Application of molecularly imprinted polymers in the anti-doping field: sample purification and compound analysis.

The problem posed by anti-doping requirements is one of the great analytical challenges; multiple compound detection at low ng ml-1 levels from complex samples, with requirements for exceptional confidence in results. This review surveys the design, synthesis and application of molecularly imprinted polymers (MIPs) in this field, focusing on the templating of androgenous anabolic steroids (AASs), as the most commonly abused substances, but also other WADA prohibited substances. Commentary on the application of these materials in detection, clean-up and sensing is offered, alongside views on the future of imprinting in this field.

Demystifying biosimilars: development, regulation and clinical use.

Biologics are an integral component in the treatment of various diseases. However, limited patient access to these medicines remains a significant global challenge, prompting development of safe and effective biosimilars. A biosimilar is 'highly similar to a reference (originator) product, for which there are no clinically meaningful differences between the two products in terms of safety, purity and potency'. Biosimilars have the potential to offer possible benefits, including lower treatment costs, thereby increasing patient access and clinical use, which may lead to better overall outcomes. Improved understanding of biosimilars may enhance confidence and trust in these agents. As increasing numbers of biosimilars achieve regulatory approval, this overview aims to address enduring knowledge gaps regarding the development and use of biosimilars.

Detection of multiple steroidal compounds in synthetic urine using comprehensive gas chromatography-mass spectrometry (GC×GC-MS) combined with a molecularly imprinted polymer clean-up protocol.

A method capable of screening for multiple steroids in urine has been developed, using a series of twelve structurally similar, and commercially relevant compounds as target analytes. A molecularly imprinted solid phase extraction clean-up step was used to make the sample suitable for injection onto a GC×GC-MS setup. Significant improvements compared to a commercially available C-18 material were observed. Each individual steroid was able to be separated and identified, using both the retention profile and diagnostic fragmentation ion monitoring abilities of the comprehensive chromatographic-mass spectrometry method. Effective LODs of between 11.7 and 27.0 pg were calculated for individual steroids, effectively equivalent to concentration levels of between 0.234 and 0.540 ng mL(-1) in urine, while the application of multiple screen was demonstrated using a 10 ng mL(-1) mixed sample. The nature of this study also removes the need for sample derivitisation which speeds up the screening process.

Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis.

RATIONALE: The phthiocerol dimycocerosates (PDIMs) are certain stable and hydrophobic waxes found in the cell membrane of Mycobacterium tuberculosis, bacteria that cause an infectious disease of growing concern worldwide. Previous studies report the analysis of derivatives of the hydrolysed PDIMs from biological samples, following complex extraction and offline derivatization of PDIMs biomarkers, prior to their analysis by gas chromatography/mass spectrometry (GC/MS). METHODS: We developed and optimized a GC/MS method based on selected ion monitoring (SIM) to detect the derivatives produced via the thermally assisted hydrolysis and methylation (THM) of the PDIMs from the cell membrane of M. tuberculosis. The extraction of PDIMs from culture is simple, and their thermochemolysis is carried out automatically online, thus avoiding the time-consuming derivatization steps of hydrolysis and esterification, usually performed offline. RESULTS: For standard PDIMs in petroleum ether, our optimized method gave an excellent linearity (R(2) = 0.99) at concentrations between 0.172 and 27.5 ng/mL, a good precision (RSD = 11.42%), and a limit of detection (LOD) of 100 pg/mL. For the PDIMs extracted from dilutions of M. tuberculosis culture, the method gave good linearity (R(2) = 0.9685) and an estimated LOD of 400 CFU/mL (CFU = colony forming units) in sterile distilled water. CONCLUSIONS: A GC/MS(SIM) method is presented for the rapid and quantitative detection of M. tuberculosis, based on the online thermochemolysis of lipidic biomarkers extracted from the bacterial culture. The method has the potential to be applied in human and veterinary clinical laboratories for the rapid diagnosis of tuberculosis in infected biological samples.

Application of comprehensive 2D chromatography in the anti-doping field: Sample identification and quantification.

