Paweł Kubica

Modern approach for determination of lactulose, mannitol and sucrose in human urine using HPLC–MS/MS for the studies of intestinal and upper digestive tract permeability

A new analytical procedure was described for the simultaneous determination of lactulose, mannitol and sucrose in urine, in which HILIC chromatography and tandem mass spectrometry detection are used. Sugars are orally administered for the estimation of intestinal permeability in children digestive tract. Samples were purified by dispersive solid phase extraction (d-SPE) using Amberlite MB150 resin. Raffinose was selected as an internal standard. The chosen chromatographic separation was carried out on ZIC(®)-HILIC column in 10 min at a flow rate of 0.3 mL/min, using mixture of acetonitrile (ACN) and ammonium acetate (NH(4)Ac) in water (H(2)O) as the mobile phase. Within-run precision (CV) measured at three concentrations was 1.08%, 0.32% and 0.49% for lactulose; 1.88%, 0.47% and 0.75% for mannitol, 2.95%, 1.31% and 0.6% for sucrose. Between-run CVs were 0.75%, 1.1% and 1.2% for lactulose; 1.1%, 1.02% and 1.01% for mannitol; 1.17%, 1.4% and 1.05% for sucrose. Analytical recovery of all three sugar probes was 95.06-99.92%. The detection limits were: 15.94 ng/mL for lactulose, 17.10 ng/mL for sucrose and 11.48 ng/mL for mannitol. The proposed method is rapid, simple, sensitive and suitable for the determination of intestinal permeability of the sugar derivatives in children.

Sensitive determination of isoprostanes in exhaled breath condensate samples with use of liquid chromatography–tandem mass spectrometry

Oxidative stress is the hallmark of various inflammatory lung diseases. Increased concentrations of reactive oxygen species in the lungs are reflected by elevated concentrations of oxidative stress markers in the breath, airways, lung tissue and blood. The aim of this work was to develop a method for the fast measurement of F2-isoprostanes in exhaled breath condensate (EBC) samples using equipment which is nowadays available and routinely exploited in analytical laboratories, liquid chromatography coupled with tandem mass spectrometry. Because of the limited volume of an EBC sample and the very low concentrations of biomarkers, we chose lyophilization as the preconcentration technique. The diastereoisomers determined show similar fragmentation patterns, which is why complete chromatographic separation with excellent peak shapes was essential for accurate quantitation. Isoprostanes were separated using a narrow-bore Agilent Extend C-18 column in isocratic elution mode using acetonitrile/methanol and water with the addition of 0.01%(v/v) formic acid. The limits of determination and quantitation for the determination of four isoprostanes in samples of EBC ranged from 1 to 3 pg/ml. The recoveries of all isoprostanes ranged from 96.7 to 101.7, with a relative standard deviation of <7%. The stability of the isoprostanes at different temperatures was measured as well.

Application of gas chromatography–tandem mass spectrometry for the determination of amphetamine-type stimulants in blood and urine

HighlightsSimple and rapid LLE‐GC–MS/MS method was developed and validated for ATS analysis.200 &mgr;L of sample and a total of 2 mL of extraction solvent was used for analysis.22 toxicological cases were investigated for ATS content. ABSTRACT Amphetamine, methamphetamine, phentermine, 3,4‐methylenedioxyamphetamine (MDA), 3,4‐methylenedioxymethamphetamine (MDMA), and 3,4‐methylenedioxy‐N‐ethylamphetamine (MDEA) are the most popular amphetamine‐type stimulants. The use of these substances is a serious societal problem worldwide. In this study, a method based on gas chromatography–tandem mass spectrometry (GC–MS/MS) with simple and rapid liquid‐liquid extraction (LLE) and derivatization was developed and validated for the simultaneous determination of the six aforementioned amphetamine derivatives in blood and urine. The detection of all compounds was based on multiple reaction monitoring (MRM) transitions. The most important advantage of the method is the minimal sample volume (as low as 200 &mgr;L) required for the extraction procedure. The validation parameters, i.e., the recovery (90.5–104%), inter‐day accuracy (94.2–109.1%) and precision (0.5–5.8%), showed the repeatability and sensitivity of the method for both matrices and indicated that the proposed procedure fulfils internationally established acceptance criteria for bioanalytical methods The procedure was successfully applied to the analysis of real blood and urine samples examined in 22 forensic toxicological cases. To the best of our knowledge, this is the first work presenting the use of GC–MS/MS for the determination of amphetamine‐type stimulants in blood and urine. In view of the low limits of detection (0.09–0.81 ng/mL), limits of quantification (0.26–2.4 ng/mL), and high selectivity, the procedure can be applied for drug monitoring in both fatal and non‐fatal intoxication cases in routine toxicology analysis.

