Achievements of supercritical fluid chromatography with mass spectrometric detection made in the field of forensic science during the last decade are reviewed. The main topics include analysis of traditional drugs of abuse (e.g. cannabis, methamphetamine) as well as new psychoactive substances (synthetic cannabinoids, cathinones and phenethylamines), doping agents (anabolic steroids, stimulants, diuretics, analgesics etc.) and chemical warfare agents. Control of food authenticity, detection of adulteration and identification of toxic substances in food are also pointed out. Main aspects of an analytical workflow, such as sample preparation, separation and detection are discussed. A special attention is paid to the performance characteristics and validation parameters of supercritical fluid chromatography-mass spectrometric methods in comparison with other separation techniques.

• MeSH
• Food Analysis MeSH
• Doping in Sports MeSH
• Mass Spectrometry methods   MeSH
• Humans MeSH
• Substance Abuse Detection MeSH
• Plant Oils chemistry   MeSH
• Psychotropic Drugs analysis   MeSH
• Chromatography, Supercritical Fluid methods   MeSH
• Illicit Drugs analysis   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Scientific and technical development contributed on the one hand greatly to easier living conditions, on the other hand there is however substantially greater danger threatening not only the life of man but also of nature, of the entire living environment. At present the problem of the possible part played by this phenomenon in reproductive disorders is in the foreground. Via the food chain heavy metals, toxic trace elements and polyhalogenic hydrocarbons penetrate into the organism. They accumulate in the organism and thus also in the reproductive tract and may have an impact on fertility. Some elements with a possible negative impact on reproductive health are in such low concentrations in tissues that only contemporary methods of their detection make it possible to map their presence in the organism. They are called trace elements. Toxic ones comprise cadmium, mercury, lead and arsenic. The mechanism of the negative action of cadmium in the organism is most probably due to its competition with the vitally important trace element - zinc. It was therefore the objective of the present investigation to trace the presence of cadmium and zinc in the organism of 100 sterile women included in an IVF programme: in blood and follicular fiuid (i.e. in a medium which surrounds the gamete - the oocyte) and to follow up their concentrations in relation to achievement pregnancy. Cadmium and zinc in blood and follicular fluid were assessed on a mass spectrometer with induction bound plasma as the source of ions (ICP-MS), Varian Co. produced in 1994. The assessed mean levels (μgf\l) of cadmium in blood (2.88 s 2.71) and foUicular fluid (1.25 s 0.55) in the group of conception cycles did not differ significantly from mean blood levels (2.82 s 2.22) and follicular levels (1.16 s 0.55) of non-conception cycles. The mean zinc levels in blood and follicular fluid did not differ either in the group of conception and non-conception cycles. Very significant are the differences in the blood and follicular fluid levels, the levels in follicular fluid being significantly lower. We may speak of a protective barrier of the oocyte formed by the follicle (probably the cells of the granulosa) against blood. Thus no relationship was found between the cadmium concentration in blood and follicular fluid of women where pregnancy was achieved and non-pregnant women. The possible cause of fertility disorders in conjunction with toxic elements is probably in damage of the granulosa cells and thus their dysfunction as regards production of steroid hormones with full impact on female fertility (hormone disruptors).

• MeSH
• Follicular Fluid analysis   MeSH
• Cadmium analysis   blood   MeSH
• Infertility, Female pathology   MeSH
• Zinc analysis   blood   MeSH

