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

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

The analysis of the home-made poppy straw extracts (so-called “compote“). Part 2 – estimation of the LD50 dose for non-purified and purified samples

resence of microbiological, inorganic and organic pollutants in home-made preserves of the poppy straw extracts – “compote”. These pollutants may influence its toxicity, however, are lacking of proper experimental studies. The authors have decided to develop the method of “compote” treatment and to determine and to compare LD50 dose for its treated form, raw form and standard morphine solution. Materials and methods: The research material was poppy straw extracts delivered by law enforcement authorities. Concentration of opiates in raw “compote” was determined using the high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Secured “compote” (1000 ml) was cleaned from plant“s ballast and other contaminations. LD50 was determined according to Litchfield-Wilcoxon method with reference to morphine. In the research thirty six 3-months-old, 200–250 g, male Sprague-Dawley rats were used. The animals were injected intraperitoneally once with appropriate extracts in three diversified doses. Results: Calculated LD50 doses of acute intraperitoneal toxicity were: for the raw extract – 83.8 mg/kg body weight; for treated extract – 51.9; for morphine hydrochloride’ solution – 61.1. Conclusions: The lowest value of LD50 for treated “compote” is attesting of its acute toxicity in comparison to the raw “compote” and morphine solution. The developed method of treating the “compote” allows for the reduction of pollutants, so it may be applied in the experimental research of both forms of drugs

Plasma homocysteine concentrations in mothers and term and preterm newborns

Abstract Aim: To assess the correlation between homocysteine concentrations and gestational age, gender, Apgar score, complications in pregnancy, delivery modalities and levels of vitamin B12 and foliate. Material and methods: Concentration of homocysteine, vitamins-B12, foliate were measured in cord blood and mother blood. There were 40 full term babies and 38 preterm babies and their mothers. Result: The homocysteine concentration in newborns correlated with homocysteine level in mothers. There was no difference in homocysteine level regardless of newborn’s gender. There was no correlation in the homocysteine concentration of mother’s blood and cord blood with the levels of vitamin B12 and foliate. In full term newborns a significant increase in homocysteine levels in comparison with premature babies was observed (7.2±1.4μmol/l vs. 6.4±1.3μmol/l; p=0.01). Additionally, negative correlation between the mothers’ age and homocysteine concentration (r=-0.23; p=0.04) and positive correlation between homocysteine concentration in cord plasma and gestation age (r=0.28; p=0.01) were found. Conclusion: Homocysteine concentration depends on gestational age, Apgar score and mother’s age. There is no correlation between homocysteine level and hypertension during pregnancy, type of delivery, levels of vitamin B12 and foliate. Determination of homocysteine level is therefore of no significant importance in newborns pathophysiology

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

Silent Bird Poisoning in Poland: Reconfirmation of Bromadiolone and Warfarin as the Proximal Causes Using GC-MS/MS-Based Methodology for Forensic Investigations

The extensive use of rodenticides poses a severe threat to non-target species, particularly birds of prey and scavengers. In this study, a GC-MS/MS-based method was used to unlock the cause of bird deaths in Poland. Organs (liver, heart, kidney, and lungs) collected during autopsies of two rooks (Corvus frugilegus) and one carrion crow (Corvus corone corone), as well as fecal samples, were analyzed for the presence of anticoagulant coumarin derivatives, i.e., warfarin and bromadiolone. As for warfarin, the highest concentration was found in crow samples overall, with concentrations in the feces and lungs at 5.812 ± 0.368 µg/g and 4.840 ± 0.256 µg/g, respectively. The heart showed the lowest concentration of this compound (0.128 ± 0.01 µg/g). In the case of bromadiolone, the highest concentration was recorded in the liver of a rook (16.659 ± 1.499 µg/g) and this concentration significantly exceeded the levels in the other samples. By revealing the reality of the threat, these discoveries emphasize the need to regulate and monitor the trade in rodenticides

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

Abstract 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