Anti-doping analysis requires an exceptional level of accuracy and precision given the stakes that are at play. Current methods rely on the application of chromatographic techniques linked with mass spectrometry to provide this. However, despite the effectiveness of these techniques in achieving good selectivity and specificity, some issues still exist. In order to reach the minimum required performance level as set by WADA, labs commonly use selective monitoring by quadrupole mass spectrometry. This can be potentially fooled through the use of masking agents or by moving the peaks, as often only a small portion of the spectrum is used for analysis. Further issues exist in the inability to detect new or modified compounds, or to reanalyse samples/spectra. One technique that could overcome these problems is that of comprehensive 2D chromatography. Here a second separation column is employed to generate greater separative power. Compared to conventional separation, GCxGC allows for a greater peak capacity (i.e., number of peaks that can be resolved within a given time) and greater separation of coeluting compounds, which makes the technique promising for the complex task required in anti-doping. When combined with Time of Flight Mass Spectrometry this technique demonstrates vast potential allowing for full mass range datasets to be obtained for retroactive analysis. Similarly, LCxLC provides improvements in resolving power compared to its 1D counterpart and can be used both online as part of the analysis or offline solely as a purification step. In this review we summarise the work in this field so far, how comprehensive chromatography has been applied to anti-doping studies, and discuss the future application for this technique.

A Comparison of ß-Carotene, Phytoene and Amino Acids Production in Dunaliella salina DF 15 (CCAP 19/41) and Dunaliella salina CCAP 19/30 Using Different Light Wavelengths.

Strains of Dunaliella salina microalgae are of considerable research and industrial interest because they hyper-accumulate ß-carotene as well as produce high-quality protein. To explore the co-production of valuable compounds in D. salina, this study compared the production of ß-carotene, phytoene and amino acids in two strains cultivated under white, red or blue light until no further nitrogen was available. D. salina DF15 (CCAP 19/41 (PLY DF15)) produced more than 12% ß-carotene (ash-free dry weight (AFDW) basis), and red light triggered the production of 9-cis ß-carotene at a 9-cis/all-trans ß-carotene ratio of 1.5. Phytoene production was also evident in D. salina DF15 under all conditions, particularly under blue light. However, the profile of essential amino acids (EAAs) and calculation of the essential amino acid index (EAAI) was less than ideal in terms of protein quality, for both strains. Umami compounds, quantified as monosodium glutamate (MSG) equivalents, indicated a higher equivalent umami concentration (EUC) in D. salina DF15 under red light (3.2 g MSG/100 g AFDW) than in D. salina CCAP19/30. Overall, D. salina DF15 demonstrates valuable traits for further exploration and product optimisation.

Ion trap mass spectrometry on a comet nucleus: the Ptolemy instrument and the Rosetta space mission.

In May 2014, the Rosetta spacecraft is scheduled to rendezvous with the comet Churyumov-Gerasimenko ('67P'). One of the instruments on board the 'Lander' which will descend on to the surface of the comet is a miniaturised GC/MS system that incorporates an ion trap mass spectrometer, specially developed for isotope ratio analysis. This article describes the development and optimisation of the ion trap for this unique application, and presents a summary of the range of pre-programmed experiments that will contribute to the characterisation of the solid and volatile cometary materials.

Methods in plant foliar volatile organic compounds research.

Plants are a major atmospheric source of volatile organic compounds (VOCs). These secondary metabolic products protect plants from high-temperature stress, mediate in plant-plant and plant-insect communication, and affect our climate globally. The main challenges in plant foliar VOC research are accurate sampling, the inherent reactivity of some VOC compounds that makes them hard to detect directly, and their low concentrations. Plant VOC research relies on analytical techniques for trace gas analysis, usually based on gas chromatography and soft chemical ionization mass spectrometry. Until now, these techniques (especially the latter one) have been developed and used primarily by physicists and analytical scientists, who have used them in a wide range of scientific research areas (e.g., aroma, disease biomarkers, hazardous compound detection, atmospheric chemistry). The interdisciplinary nature of plant foliar VOC research has recently attracted the attention of biologists, bringing them into the field of applied environmental analytical sciences. In this paper, we review the sampling methods and available analytical techniques used in plant foliar VOC research to provide a comprehensive resource that will allow biologists moving into the field to choose the most appropriate approach for their studies.

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography-mass spectrometry.

A proof of principle gas chromatography-mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36-68% for PDIMs, allowing some superior detection of the target analytes.

Detection of Mycobacterium tuberculosis in sputum by gas chromatography-mass spectrometry of methyl mycocerosates released by thermochemolysis.

