Implementation and Performance of the Gas Chromatography/Combustion/Isotope Ratio Mass Spectrometry-Based Method for the Confirmatory Analysis of Endogenous Anabolic Steroids during the Rio de Janeiro Olympic and Paralympic Games 2016.

Carbon isotope ratio (CIR) confirmation is one of the most complex and delicate analyses in the doping control field, due to the nature of the molecules to be confirmed, normally present in urinary samples as a consequence of an endogenous production. The requirements for method validation established by the World Anti-Doping Agency (WADA) have been pushing the accredited laboratories to improve their methods. The choice of the method is always a cost benefit ratio involving a hard-working and time-consuming analysis and the guarantee of reporting of reliable results. This work presents the method fully validated by the Brazilian Doping Control Laboratory as part of the preparation for the Rio de Janeiro Summer Olympic and Paralympic Games 2016. Sample preparation encompassed solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, acetylation, and purification by preparative high-performance liquid chromatography, and analyses were performed by gas chromatography/combustion/isotope ratio mass spectrometry. This proved to be a robust method to CIR confirmation in a big event, as demonstrated by the analysis of 179 samples during the Games 2016, from clearly negative results and adverse findings for testosterone (T) and related substances, boldenone and its metabolite, 19-norandrosterone and formestane. Two atypical findings were also reported for T and metabolites.

Development and validation of a multidimensional gas chromatography/combustion/isotope ratio mass spectrometry-based test method for analyzing urinary steroids in doping controls.

The misuse of the steroid hormone testosterone for performance enhancement has been frequently reported in the past, and its administration is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Testosterone is produced endogenously in human. Endogenous and exogenous testosterone together with their metabolites can be unambiguously distinguished by means of their carbon isotope ratios if compared to endogenous reference compounds. Established isotope ratio mass spectrometry methods for analyzing urinary steroids for doping control purposes consist of up to two time-consuming HPLC purification steps to achieve the required purity of all analytes. In order to accelerate the sample preparation, multidimensional gas chromatography was applied. This technique is known to be suitable for the separation of complex matrices. Multidimensional gas chromatography consists of two gas chromatographs connected by a pressure-controlled heart-cutting device. In the first dimension, a less polar capillary column was installed for peak purification. In the second dimension, separation was achieved employing a column of medium polarity. Retention time stability and transfer windows were monitored by a flame ionization detector. Detection was performed simultaneously by isotope ratio mass spectrometry and a single quadrupole mass spectrometer for analyte identity confirmation and assessment of peak purity. Instead of two working days required for the HPLC-based routine method, the sample preparation is shortened by the herein presented approach to one working day. For glucuronic acid-conjugated steroids, sample pretreatment is based on solid-phase extraction, liquid-liquid extraction, enzymatic hydrolysis, and derivatization of the target analytes to their corresponding acetates. These steroid acetates are divided according to their polarity into two fractions by solid phase extraction. Further, sulfoconjugated steroids are processed by Pseudomonas aeruginosa arylsulfatase and extracted following a recently established procedure. Following WADA guidelines, the method was validated by determining the parameters linear range, limit of quantification, intra- and interday precision, accuracy and specificity utilizing linear mixing models. Additionally, a reference population (n = 74) was investigated and the obtained data were compared to the established method. An excretion study was also conducted with 4-androstenedione to prove the fit for purpose of the methodology. The results demonstrate that the method is suitable for an application in routine doping control analysis.

Doping control analysis at the Rio 2016 Olympic and Paralympic Games.

This paper summarises the results obtained from the doping control analyses performed during the Summer XXXI Olympic Games (August 3-21, 2016) and the XV Paralympic Games (September 7-18, 2016). The analyses of all doping control samples were performed at the Brazilian Doping Control Laboratory (LBCD), a World Anti-Doping Agency (WADA)-accredited laboratory located in Rio de Janeiro, Brazil. A new facility at Rio de Janeiro Federal University (UFRJ) was built and fully operated by over 700 professionals, including Brazilian and international scientists, administrative staff, and volunteers. For the Olympic Games, 4913 samples were analysed. In 29 specimens, the presence of a prohibited substance was confirmed, resulting in adverse analytical findings (AAFs). For the Paralympic Games, 1687 samples were analysed, 12 of which were reported as AAFs. For both events, 82.8% of the samples were urine, and 17.2% were blood samples. In total, more than 31 000 analytical procedures were conducted. New WADA technical documents were fully implemented; consequently, state-of-the-art analytical toxicology instrumentation and strategies were applied during the Games, including different types of mass spectrometry (MS) analysers, peptide, and protein detection strategies, endogenous steroid profile measurements, and blood analysis. This enormous investment yielded one of the largest Olympic legacies in Brazil and South America. Copyright © 2017 John Wiley & Sons, Ltd.

