spanish birth cohort study. Methods We assessed exposure using the average of two phthalate metabolite spot-urine concentrations collected from the mothers in the first and third pregnancy trimesters (creatinine-adjusted, n = 391). Study outcomes were the difference in age- and sex-specific z-scores for weight between birth and 6 months of age; and repeated age- and sex-specific z-scores for body mass index (BMI) at 1, 4, and 7 years; waist-to-height ratio at 4 and 7 years; and age- and height-specific z-scores for systolic and diastolic blood pressure at 4 and 7 years. Results The sum of five high-molecular-weight phthalate metabolites (ΣHMWPm) was associated with lower weight z-score difference between birth and 6 months (β per doubling of exposure = –0.41; 95% CI: –0.75, –0.06) and BMI z-scores at later ages in boys (β = –0.28; 95% CI: –0.60, 0.03) and with higher weight z-score difference (β = 0.24; 95% CI: –0.16, 0.65) and BMI z-scores in girls (β = 0.30; 95% CI: –0.04, 0.64) (p for sex interaction = 0.01 and 0.05, respectively). The sum of three low-molecular-weight phthalates (ΣLMWPm) was not significantly associated with any of the growth outcomes. ΣHMWPm and ΣLMWPm were associated with lower systolic blood pressure z-scores in girls but not in boys. Conclusions This study suggests that prenatal phthalate exposure may be associated with postnatal growth and blood pressure in a sex-specific manner. Inconsistencies with previous cross-sectional findings highlight the necessity for evaluating phthalate health effects in prospective studies. Citation Valvi D, Casas M, Romaguera D, Monfort N, Ventura R, Martinez D, Sunyer J, Vrijheid M. 2015. Prenatal phthalate exposure and childhood growth and blood pressure: evidence from the Spanish INMA-Sabadell birth cohort study. Environ Health Perspect 123:1022–1029; http://dx.doi.org/10.1289/ehp.1408887

Diagnostic evidence for the presence of β-agonists using two consecutive derivatization procedures and gas chromatography–mass spectrometric analysis

A GC-MS procedure for the detection of different beta-agonists in urine samples based on two consecutive derivatization steps is described. The derivatization procedure is based on the consecutive formation of cyclic methylboronate derivatives followed by a second derivatization step with MSTFA on the same extract, forming TMS derivatives. Injections in the GC-MS system may be carried out after each one of the derivatization steps, obtaining enough information for unambiguous identification. Limits of detection for the two derivatization steps ranged from 0.5 to 5 ng/ml. This procedure was tested with the beta-agonists bambuterol, clenbuterol, fenoterol, formoterol, salbutamol, salmeterol, alpha-hydroxy-salmeterol and terbutaline.

Alternative long-term markers for the detection of methyltestosterone misuse.

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17β-diol and 17α-methyl-5β-androstan-3α,17β-diol. The detection of these metabolites is normally performed by gas chromatography-mass spectrometry, after previous hydrolysis with β-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5β-androstan-3α,17β-diol 3α-sulphate; S2, 17β-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17β-methyl-5β-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17β-methylandrostan-4,6-dien-3-one.