Tuberculosis requires rapid diagnosis to prevent further transmission and allow prompt administration of treatment. Current methods for diagnosing pulmonary tuberculosis lack sensitivity are expensive or are extremely slow. The identification of lipids using gas chromatography- electron impact mass spectrometry (GC-EI/MS) could provide an alternative solution. We have studied mycocerosic acid components of the phthiocerol dimycocerosate (PDIM) family of lipids using thermochemolysis GC-EI/MS. To facilitate use of the technology in a routine diagnostic laboratory a simple extraction procedure was employed where PDIMs were extracted from sputum using petroleum ether, a solvent of low polarity. We also investigated a method using methanolic tetramethylammonium hydroxide, which facilitates direct transesterification of acidic components to methyl esters in the inlet of the GC-MS system. This eliminates conventional chemical manipulations allowing rapid and convenient analysis of samples. When applied to an initial set of 40 sputum samples, interpretable results were obtained for 35 samples with a sensitivity relative to culture of 94% (95%CI: 69.2,100) and a specificity of 100% (95%CI: 78.1,100). However, blinded testing of a larger set of 395 sputum samples found the assay to have a sensitivity of 61.3% (95%CI: 54.9,67.3) and a specificity of 70.6% (95%CI: 62.3,77.8) when compared to culture. Using the results obtained we developed an improved set of classification criteria, which when applied in a blinded re-analysis increased the sensitivity and specificity of the assay to 64.9% (95%CI: 58.6,70.8) and 76.2% (95%CI: 68.2,82.8) respectively. Highly variable levels of background signal were observed from individual sputum samples that inhibited interpretation of the data. The diagnostic potential of using thermochemolytic GC-EI/MS of PDIM biomarkers for diagnosis of tuberculosis in sputum has been established; however, further refinements in sample processing are required to enhance the sensitivity and robustness of the test.

Qualitative drug analysis of hair extracts by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GCxGC/TOFMS) is applied to a qualitative analysis of three sample extracts from hair suspected of containing various drug compounds. The samples were also subjected to a quantitative target analysis for codeine, morphine, 6-monoacetylmorphine (6-MAM), amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethylamphetamine (MDMA), methadone, and benzylpiperazine (BZP) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). GCxGC/TOFMS provided a non-specific procedure that identified various drugs, metabolites, and impurities not included in the target analysis. They included cocaine, diazepam, and methaqualone (quaalude). Comprehensive GCxGC separation was achieved using twin-stage cryo-modulation to focus eluant from a DB-5 ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The TOF mass spectrometer provided unit mass resolution in the mass range m/z 5-1000 and rapid spectral acquisition (< or = 500spectra/s). Clean mass spectra of the individual components were obtained using mass spectral deconvolution software. The 'unknown' components were identified by comparison with mass spectra stored in a library database.

Determining the levels of volatile organic pollutants in urban air using a gas chromatography-mass spectrometry method.

The paper presents the application of a method based on coupled gas chromatography-mass spectrometry, using an isotopically labelled internal standard for the quantitative analysis of benzene (B), toluene (T), ethyl benzene (E), and o-, m-, p-xylenes (X). Their atmospheric concentrations were determined based on short-term sampling, in different sites of Cluj-Napoca, a highly populated urban centre in N-W Romania, with numerous and diversified road vehicles with internal combustion engines. The method is relatively inexpensive and simple and shows good precision and linearity in the ranges of 7-60 mug/m(3) (B), 13-90 mug/m(3) (T), 7-50 mug/m(3) (E), 10-70 mug/m(3) (X-m,p), and 20-130 mug/m(3) (X-o). The limits of quantitation/detection of the method LOQ/LOD are of 10/5 mug/m(3) (Xo), 5/3 mug/m(3) (B, E, X-m,p), and of 3/1 mug/m(3) (T), respectively.

The detection of various opiates and benzodiazepines by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS) is applied to qualitative and quantitative drug testing. Human serum was 'spiked' with known quantities of benzodiazepines and a 'street heroin' mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid-phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N-methyl-N-(tert-butyldimethyl)trifluoroacetamide (MTBSTFA) to improve the chromatographic performance. An orthogonal separation of the matrix constituents was achieved by coupling a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The eluant was focused onto the second column by a twin-stage cryo-modulator. Rapid 6 s modulation times were achieved by transfer from a 30 m x 0.25 mm (length x internal diameter) to a 2 m x 0.1 mm column. TOFMS with rapid spectral acquisition (< or =500 spectra/s) was employed in the mass range m/z 40-650. A clean mass spectrum was obtained for each analyte using mass spectral deconvolution software. The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for two benzodiazepines, flunitrazepam and its major metabolite 7-aminoflunitrazepam (7-amino-FN), in the concentration range 5-1000 ng/mL. The limits of detection (LODs) and limits of quantitation (LOQs), calculated by repeat injections (x10) of the lowest standard, were 1.6 and 5.4 ng/mL (flunitrazepam); 2.5 and 8.5 ng/mL (7-amino-FN), respectively. There is scope to extend this protocol to screen a large number of drugs and metabolites stored in a library database.