Optimization of an online heart-cutting multidimensional gas chromatography clean-up step for isotopic ratio mass spectrometry and simultaneous quadrupole mass spectrometry measurements of endogenous anabolic steroid in urine.

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5ß-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd.

Thermodynamic-based retention time predictions of endogenous steroids in comprehensive two-dimensional gas chromatography.

This work evaluates the application of a thermodynamic model to comprehensive two-dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometry for anabolic agent investigation. Doping control deals with hundreds of drugs that are prohibited in sports. Drug discovery in biological matrices is a challenging task that requires powerful tools when one is faced with the rapidly changing designer drug landscape. In this work, a thermodynamic model developed for the prediction of both primary and secondary retention times in GC × GC has been applied to trimethylsilylated hydroxyl (O-TMS)- and methoxime-trimethylsilylated carbonyl (MO-TMS)-derivatized endogenous steroids. This model was previously demonstrated on a pneumatically modulated GC × GC system, and is applied for the first time to a thermally modulated GC × GC system. Preliminary one-dimensional experiments allowed the calculation of thermodynamic parameters (ΔH, ΔS, and ΔC p ) which were successfully applied for the prediction of the analytes' interactions with the stationary phases of both the first-dimension column and the second-dimension column. The model was able to predict both first-dimension and second-dimension retention times with high accuracy compared with the GC × GC experimental measurements. Maximum differences of -8.22 s in the first dimension and 0.4 s in the second dimension were encountered for the O-TMS derivatives of 11ß-hydroxyandrosterone and 11-ketoetiocholanolone, respectively. For the MO-TMS derivatives, the largest discrepancies were from testosterone (9.65 ) for the first-dimension retention times and 11-keto-etiocholanolone (0.4 s) for the second-dimension retention times.

Comparative analysis of three Australian finger lime (Citrus australasica) cultivars: identification of unique citrus chemotypes and new volatile molecules.

The volatile constituents of the peel of three cultivars of Australian finger lime (Citrus australasica) were investigated: Alstonville, Judy's Everbearing and Durham's Emerald. Both qualitative and quantitative GC-MS analyses were performed on their peel solvent extract. The results showed that the unique phenotypes of finger lime are also correlated to unique molecular compositions. Each cultivar revealed a different chemotype: limonene/sabinene for cv. Alstonville, limonene/citronellal/isomenthone for cv. Judy's Everbearing, and limonene/citronellal/ citronellol for cv. Durham's Emerald. To the best of our knowledge, these chemotypes have never been reported in any other citrus species. Furthermore, the amounts of some volatile constituents (γ-terpinene, α-pinene, ß-pinene, citral), which are generally the major constituents besides limonene in lime species, were surprisingly low in the three cultivars. Comparative GC-MS analysis also showed that some volatile molecules tended to be specific to one cultivar and could therefore be considered as markers. Moreover six molecules were reported for the first time in a citrus extract and confirmed by synthesis. Heart-cutting enantioselective two-dimensional GC-MS was performed to determine the enantiomeric distribution of the major chiral constituents. The combined data on three finger lime cultivars gives evidence of their divergence from other citrus species.

Multidimensional gas chromatography hyphenated to mass spectrometry and olfactometry for the volatile analysis of citrus hybrid peel extract.

In this work, the volatile composition of the peel extract of limequat, a natural citrus hybrid, was investigated by using both conventional (gas chromatography-mass spectrometry [GC-MS], deconvolution) and more advanced (multidimensional GC-MS/olfactometry [MDGC-MS/O] and enantioselective [Es]-MDGC-MS/O) analytical techniques. Although the GC-MS analysis identified most of the components, some peaks remained unidentified. These unknown peaks were elucidated by deconvolution processing and using a selectable one-dimensional or two-dimensional GC-MS system equipped with an olfactometry port. The MDGC results, with both polar and chiral second dimensions, hyphenated to mass spectrometry and olfactometry, proved to be particularly useful for the identification and chiral characterization of coeluted trace compounds. In particular, the application of the Es-MDGC-MS/O configurations was used to confirm, by on-line enrichment, the presence of cis-δ-jasminlactone. This technique was fundamental for the reliable identification and exact determination of the enantiomeric distribution of this chiral compound, as well as for the sensorial evaluation of each enantiomer at the sniffing port.