Cíl práce: Cílem práce bylo prokázat přítomnost organochlorovaných pesticidů (OCP)- DDT a jeho metabolitů a lindanu a jeho optických konformerů (alfa, beta, gama a delta konformerů hexachlorocyklohexanu – HCH), hexacyklobenzenu (HCB) v krvi a ve folikulární tekutině neplodných žen zařazených do programu in vitro fertilizace a embryotransferu (IVF + ET). Při průkazu jejich přítomnosti potvrdit nebo vyloučit jejich možnou kumulaci ve folikulární tekutině. Typ studie: Pilotní studie. Název a sídlo pracoviště: Gynekologicko-porodnická klinika 1. LF a VFN, Praha, AXYS Varilab s.r.o., Vrané nad Vltavou, Ústav hygieny a epidemiologie 1. LF UK, Praha, Euromise Centrum, Karlova univerzita a Akademie věd, Praha. Metodika: Hladiny organochlorovaných pesticidů byly stanoveny v krvi a ve folikulární tekutině 30 žen zařazených do programu IVF+ET. Pod ultrazvukovou kontrolou byl proveden transvaginální odběr folikulární tekutiny. Před zahájením anestezie byl odebrán vzorek žilní krve. Po zpracování embryologem byla zbylá folikulární tekutina a odebraná krev zamražena a odeslána do laboratoře. Vzorky byly zpracovány metodou vysokorozlišující plynové chromatografie a hmotnostní spektrometrie (HR GC-MS). Výsledky: U většiny vyšetřených žen jsme prokázali přítomnost organochlorovaných pesticidů v krvi i ve folikulární tekutině. Statistické vyhodnocení rozdílů v koncentraci prokázalo jejich kumulaci ve folikulární tekutině. Hladiny DDT a jeho metabolitů v krvi se pohybovaly od 2,8 do 6399,3 ng/g tuku, ve folikulární tekutině od 1,4 do 4 099,8 ng/g tuku. Hladiny HCB se pohybovaly v krvi od 213,5 do 5290,4 ng/g tuku, ve folikulární tekutině od 85,9 do 3255,3 ng/g tuku. Hladiny konformerů HCH byly v krvi v rozmezí od 1,0 do 259,9 ng/g tuku, ve folikulární tekutině v rozmezí 0,1 až 197,9 ng/g tuku. Medián rozdílů hladin ve folikulární tekutině a krvi je 1,47–670,13 ng/g tuku. Průměr rozdílů hladin ve folikulární tekutině a v krvi je 4,08–936,71 ng/g tuku. Závěr: Organochlorované pesticidy je možné prokázat v krvi i ve folikulární tekutině. Byla prokázána jejich kumulace ve folikulární tekutině.

Objective: The aim of this study was to detect DDT and its metabolites, lindane and its conformers (alfa, beta, gama and delta conformers of hexachlorocyklohexane – HCH) and hexacyclobenzene (HCB) in blood and follicular fluid of infertile women undergoing IVF+ET program. In the case if their detection, to confirm their cumulation in follicular fluid. Design: Pilot study. Setting: Department of Obstetrics and Gynecology, 1st Faculty of Medicine, Charles University and General Faculty Hospital, Prague, AXYS Varilab s.r.o., Vrané nad Vltavou, Institute of Hygiene and Epidemiology, 1st Faculty of Medicine Charles University and General Faculty Hospital, Prague, Euromise Centrum, Charles University and Academy of Arts, Prague, Czech republic. Methods: We detected the level of DDT, DDE, DDD, lindane and its conformers (alfa, beta, gama and delta conformers of hexachlorocyklohexane – HCH) and hexacyclobenzene (HCB) in blood and folicular fluid of 30 infertile women undergoing IVF + ET program. We recieved the follicular fluid by transvaginal punction of follicular fluid under ultrasonography control. The venous blood was taken before begining of anestesia. The follicular fluid and blood were frozen and transported to the laboratory. There the samples were examinated by the methods of gass chromatography and mass spectrometry. Results: We confirmed the possibility of detection of DDT, DDE, DDD, lindane and its conformers (alfa, beta, gama and delta conformers of hexachlorocyclohexane – HCH) and hexacyclobenzene (HCB) in blood and follicular fluid of infertile women. The differences in concentrations in blood and follicular fluid were statistically analysed. We confirmed the cumulation of DDT, DDE, DDD, lindane and its conformers (alfa, beta, gama and delta conformers of hexachlorocyclohexane – HCH) and hexacyclobenzene (HCB) in follicular fluid of infertile women. The levels of these compounds in blood differed from 2.8 to 6399.3 ng/g of fat, in follicular fluid from 1.4 to 4 099.8 ng/g of fat. Conclusion: It is possible to detect DDT, DDE, DDD, lindane and its conformers (alfa, beta, gama and delta conformers of hexachlorocyclohexane – HCH) and hexacyclobenzene (HCB) in blood and follicular fluid of infertile women. The cumulation of these xenobiotics in follicular fluid was found.

