Development, Validation, and Two-Year Application of Rapid and Simple LC-MS/MS-Based Method for the Determination of K2MK-7 in Blood Samples.

Biological properties of menaquinone-7, one of the vitamin K2 vitamers (K2MK-7), both those proven and those that remain to be investigated, arouse extensive interest that goes beyond the strictly scientific framework. The most important of them is the prevention of age-related diseases, considering that we live in the times identified as the era of aging societies and many people are exposed to the vitamin K2MK-7 deficiency. Therefore, an effective analytical protocol that can be adopted as a diagnostic and preventive analytics tool is needed. Herein, a simple sample preparation method followed by the liquid chromatography-tandem mass spectrometry-based method (LC-MS/MS), was used for the selective and sensitive determination of K2MK-7 in serum samples. Under the optimized conditions, using 500 µL of serum and the same amount of n-hexane, the reproducibility and the accuracy were obtained in the ranges of 89-97% and 86-110%, respectively, and the limit of detection value was 0.01 ng/mL. This method was used for the routine analysis. Statistical interpretation of the data from 518 samples obtained during 2 years of practice allowed for obtaining information on the content and distribution of K2MK-7 in the Polish population, broken down by the sex and age groups.

Development of innovative methodology for determination of 6-thioguanine in whole blood erythrocytes by HPLC-PDA-based technique for medical diagnostics purposes.

6-Thioguanine is an immunosuppressive drug, an analogue of guanine, applied to treat acute leukemia and inflammatory bowel disease. Excessive use of 6-thioguanine during clinical treatment may cause side effects. Moreover, providing a dose too low will be ineffective. Therefore, there is a critical need for a rapid, selective and routine approach to quantifying 6-thioguanine in body fluids to support a clinical application. A fully validated HPLC method has been developed to determine 6-thioguanine in whole blood samples using 5-bromouracil as an internal standard. 6-Thioguanine nucleotides were released from erythrocytes by perchloric acid, and then hydrolysed at 100 °C to the parent thiopurine, 6-thioguanine. The following validation parameters of the method were determined: specificity/selectivity, linearity range (479-17,118 ng/mL, R > 0.992), limits of detection (150 ng/mL) and quantification (479 ng/mL), accuracy (- 5.6 < Bias < 14.7), repeatability (CV 1.30-3.24%), intermediate precision (CV 4.19-5.78%), extraction recovery (79.1-103.6%) and carryover. Furthermore, the stability of the drug in whole blood samples under various storage conditions was investigated. The suggested method is suitable for determining 6-thioguanine in whole blood erythrocyte samples for drug level monitoring, thus correct dosing.

The Development of New Methodology for Determination of Vincristine (VCR) in Human Serum Using LC-MS/MS-Based Method for Medical Diagnostics.

In this article, we have presented the development and validation of a rapid and sensitive reversed-phase liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of vincristine (VCR) in patient serum samples. Chromatographic separation was achieved on a Kinetex® (Singapore) column using a mobile phase consisting of 25 mM acetic acid and 0.3% formic acid (A) and methanol (B) in a gradient elution mode at a flow rate of 0.3 mL/min. The VCR and internal standard (vinblastine) were monitored using the multiple reaction monitoring mode under positive electrospray ionization. The lower limit of quantification (LLOQ) was 0.67 ng/mL, and the upper limit of quantification (ULOQ) was 250 ng/mL for VCR. The calculated values of LOD and LOQ for VCR were 0.075 and 0.228 ng/mL, respectively. The calibration curve was linear over the VCR concentration range of 1.0−250 ng/mL in serum. The intra- and inter-day precision and precision were within the generally accepted criteria for the bioanalytical method (<15%). The method was successfully applied to the analysis of serum samples in clinical practice.

Comparison of the antioxidant properties of serum and plasma samples as well as glutathione under environmental and pharmacological stress factors involving different classes of drugs.