Reliable identification of pesticides using linear retention indices as an active tool in gas chromatographic-mass spectrometric analysis.

The present research is focused on the current development and employment of a dedicated pesticide mass spectral library, characterized by a double-filter search procedure: the first is based on the degree of spectral similarity and the second on chromatographic retention information (linear retention indices). The highly pure mass spectra contained in the library have been attained by subjecting mixtures of widely used pesticides to comprehensive GC-MS analysis. Linear retention indices for conventional GC-MS analyses were calculated by injecting the same compounds on a single-column GC-MS system. The effectiveness of this approach was verified by analysing a white wine sample spiked with 10 structurally similar pyrethroid compounds, through automated solid-phase microextraction-GC-MS.

Fast gas chromatography-full scan quadrupole mass spectrometry for the determination of allergens in fragrances.

The Scientific Committee for Cosmetics and NonFood Products in the 7th Amendment to the European Cosmetics Directive established 26 fragrance components, widely used in cosmetic products, as being responsible for a series of contact allergies (Directive 2003/15/EC, Official Journal of the European Union, L66/26, 11.3.2003). The regulation foresees that any allergen, present in excess of 100 mg/kg in rinse-off and of 10 mg/kg in leave-on formulations, must be reported on the label of the product. The present research reports a fast GC-full scan quadrupole mass spectrometric method (under 5 min) for the qualitative/quantitative analysis of allergens in perfumes. Reliable peak identification was achieved through a twin-filtered MS library matching procedure, considering a minimum degree of spectral similarity (90%) and retention data (a linear retention index window was applied). Peak quantification was carried out by using a specific extracted ion. In case a suspected allergen fell within its retention time window but presented a low degree of spectral purity (< 90%), analyte determination was achieved by using three extracted ions (one quantifier and two qualifiers). The fast GC-MS method was validated in terms of intraday retention time and peak area precision, LODs and LOQs, and method linearity. Finally, peak skewing was also evaluated and was within more than acceptable limits.

Comprehensive gas chromatography coupled to mass spectrometry for the separation of pesticides in a very complex matrix.

The present research is focused on the development of a comprehensive two-dimensional gas chromatography-rapid scanning quadrupole mass spectrometric (GC x GC-qMS) methodology for the analysis of trace-amount pesticides contained in a complex real-world sample. Reliable peak assignment was carried out by using a recently developed, dedicated pesticide MS library (for comprehensive GC analysis), characterized by a twin-filter search procedure, the first based on a minimum degree of spectral similarity and the second on the interactive use of linear retention indices (LRI). The library was constructed by subjecting mixtures of commonly used pesticides to GC x GC-qMS analysis and then deriving their pure mass spectra and LRI values. In order to verify the effectiveness of the approach, a pesticide-contaminated red grapefruit extract was analysed. The certainty of peak assignment was attained by exploiting both the enhanced separation power of dual-oven GC x GC and the highly effective search procedure.

Generation of improved gas linear velocities in a comprehensive two-dimensional gas chromatography system.

A comprehensive two-dimensional gas chromatography (GC x GC) system (for convenience defined as "split flow" GC x GC), which may be operated at improved gas linear velocities in both dimensions, has been developed. The setup is formed of an apolar 30 m x 0.25 mm i.d. column connected, by means of a Y press fit, to a detector-linked 1 m x 0.1 mm i.d. polar analytical column, which passes through the (cryogenic) modulator, and to a 0.3 m x 0.1 mm i.d. retention gap, which is connected to a manually operated split valve. The latter enables the regulation of gas flows through the second analytical column [e.g., 60:40 (FID) ratio, 50:50 ratio, 40:60 (FID) ratio, etc.], in order to generate the most appropriate gas linear velocity, which is related to each specific analysis. In the pre-sent investigation, two sets of traditional and split flow GC x GC analyses were carried out on a cod liver oil fatty acid methyl ester sample by using the same temperature programs [180-250 degrees C at (a) 3 degrees C/min and at (b) 1.3 degrees C/min] and at an average first-dimension linear velocity of approximately 35.0 cm/s; thus, primary column retention times (and therefore elution temperatures) were essentially maintained. The second-dimension linear velocity was calculated to be approximately 333 cm/s in the traditional applications, while it was split valve-regulated until the most appropriate values [(a) approximately 213 cm/s; (b) approximately 264 cm/s] were attained in the alternative applications. Substantial improvements were observed and measured in the chromatography along the y-axis, while the contour plot chemical class structure was maintained.