• MeSH
• Chromatography, Gas methods   statistics & numerical data   MeSH
• DDT chemistry   blood   metabolism   MeSH
• Fertilization in Vitro MeSH
• Research Support as Topic MeSH
• Follicular Fluid chemistry   MeSH
• Pesticides chemistry   blood   metabolism   MeSH
• Embryo Transfer MeSH
• Spectrometry, Mass, Secondary Ion statistics & numerical data   MeSH
• Xenobiotics chemistry   blood   MeSH
• Infertility, Female diagnosis   therapy   MeSH

The applicability of ultrahigh-performance supercritical fluid chromatography coupled with mass spectrometry (UHPSFC/MS) for the qualitative analysis of metabolites with a wide polarity range (log P: -3.89-18.95) was evaluated using a representative set of 78 standards belonging to nucleosides, biogenic amines, carbohydrates, amino acids, and lipids. The effects of the gradient shape and the percentage of water (1, 2, and 5%) were investigated on the Viridis BEH column. The screening of eight stationary phases was performed for columns with different interaction sites, such as hydrogen bonding, hydrophobic, π-π, or anionic exchange type interactions. The highest number of compounds (67) of the set studied was detected on the Torus Diol column, which provided a resolution parameter of 39. The DEA column had the second best performance with 58 detected standards and the resolution parameter of 54. The overall performance of other parameters, such as selectivity, peak height, peak area, retention time stability, asymmetry factor, and mass accuracy, led to the selection of the Diol column for the final method. The comparison of additives showed that ammonium acetate gave a superior sensitivity over ammonium formate. Moreover, the influence of the ion source on the ionization efficiency was studied by employing atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). The results proved the complementarity of both ionization techniques, but also the superior ionization capacity of the ESI source in the negative ion mode, for which 53% of the analytes were detected compared to only 7% for the APCI source. Finally, optimized analytical conditions were applied to the analysis of a pooled human plasma sample. 44 compounds from the preselected set were detected in human plasma using ESI-UHPSFC/MS in MSE mode considering both ionization modes.

• MeSH
• Amino Acids MeSH
• Atmospheric Pressure MeSH
• Chromatography, Liquid MeSH
• Mass Spectrometry MeSH
• Humans MeSH
• Chromatography, Supercritical Fluid * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

In the past two decades, supercritical fluid chromatography has evolved from a niche application to a comprehensive technology and a fully-fledged alternative to conventional high-performance liquid chromatography. In this study, we have focused on chiral separation of synthetic cathinones in gradient supercritical fluid chromatography coupled to mass spectrometry using an inverse gradient of a make-up solvent. Synthetic cathinones possess an amphetamine-like effect and, therefore, are frequently being offered on the Internet as a replacement for illicit drugs. Cathinones are chiral compounds, however, they are usually marketed and used as racemic mixtures. Since the effect of individual enantiomers can significantly vary, there is a need for the development of enantioseparation methods enabling to study the biological effects of individual enantiomers. Since cathinones are basic molecules, they are easily protonated (positively charged) under weakly acidic mobile phase conditions, which is a typical feature of supercritical mobile phases with an alcohol as an organic modifier. The positively charged species represent ideal analytes for ion exchangers, such as chiral zwitterion ion exchangers Chiralpak ZWIX (+) and Chiralpak ZWIX (-), which possess a positively and negatively charged unit in the molecular structure of the selectors. The presence of the positive charge in the selector's structure, functioning as a counter-ion for the positively charged analytes, significantly reduces the required amount of a buffer, which is plausible for hyphenation of such a separation system with mass spectrometry. For mass spectrometry hyphenated to supercritical fluid chromatography, the use of a make-up solvent is required to avoid analyte precipitation when using a low concentration of an organic co-solvent (modifier) in the super-/subcritical mobile phase. Hereby, we introduce a unique approach, which is based on the gradient introduction of the make-up to the post-column effluent. Using this approach, it is possible to keep constant the overall amount of the organic solvent (modifier and make-up) introduced into the mass spectrometer when using a gradient of the organic modifier. We show that the developed gradient elution method facilitates the chiral separation of all employed analytes, while the mobile-phase gradient compensation by the inverse make-up gradient enables their detection with high signal intensities.