We compared the antioxidant activity of serum and plasma samples of a known glutathione content with the activity of glutathione, whilst determining to what extent various stress factors might change the activity of the tested samples. Copper ions and benzene were used as examples of environmental stress factors, and xenobiotics in the form of representatives of various groups of drugs, were used as examples of pharmacological stressors at therapeutic ranges. The activity was assessed by the ABTS, ORAC, FRAP and CUPRAC methods. Glutathione content was measured by the HPLC-FD method. During the experiments, plasma samples were shown to be more resistant to oxidative stress. Moreover, the important role of environmental xenobiotics in oxidative stress was revealed, as well as the differentiated influence of pharmaceutical xenobiotics. Among all pharmaceutical xenobiotics tested, including representatives of antiarrhythmic, antiepileptic, cytostatic and mucolytic drugs, the greatest stress was shown for antiarrhythmic drugs and cytostatics.

A fully validated HPLC-UV method for determination of sulthiame in human serum/plasma samples.

Sulthiame is an old antiepileptic medicine with controversial history, whose effectiveness and safety in use have been stated in some current studies. However, there is still a need for further clinical examinations for confirmation of its usefulness and tolerability in monotherapy and add-on therapy for epilepsy of various etiologies. A fully validated RP HPLC-UV method for determination of sulthiame in serum/plasma samples using desethylatrazine as the internal standard was developed. The biological fluid was prepared for analysis by a simple precipitation method with acetonitrile. The following validation parameters of the method were determined: selectivity/specificity, linearity range (0.2-50.0 µl/ml, R2 > 0.9999), limits of detection (0.19 µl/ml) and quantification (0.58 µl/ml), precision (intra-day CV 1.06% and inter-day CV 1.25%), extraction recovery (~100%), accuracy (bias, -4.61-0.80%), carryover and ruggedness. Moreover, the stability of the medicine in plasma samples under different storage conditions was also tested. The usability of the method for clinical examinations was checked by analysis of serum samples originating from 19 patients treated with sulthiame. The proposed method is appropriate for determination of sulthiame in serum/plasma samples for drug monitoring purposes, as well as for pharmacokinetic studies.

Analysis of serum homocysteine in the laboratory practice - comparison of the direct chemiluminescence immunoassay and high performance liquid chromatography coupled with fluorescent detection.

INTRODUCTION: Effective diagnosis of cardiovascular diseases requires the right tools to be used enabling selective and sensitive analysis of their biomarkers. One of them is homocysteine (Hcy), nowadays determined by immunoassays and chromatographic methods. This study aims to compare the results obtained by direct chemiluminescence immunoassay (CLIA) and high performance liquid chromatography with fluorescent detection (HPLC-FD) using commercial kits. MATERIALS AND METHODS: Homocysteine concentration was determined in serum samples obtained from 101 individuals, using Atellica IM HCY (Siemens Healthineers, Erlangen, Germany) and HCY in plasma/serum - HPLC-FD (Chromsystems Instruments & Chemicals GmbH, Gräfelfing, Germany) tests validated for routine analysis. The latter was applied as a reference method. The comparability and agreement between the tested methods were evaluated using the Passing-Bablok (PB) regression analysis and the Bland-Altman (BA) method of the differences analysis. RESULTS: Studies showed that CLIA gives higher Hcy concentrations (15.7 ± 4.14 µmol/L). Passing-Bablok regression analysis of the results obtained with CLIA (y) compared with HPLC-FD (x) yielded an intercept of 0.22 (95%CI: - 2.16 to 2.46) and slope of 1.58 (95%CI: 1.33 to 1.87). Bland-Altman analysis demonstrated a systematic positive bias for CLIA of 5.85 ± 2.77 µmol/L. CONCLUSIONS: Methods disagreement precludes their interchangeability. Lower Hcy values by HPLC-FD result from its greater selectivity. High performance liquid chromatography with fluorescent detection should be considered as preferential method for analysing Hcy in blood serum as well as the recommended reference method for routine clinical analysis. This fact, however, imposes the need to establish new reference ranges.