Sensitive simultaneous determination of 19 fluorobenzoic acids in saline waters by solid-phase extraction and liquid chromatography–tandem mass spectrometry

A solid-phase extraction (SPE) procedure using C18 stationary phase was optimized for the preconcentration of 19 fluorinated derivatives of benzoic acid (FBA): mono-, di-, tri-, and tetrafluorosubstituted in the ring, trifluoromethylbenzoic acid and 3,5-bistrifluoromethyl benzoic acid from undiluted salt-rich (>20%) reservoir waters. Quantitative (>90%) retention/elution of 16 out of 19 analyte compounds was achieved allowing a fourfold preconcentration factor accompanied by the elimination of >99% of salt. For the three most polar compounds (2,6-dFBA, 2,3,6-tFBA, and 2,4,6-tFBA) the non-quantitative recoveries (>70%) were corrected by dedicated custom-synthesized deuterated internal standards. The FBAs were determined by HPLC - MS/MS revisited in terms of a choice of column, elution conditions and MS/MS signal acquisition parameters allowing the baseline separation and a gain in sensitivity. For a sample intake of 4 mL, detection limits for all the compounds in a reservoir water sample containing more than 20% salt were between 0.01 and 0.05 ng/mL which represents a gain of a factor of 10-20 in comparison with the state-of the art LC-MS/MS procedures for samples of similar complexity.

Analytical studies on ascosin, candicidin and levorin multicomponent antifungal antibiotic complexes. The stereostructure of ascosin A2.

In the class of polyene macrolides, there is a subgroup of aromatic heptaenes, which exhibit the highest antifungal activity within this type of antibiotics. Yet, due to their complex nature, aromatic heptaenes were not extensively studied and their potential as drugs is currently underexploited. Moreover, there are many inconsistencies in the literature regarding the composition and the structures of the individual components of the aromatic heptaene complexes. Inspired by one of such cases, herein we conducted the analytical studies on ascosin, candicidin and levorin using HPLC-DAD-(ESI)Q-TOF techniques. The resulting chromatograms and the molecular masses of the individual components of these three complexes strongly indicated that the major components of ascosin, candicidin and levorin are structurally identical. In order to validate these results, the main component of previously structurally uncharacterized ascosin was derivatized, isolated and subjected to 2D NMR studies. The resulting structure of the ascosin’s main component, herein named ascosin A2, was shown to be identical with the earlier reported structures of the main components of candicidin and levorin complexes: candicidin D and levorin A2. In the end, all the structural knowledge regarding these three antibiotic complexes was gathered, systematized and completed, and the new nomenclature was proposed.

Evaluation of flavour profiles in e-cigarette refill solutions using gas chromatography–tandem mass spectrometry

Many flavour compounds that are present in e-liquids for e-cigarettes are responsible for specific tastes and smoking sensations for users. Data concerning content and specific types of flavours is often limited and unknown to users. The aim of the research was to define and compare flavour profiles of e-liquids with the same group taste from different manufacturers. Gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) was used to separate and identify 90 popular compounds (98, including isomers) of interest. The developed method was validated in terms of accuracy (88-113%) for three spiking levels and the intra-day (0.2-13%) and inter-day precision (1-10%). Limits of quantitation were in the range of 10-816 ng/mL, while the matrix effects for 80% of the compounds were at negligible levels. The proposed method is rapid, simple and reliable and uses a green and modern GC-MS/MS technique. Twenty-five samples of five different flavours (tobacco, strawberry, cherry, menthol and apple) from five different producers were analysed, and the determined compounds were categorized and differentiated. The approach proposed in this study allowed for the evaluation of which compounds/group of compounds are responsible for taste and to distinguish common flavour compounds among the investigated brands for each flavour. Furthermore, the presented research can be considered in future toxicological studies.