• MeSH
• Alkaloids chemical synthesis   chemistry   isolation & purification   MeSH
• Mass Spectrometry methods   MeSH
• Rheology * MeSH
• Solvents chemistry   MeSH
• Stereoisomerism MeSH
• Chromatography, Supercritical Fluid methods   MeSH
• Temperature MeSH
• Pressure MeSH
• Chromatography, High Pressure Liquid MeSH
• Publication type
• Journal Article MeSH

For the authentication and adulteration control the lipidomic approach was used. The samples of cow and/or soyabean milk and their mixtures were analyzed by supercritical fluidic chromatography coupled with highresolution mass spectrometry. The obtained data were processed by statistical analysis. The results showed that the above approach and methods can be used for the determination of authenticity and adulteration control of cow and soyabean milk. The amount of foreign milk to the cow or soyabean milk can be also determined from the characteristic triacylglycerol content. This procedure, which is described above, is able to is able to detect 1 % of cow milk in soyabean milk and vice versa.

• MeSH
• Food Analysis * MeSH
• Mass Spectrometry * methods   utilization   MeSH
• Food Inspection MeSH
• Milk * MeSH
• Multivariate Analysis MeSH
• Chromatography, Supercritical Fluid * methods   utilization   MeSH
• Triglycerides analysis   MeSH
• Investigative Techniques MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH

The approaches to matrix effects determination and reduction in ultra-high performance supercritical fluid chromatography with mass spectrometry detection have been evaluated in this study using different sample preparation methods and investigation of different calibration models. Five sample preparation methods, including protein precipitation, liquid-liquid extraction, supported liquid extraction, and solid phase extraction based on both "bind and elute" and "interferent removal" modes, were optimized with an emphasis on the matrix effects and recovery of 8 forms of vitamin E, including α-, β-, γ-, and δ-tocopherols and tocotrienols, from plasma. The matrix effect evaluation included the use and comparison of external and internal calibration using three models, i.e., least square with no transformation and no weighting (1/x0), with 1/x2 weighting, and with logarithmic transformation. The calibration model with logarithmic transformation provided the lowest %-errors and the best fits. Moreover, the type of the calibration model significantly affected not only the fit of the data but also the matrix effects when evaluating them based on the comparison of calibration curve slopes. Indeed, based on the used calibration model, the matrix effects calculated from calibration slopes ranged from +92% to - 72% for α-tocopherol and from -77% to +19% in the case of δ-tocotrienol. Thus, it was crucial to calculate the matrix effect by Matuszewski's post-extraction approach at six concentration levels. Indeed, a strong concentration dependence was observed for all optimized sample preparation methods, even if the stable isotopically labelled internal standards (SIL-IS) were used for compensation. The significant differences between individual concentration levels and compounds were observed, even when the tested calibration range covered only one order of magnitude. In methods with wider calibration ranges, the inappropriate use of calibration slope comparison instead of the post-extraction addition approach could result in false negative results of matrix effects. In the selected example of vitamin E, solid-phase extraction was the least affected by matrix effects when used in interferent removal mode, but supported liquid extraction resulted in the highest recoveries. We showed that the calibration model, the use of a SIL-IS, and the analyte concentration level played a crucial role in the matrix effects. Moreover, the matrix effects can significantly differ for compounds with similar physicochemical properties and close retention times. Thus, in all bioanalytical applications, where different analytes are typically determined in one analytical run, it is necessary to carefully select the data processing in addition to the method for the sample preparation, SIL-IS, and chromatography.