Development and validation of GC-MS/MS method useful in diagnosing intestinal dysbiosis.

Dysbiosis is a disorder of the bacterial flora of the human digestive tract. It is usually diagnosed clinically by direct detection of an abnormal pattern of the intestinal microbiota. The intermediate diagnosis based on determining the content of microflora metabolites, considered as chemical markers of this disorder, is still rarely used. This is, among others, due to the variety of properties of compounds recognised as dysbiosis markers and as a consequence, the use of different methods for their analysis. To the best of our knowledge, there is still no analytical procedure that would allow unambiguous determination of all compounds in one procedure. In the present study, we have established a detailed method for the quantitative analysis of hydrocinnamic, citramalic, p-hydroxybenzeneacetic, tartaric, hippuric, 4-hydroxybenzoic, indoxylsulfuric, tricarballylic, 3,4-dihydroxyhydrocinnamic and benzoic acids along with DL-arabitol that employs the direct derivatization of compounds in a small volume of urine sample followed by gas chromatography - tandem mass spectrometry (GC-MS/MS). To show that the optimised method is a useful tool for chemical diagnosis of dysbiosis, it was applied for determination of the dysbiosis markers in the authentic urine samples.

Influence of the type of tree habitat on the character of co-occurrence of Fe, Mn, Zn, Cu, Pb, Ni, Cr and Co in the soil of the Tatra Mountain National Park.

The objective of the research was to determine the effect of habitat type of selected species of trees on the nature of co-occurrence of Fe, Mn, Zn, Cu, Pb, Cd, Ni, Cr and Co. The presence of speciation forms of these metals was investigated, with reference to the species composition of tree stands in selected areas of the Tatra Mountain National Park (Chocholowska Valley, Strazyska Valley, Koscieliska Valley, as well as Mala Laka Valley).Contents of selected metals in samples were determined by the flame ASA method, with an accuracy of 0.1 µg/g. In habitats dominated by maples, the Pb content in the Chocholowska Valley, unlike Koscieliska Valley covered with beeches, the Pb content in the form directly bioavailable, was twice as high. This was clearly proved in the case of Strazyska Valley where the soil in beech tree habitats contained larger quantities of exchangeable forms of Pb, than that in the Chocholowska Valley. The soil of the valleys, including the Mala Laka Valley, showed peculiar characteristic averaging of the contents of selected speciation forms of metals in the soil. Content corresponding to 10 percentile and geometrical average may be regarded as benchmarks in future studies of the Tatra Mountain National Park, or other protected areas.

[Changing the contents of Fe and Mn in the tonsils of children exposed to passive smoking and their local imission example Chorzow]. / Zmiana zawartosci Fe i Mn w migdalkach dzieci narazonych na bierne palenie i ich lokalna imisje na przykladzie Chorzowa.

The subject of the research were samples of overgrown adenoids removed by adenoidectomy 56 children, including 30 boys and 26 girls, exposure and unexposure from passive smoking, living in the administrative area of Chorzów. The statistic characteristic of Fe and Mn occurrence is presented in the thesis. The studies were carried out on the changes of Fe and Mn and other elements, (B, Al, La, Cd, Co, Cr, Cu, Ni, Pb, Zn, As, Se, Hg, V, Be, Mo, Sn, V, Ti, Sb, Bi, TI, Zr, Ca, Mg, Na, Ba, Sr, Li) respectively. The elemental composition of adenoids was determined with ICP-AES method (Inductively Coupled Plasma - Atomic Emission Spectroscopy). The studies on Fe and Mn occurrence in adenoids showed the presence of its higher concentrations in exposure boys (Fe - 116.13 microg/g; Mn - 0.70 microg/g), in comparison with exposure girls from passive smoking (93.06 microg/g; Mn - 0.57 microg/g).