Determination of trace levels of eleven bisphenol A analogues in human blood serum by high performance liquid chromatography–tandem mass spectrometry

Chemicals showing structural or functional similarity to bisphenol A (BPA), commonly called BPA analogues, have recently drawn scientific attention due to their common industrial and commercial application as a substitute for BPA. In the European Union, the use of BPA has been severely restricted by law due to its endocrine disrupting properties. Unfortunately, it seems that all BPA analogues show comparable biological activity, including hormonal disruption, toxicity and genotoxicity. Until now, the knowledge about human exposure to BPA analogues is scarce, mainly due to the lack of the data concerning their occurrence in human derived biological samples. This study presents the development of an analytical method for determination of trace levels of eleven BPA analogues in human blood serum samples. The method involves fast and simple liquid-liquid extraction, using low sample and solvent volumes. Chromatographic separation of analytes was optimized using one-factor-at-a-time approach (mobile phase composition, gradient shape, chromatographic column selection, separation temperature, etc.). The method allows for effective separation of the analytes, even in the case of configurational isomers (bisphenol M and bisphenol P). The calibration curves for all analytes were linear in the range tested. The limits of detection and quantitation were in the range of 0.0079÷0.039ng/mL and 0.024÷0.12ng/mL respectively. Compound-dependent recovery values were in the rage of 88÷138%. Matrix effects were mitigated with the help of matrix-matched calibration curves prepared for every batch of samples. Results obtained after the analysis of 245 real human blood serum samples indicate that human beings are exposed to different BPA analogues, that are present in the environment and in common, daily use products.

Serum bisphenol A concentrations correlate with serum testosterone levels in women with polycystic ovary syndrome

The aim of this study was to determine serum bisphenol A (BPA) concentrations using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) in women with polycystic ovary syndrome (PCOS) (n = 106, age range 18-40 yrs) and to evaluate its potential impact on their hormonal and metabolic profile. The control group consisted of age- and BMI-matched 80 eumenorrheic women with no clinical or biochemical hyperandrogenism. Our results showed that women with PCOS had significantly higher serum BPA concentrations than healthy controls (geometric mean and [95% CI]: 0.202 ng/mL [0.150; 0.255] vs. 0.154 ng/mL [0.106; 0.201], P = 0.035), which correlated positively with serum total testosterone (TST) (R=0.285, P = 0.004) and the free androgen index (FAI) (R = 0.196, P = 0.049). There were no significant correlations between serum BPA and BMI, waist circumference, serum glucose, insulin and lipids. These results point to the potential role of BPA in the pathogenesis of the ovarian hyperandrogenism in women with PCOS.

New approach for e-cigarette aerosol collection by an original automatic aerosol generator utilizing melt-blown non-woven fabric

Currently, there is lack of standardized conditions for the collection and analysis of e-cigarette (EC) aerosol. Considering the urgent need for the development of these guidelines, a procedure for EC aerosol analysis was developed. A novel automatic e-cigarette aerosol generator was designed. For the first time, melt-blown non-woven fabric was applied for the effective uptake of compounds released from vaporized e-liquid. The extraction procedure was optimized in terms of type of extraction solvent, amount of sorbent and solvent volume. For optimization, a model e-liquid containing flavour additives belonging to various chemicals group with various chemical properties was investigated. The aerosol trapping efficiency was satisfactory and was equal to 92 ± 7%. Final determination was performed by GC-MS/MS. Quantitation was based on the mass change tracking approach (MCT), which assumes the monitoring of e-liquid mass changes before and after vaping. The combination of non-woven fabric and sampling approach (MCT) was proven to be effective in acquisition of reliable data. Thus, the concentrations in aerosol and emission factors were calculated for aerosols collected during the vaping of both model e-liquids and real samples. Validation was performed by evaluating key analytical parameters, such as linearity, accuracy, precision, limit of detection (LOD) and quantitation (LOQ). For all investigated compounds, recoveries from 70% to 118% together with precision and reproducibility below 12% were achieved. The applicability of the described approach was examined by analysing EC refill solutions commercially available on the Polish market.

Intramolecular transformation of an antifungal antibiotic nystatin A1 into its isomer, iso-nystatin A1 - structural and molecular modeling studies.

Nystatin A1, a polyene macrolide antifungal antibiotic, in a slightly basic or acidic solution undergoes an intramolecular transformation, yielding a structural isomer, the translactonization product, iso‐nystatin A1 with lactone ring diminished by two carbon atoms. Structural evidence is provided by advanced NMR and Mass Spectrometry (MS) studies. Molecular dynamics simulations and quantum mechanics calculations gave the insight into the course and mechanism of the transformation and its effect on the conformation of the subject molecule. Copyright