• MeSH
• Solid Phase Extraction methods   MeSH
• Mass Spectrometry * methods   MeSH
• Calibration MeSH
• Humans MeSH
• Chromatography, Supercritical Fluid * methods   MeSH
• Vitamin E * blood   analysis   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Cíl práce: Cílem studie bylo prokázat přítomnost toxických polychlorovaných bifenylů v krvi a ve folikulární tekutině žen zařazených do programu IVF+ET a určit hladiny jednotlivých kongenerů. Prokázat, zda se toxické bifenyly nekumulují ve folikulární tekutině. Typ studie: Pilotní studie. Název a sídlo pracoviště: Gynekologicko-porodnická klinika 1. LF a VFN, Praha, AXYS Varilab s.r.o., Vrané nad Vltavou, Ústav hygieny a epidemiologie 1. LF UK, Praha, Euromise Centrum, Karlova Univerzita a Akademie věd, Praha. Metodika: Hladiny toxických polychlorovaných bifenylů byly stanoveny v krvi a folikulární tekutině 30 žen zařazených do programu IVF+ET. Folikulární tekutina byla odebrána transvaginálně pod ultrazvukovou kontrolou. Před úvodem do anestezie byl odebrán vzorek venózní krve. Folikulární tekutina a krev byly zamraženy a předány do laboratoře. Vzorky byly zpracovány metodou vysokorozlišující hmotnostní spektrometrie a plynové chromatografie. Analytická metoda zachycovala všechny existující kongenery PCB s 3 až 7 atomy chloru. Výsledky: Vyšetřeny byly hladiny PCB 77, 81, 105, 114, 118+123, 126, 156, 157, 167, 169, 189. Hladiny toxických bifenylů byly stanoveny v ng na gram tuku navážky. Hladiny se pohybovaly od nedetekovatelných hladin až do 400 ng/g tuku navážky. Statistické vyhodnocení bylo provedeno t testem a Wilcoxonovým testem. Všechny bifenyly (kromě PCB 126) jsou více zastoupeny ve folikulární tekutině. Závěr: PCB jsou přítomny v krvi i ve folikulární tekutině neplodných pacientek zařazených do programu IVF+ET. Většina stanovovaných xenobiotik se kumuluje ve folikulární tekutině. V dalším sledování bude analyzován vliv těchto látek na úspěšnost programu IVF+ET.

Objective: The aim of this study was to confirm the possibility of detection of toxic polychlorinated biphenyls in blood and follicular fluid of infertile women undergoing IVF+ET program and determine the levels of some congeners. To confirm their cumulation in follicular fluid. Design: Pilot study. Setting: Department of Obstetrics and Gynecology, 1st Faculty of Medicine, Charles University and General Faculty Hospital, Prague, AXYS Varilab s.r.o., Vrané nad Vltavou, Institute of Hygieny and Epidemiology, 1st Faculty of Medicine Charles University and General Faculty Hospital, Prague, Euromise Centrum, Charles University, Prague. Methods: We detected the level of polychlorinated biphenyls in blood and follicular fluid of infertile women undergoing IVF+ET program. We recieved the follicular fluid by transvaginal punction of follicles under ultrasonography control. The blood was taken before begining of anestezia. The follicular fluid and blood were frozen and transported to the laboratory. The samples were examined there by methods of gas chromatography and mass spectrometry. We were able to find all PCBs with 3–7 atoms of chlorine. Results: We confirmed the possibility of detection of polychlorinated biphenyls (PCBs) in blood and follicular fluid of infertile women. We examineted the levels of PCB 77, 81, 105, 114, 118+123, 126, 156, 157, 167, 169, 189. The levels of PCBs were in ng/gram of fat. The levels of polychlorinated biphenyls differed from 0 to 400 ng/g of fat. Statistical analysis was made by t test a Wilcox test. All PCBs are cumulated in follicular fluid, except of PCB 126. Conclusion: The possibility of detection of PCBs in blood and follicular fluid of infertile women undergoing IVF+ET program was confirmed. The cumulation of these xenobiotics in follicular fluid was found. In the future we will analyse the relationship between the presence of these xenobiotics and achieving succesful pregnancy.

• MeSH
• Chromatography, Gas methods   instrumentation   statistics & numerical data   MeSH
• Research Support as Topic MeSH
• Follicular Fluid chemistry   ultrasonography   MeSH
• Humans MeSH
• Pilot Projects MeSH
• Polychlorinated Biphenyls blood   MeSH
• Reference Values MeSH
• Spectrometry, Mass, Fast Atom Bombardment methods   instrumentation   statistics & numerical data   MeSH
• Xenobiotics chemistry   blood   MeSH
• Infertility, Female diagnosis   etiology   therapy   MeSH
• Check Tag
• Humans MeSH
• Female MeSH
• Publication type
• Comparative Study MeSH