Odour fingerprint acquisition by means of comprehensive two-dimensional gas chromatography-olfactometry and comprehensive two-dimensional gas chromatography/mass spectrometry.

The analysis of complex matrices, such as perfumes, by means of gas chromatography-olfactometry (GC-O) can be rather imprecise due to the co-elutions, leading to a possible masking of odour-active trace-level compounds by major interferences or agglomeration of olfactive impressions resulting in unreliable olfactive characterization. To overcome these limits an innovative technique, comprehensive two-dimensional gas chromatography-olfactometry (GC x GC-O), was applied, revealing several relevant co-elutions, as in the linalool and linalyl acetate zones. A total of 177 compounds, out of these 135 odour-active, were detected by GC-O, while about 481 out of 818 compounds presented odour-activity through GC x GC-O analyses. In addition, GC/mass spectrometry (GC/MS) and GC x GC/MS analyses were also performed. Peak assignment was achieved by means of different information sources, such as GC/MS, GC x GC/MS, LRI, injection of standards and olfactive impressions.

Fast enantiomeric analysis of a complex essential oil with an innovative multidimensional gas chromatographic system.

The present research is focussed on the evaluation of a recently developed high performance multidimensional gas chromatographic (MDGC) system employed in the fast analysis of a series of chiral compounds contained in rosemary essential oil. The heart of the MDGC system consists in a simple transfer device for the rapid sequential re-injection of analyte "heart-cuts" from the first to the second dimension. The transfer system has no temperature restrictions, presents very low dead volumes and achieves multidimensional analysis through a pressure-balance mechanism. The MDGC set-up is characterized by two GC ovens (enabling independent temperature programming) and the possibility of mass spectrometric (MS) and/or flame ionization detection (FID). Multiple-cut conventional and fast MDGC-FID methods were developed and the results obtained compared, in order to evaluate the effectiveness of the system. In this respect, the rapid method provided the same analytical result in a greatly reduced time (approximately five times less). Furthermore, quali/quantitative data reproducibilty was very good. Fast MDGC was achieved by using micro-bore (0.1mm I.D.) columns in both dimensions.

Analysis of roasted coffee bean volatiles by using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

The volatile headspace from two coffee bean varieties, namely Arabica (Coffea arabica) and Robusta (Coffea canephora ex Froehner), were sampled by using solid-phase microextraction (SPME), and then analysed with comprehensive two-dimensional gas chromatography interfaced to a time-of-flight mass spectrometer (GC x GC-TOFMS). Two distinct column set combinations were investigated--an apolar-polar and polar-apolar configuration--and the separation achieved from each set was compared. Results were compared with a previous literature report for coffee analysed by GC x GC-FID, using an analogous polar-apolar column set combination, where authentic standards were used to confirm the position of the selected components in the 2D separation space. The present study provides independent mass spectral confirmation of component identity, and demonstrates that the relative, structured position of these components is comparable in the two experiments. Total ion current (TIC) chromatograms were processed using ChromaTOF automated data processing software. It was necessary to restrict the number of processed peaks to 1000 (S/N > 100), which required approximately 8 h for processing. Extracted ion chromatograms were generated using prominent fragment ions, and unique masses, to aid in analyte identification process, and was particularly useful in instances of component peak overlap, and for the identification of pyrazine analytes (e.g. 44, 88, 122 u). Semi-quantitative analysis was restricted to the 44 selcted components; however, the omission of peaks with S/N < 100, limiting the processed peaks to 1000, reduced the semi-quantitative application of the GC x GC-TOFMS method developed. Finally, results gained from GC x GC-TOFMS and GC x GC-qMS analyses were comparable with respect to spectral similarity assignments for the 44 target analytes.

Fast GC analysis with a 50 microm ID column: theory, practical aspects, and application to a highly complex sample.

This research focuses on the minimization of GC analysis times through the use of a 5 m x 0.05 mm ID x 0.05 microm (film thickness) column. Experimental minimum plate height (Hmin) and optimum linear velocity values were derived from standard compound applications, under various analytical conditions, and then related to classical chromatographic theory. Deviations from the latter are measured and discussed. Practical aspects linked to the use of such capillaries, such as column sample capacity and detector acquisition rates, are also considered. Furthermore, a fast, and what can be considered a very fast method, were applied to the separation of a fuel sample. Coefficients of variation of elution times and relative peak areas were calculated in the very fast application. All analytical results are compared with those obtained by conventional 0.25 mm ID column applications.

Fast GC for the analysis of citrus oils.

In this investigation, the gas chromatographic (GC) analysis of citrus essential oils is carried out in 3.3 min, with a speed gain of almost 14 times in comparison with traditional GC procedures. The fast method that is developed requires the application of severe experimental conditions (accelerated temperature program rates, high inlet pressures, and split ratios) and, thus, the support of adequate instrumentation. The samples investigated can be considered to be rather complex and, although a slight loss in peak resolution is observed, the overall analytical result is excellent. All data obtained are compared with that of a conventional application on the same matrices. This is done in order to evaluate the effectiveness and advantages of fast GC achieved with narrow bore columns.

Ultra-fast essential oil characterization by capillary GC on a 50 microm ID column.

A 5 m x 50 microm capillary column with 0.05 microm stationary phase film thickness, with a calculated efficiency of almost 20,000 plates per metre (under optimum conditions), was used for very fasthigh resolution GC analysis of lime essential oil. The total analysis time of this volatile essential oil was less than 90 s. Fast GC is shown to be appropriate for essential oil quality assurance analysis, and quantitative results of key components are comparable with those obtained by using conventional GC analysis. The fast GC analysis is approximately 33 times faster than the conventional GC method.

Comprehensive multidimensional GC for the characterization of roasted coffee beans.

The present investigation is based on the separation of one of the most complex food matrices: the roasted coffee bean volatile fraction. Analysis of the two main species of coffee (Arabica/Robusta) was achieved through an effective and simple sampling procedure, headspace solid-phase microextraction (SPME), and the unprecedented resolving power of comprehensive gas chromatography (GC x GC). The combination of these two techniques proved to be a powerful tool for the extraction and separation of coffee volatiles. In fact, thousands of compounds that play various roles in the constitution of coffee aroma profile were resolved in the 2-D contour plot, each occupying a specific position pinpointed by two retention time coordinates. The potential use of this method for the assessment of coffee quality and the detection of commercial fraud is discussed. The potential of GC x GC for identification and classification of unknowns was also demonstrated, as the formation of characteristic patterns for structurally related compounds was observed in the bidimensional chromatogram. Moreover, reproducibility results were supported by the use of an autosampler for SPME applications that allowed any inaccuracy arising from manual handling to be avoided.

Evaluation of fast gas chromatography and gas chromatography-mass spectrometry in the analysis of lipids.

Fast and conventional gas chromatography (GC) techniques were applied to nine different lipidic matrices (butter, lard, tallow, and peanut, corn, sunflower, soya, olive, menhaden oils). Simultaneous methylic transesterification was performed on all samples prior to GC analysis. Several practical aspects concerning high speed analysis were investigated, such as the great increase in linear velocity, the use of fast temperature ramps, column sample capacity and detection systems. Analytical results showed certain losses in resolution, balanced by a consistent reduction in analysis time. The actual time savings were variable (60-70 min) as they were dependent on the complexity of the sample while the speed enhancement factor was equal to 10.5. Peak identification was achieved by means of different information sources, such as fast GC-mass spectrometry (MS), linear retention indices and comprehensive two-dimensional (2D) gas chromatography group patterns. The method developed was shown to be applicable in routine applications on complex natural samples.

Detailed analysis and group-type separation of natural fats and oils using comprehensive two-dimensional gas chromatography.

Comprehensive gas chromatography (GC x GC) is an adequate methodology for the separation and identification of very complex samples. It is based on the coupling of two capillary columns that each give a different but substantial contribution to the unprecedented resolving power of this technique. The 2D space chromatograms that derive from GC x GC analysis have great potential for identification. This is due to the fact that the contour plot positions, pinpointed by two retention time coordinates, give characteristic patterns for specific families of compounds that can be mathematically translated. This investigation concerned the application of this principle to fatty acid methyl esters that were grouped on an equal double bond number basis. The ester samples were derived from various lipids and all underwent bidimensional analysis on two sets of columns. Peak attribution was supported by mass spectra, linear retention indices and information reported in the literature.