The new ultra-high performance liquid chromatography method with tandem mass spectrometry and fluorescence detection allowing fast, selective, and high-throughput analysis of neopterin, kynurenine, tryptophan, and creatinine in gingival crevicular fluid (GCF) has been optimized. Defining the pathophysiology of periodontal disease and identification of potential diagnostic test for active periodontitis remains a significant challenge in the field of oral disease diagnosis. Analysis of GCF provides a non-invasive means of evaluating the role of the host response in periodontal disease. In addition, the analysis of GCF provides an information about current inflammation level of sampled site/tooth. Determination of GCF inflammatory biomarkers such as neopterin, kynurenine, and tryptophan can contribute to diagnosis, evaluation of treatment, and progression of periodontal diseases such as gingivitis and periodontitis. The separation of target analytes was carried out using a column KinetexTM Polar C18 100 Å, (100 × 3.0 mm) packed with 2.6 μm core-shell particles applying an elution with a gradient formed from 0.2% aqueous formic acid and 90% aqueous acetonitrile. Kynurenine, tryptophan, and creatinine were detected using mass spectrometry with electrospray ionization to improve the sensitivity while neopterin was detected using fluorescence detection. The separation of these four substances was achieved after using a very simple sample preparation technique convenient for small amount of biological sample. Only less than 20 μL sample was needed and the separation was completed in 4 min. MS/MS analysis was performed using multiple reaction monitoring (MRM) under a positive ionization mode. Deuterium labeled internal standard was used for the more precise quantification. The lower limits of quantification (LLOQ) for target analytes were 0.50 × 10-3 μmol/L for neopterin, 0.10 μmol/L for kynurenine, and 0.20 μmol/L for tryptophan and creatinine. The within-run and between-run accuracy were in a range of 96.67-114.77% for all quality controls and LLOQ of all analytes. Matrix effect, extraction recovery, and stability testing have all been investigated. The method was tested with real-life samples using GCF collected from patients suffering from periodontitis and from healthy controls. Neopterin levels in patients were significantly higher (P = 0.020) than in healthy subjects and indicate good potential of this method for using in evaluation of periodontal pathogenesis and healing outcomes following a treatment.

• MeSH
• Biomarkers analysis   MeSH
• Chromatography, Liquid MeSH
• Gingival Crevicular Fluid chemistry   MeSH
• Humans MeSH
• Periodontitis * diagnosis   MeSH
• Tandem Mass Spectrometry * MeSH
• Chromatography, High Pressure Liquid MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

New psychoactive substances represent serious social and health problem as tens of new compounds are detected in Europe annually. They often show structural proximity or even isomerism, which complicates their analysis. Two methods based on ultra high performance supercritical fluid chromatography and ultra high performance liquid chromatography with mass spectrometric detection were validated and compared. A simple dilute-filter-and-shoot protocol utilizing propan-2-ol or methanol for supercritical fluid or liquid chromatography, respectively, was proposed to detect and quantify 15 cathinones and phenethylamines in human urine. Both methods offered fast separation (<3 min) and short total analysis time. Precision was well <15% with a few exceptions in liquid chromatography. Limits of detection in urine ranged from 0.01 to 2.3 ng/mL, except for cathinone (5 ng/mL) in supercritical fluid chromatography. Nevertheless, this technique distinguished all analytes including four pairs of isomers, while liquid chromatography was unable to resolve fluoromethcathinone regioisomers. Concerning matrix effects and recoveries, supercritical fluid chromatography produced more uniform results for different compounds and at different concentration levels. This work demonstrates the performance and reliability of supercritical fluid chromatography and corroborates its applicability as an alternative tool for analysis of new psychoactive substances in biological matrixes.

• MeSH
• Alkaloids MeSH
• Urinalysis methods   MeSH
• Phenethylamines urine   MeSH
• Mass Spectrometry MeSH
• Calibration MeSH
• Humans MeSH
• Limit of Detection MeSH
• Methanol urine   MeSH
• Psychotropic Drugs analysis   MeSH
• Reproducibility of Results MeSH
• Solvents MeSH
• Chromatography, Supercritical Fluid * MeSH
• Chromatography, High Pressure Liquid * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH