An alternative approach to validation of liquid chromatography-mass spectrometry methods for the quantification of endogenous compounds.

Despite the progress in the quantification of xenobiotics, the development and validation of methods designed for endogenous substances still remain challenging due to the natural presence of the analytes in a biological matrix, leading to the inability to obtain a blank sample. Several generally recognized procedures are described to solve this issue, like using surrogate or analyte-depleted matrices or surrogate analytes. However, the workflows used do not always meet the requirements for developing a reliable analytical method or are cost-intensive. This study aimed to design an alternative approach for preparing validation reference samples using authentic analytical standards while preserving the nature of the biological matrix and solving the problem with the inherent presence of analyzed compounds in a studied matrix. The methodology used is based on the standard-addition type procedure. However, unlike the original method, the addition is modified according to a previously measured basal concentration of monitored substances in the pooled biological sample to obtain a predefined concentration in reference samples according to the European Medicines Agency (EMA) validation guideline. The study shows the advantages of described approach on an example of LC-MS/MS analysis of 15 bile acids in human plasma and compares it with other methods commonly used in this field. The method was successfully validated according to the EMA guideline with lower limit of quantification of 5 nmol/L and linearity in the range of 5 - 2000 nmol/L. Finally, the method was used in a metabolomic study on a cohort of pregnant women (n = 28) to confirm intrahepatic cholestasis, the major liver disease observed in pregnancy.

Effect of titanium nanostructured surface on fibroblast behavior.

As the consumption of implants increases, so do the requirements for individual types of implants, for example, improved biocompatibility or longevity. Therefore, the nano-modification of the titanium surface is often chosen. The aim was to characterize the modified surface with a focus on medical applications. The titanium surface was modified by the anodic oxidation method to form nanotubes. Subsequently, the material was characterized and analyzed for medical applications-surface morphology, surface wettability, chemical composition, and release of ions into biological fluids. A human gingival fibroblasts (HGFb) cell line was used in the viability study. A homogeneous layer of nanotubes of defined dimensions was formed on the titanium surface, ensuring the material's biocompatibility-the preparation conditions influence the resulting properties of the nanostructured surface. Nanostructured titanium exhibited more suitable characteristics (e.g., wettability, roughness, ion release) for biological applications than compared to pure titanium. It was possible to understand the behavior of the modified layer on the titanium surface and its effect on cell behavior. Another contribution of this work is the combination of material characterization (ion release) with the study of cytocompatibility (direct contact of cells with metals).

High-throughput salting-out assisted liquid-liquid extraction using a 3D printed device and its application in the quantification of ibrutinib and its metabolite PCI-45227 in human serum.

Liquid-liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid-liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1-200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of - 1.67-5.67 %, while the inter-day accuracy was in the range of 0.20-6.90 %. The intra-day precision for IBT was in the range of 3.20-4.37 % and 3.13-4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of - 11.2-3.71 % and the inter-day accuracy in the range of - 5.76-0.92 %. The intra-day precision for PCI was in the range of 3.49-7.64 % and 3.63-8.61 % for the measurement between days. In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63-3.78 ng/mL, PCI-45227 concentration range: 1.84-14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34-89.0 ng/mL, PCI-45227 concentration range: 5.64-124 ng/mL).

Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairment of Bile Acid Metabolomics in Rats.

Multidrug resistance-associated protein 2 (Mrp2) mediates biliary secretion of anionic endobiotics and xenobiotics. Genetic alteration of Mrp2 leads to conjugated hyperbilirubinemia and predisposes to the development of intrahepatic cholestasis of pregnancy (ICP), characterized by increased plasma bile acids (BAs) due to mechanisms that are incompletely understood. Therefore, this study aimed to characterize BA metabolomics during experimental Mrp2 deficiency and ICP. ICP was modeled by ethinylestradiol (EE) administration to Mrp2-deficient (TR) rats and their wild-type (WT) controls. Spectra of BAs were analyzed in plasma, bile, and stool using an advanced liquid chromatography-mass spectrometry (LC-MS) method. Changes in BA-related genes and proteins were analyzed in the liver and intestine. Vehicle-administered TR rats demonstrated higher plasma BA concentrations consistent with reduced BA biliary secretion and increased BA efflux from hepatocytes to blood via upregulated multidrug resistance-associated protein 3 (Mrp3) and multidrug resistance-associated protein 4 (Mrp4) transporters. TR rats also showed a decrease in intestinal BA reabsorption due to reduced ileal sodium/bile acid cotransporter (Asbt) expression. Analysis of regulatory mechanisms indicated that activation of the hepatic constitutive androstane receptor (CAR)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by accumulating bilirubin may be responsible for changes in BA metabolomics in TR rats. Ethinylestradiol administration to TR rats further increased plasma BA concentrations as a result of reduced BA uptake and increased efflux via reduced Slco1a1 and upregulated Mrp4 transporters. These results demonstrate that Mrp2-deficient organism is more sensitive to estrogen-induced cholestasis. Inherited deficiency in Mrp2 is associated with activation of Mrp3 and Mrp4 proteins, which is further accentuated by increased estrogen. Bile acid monitoring is therefore highly desirable in pregnant women with conjugated hyperbilirubinemia for early detection of intrahepatic cholestasis.

Metformin impairs bile acid homeostasis in ethinylestradiol-induced cholestasis in mice.

Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

In vitro and in vivo metabolism of 3-quinuclidinyl benzilate by high-resolution mass spectrometry.

3-Quinuclidinyl benzilate (QNB) is an anticholinergic compound that affects the nervous system. Its hallucinogenic action has led to its potential utility as an incapacitating warfare agent, and it is listed in Schedule 2 by the Organization for the Prohibition of Chemical Weapons. Although this compound has been known for a long time, limited information is available regarding its metabolism and mass spectrometric data of the metabolites, the information that could facilitate the identification of QNB in case of suspected intoxication. To the best of our knowledge, the analytical methods previously described in the literature are based on outdated procedures, which may result in a significantly lower number of observable metabolites. The aim of this work was to obtain deeper insight into QNB biotransformation using a combination of in vitro and in vivo approach. The development of a suitable method for the separation and detection of metabolites using mass spectrometry together with the identification of reliable diagnostic fragments for the unambiguous identification of QNB metabolites in the different biological matrices are also presented in this work. A screening of rat plasma, urine and tissue homogenates revealed 26 new metabolites related to the cytochrome P450 biotransformation pathway, which involves N-oxidation and hydroxylation(s) followed by O-methylation and O-glucuronosylation within phase II of the metabolism. A study showed that the brain is not metabolically active in the case of QNB and that the metabolites do not cross the blood-brain barrier; thus, the toxicodynamic effects are due to QNB itself. In addition, in vitro experiments performed using isolated human liver microsomes revealed N-oxidation as the principal metabolic pathway in human tissue. In light of current global events, the abuse of QNB by terrorists or para-military groups is a real possibility, and our findings may improve the detection systems used in laboratories involved in postexposure investigations.

[Prevention of infectious complications of central venous catheters in children]. / Prevencia infekcných komplikáclí centrálnych zilových katétrov u detí.

According to literary data the use of central venous catheters (CVC) is burdened with a significantly higher number of complications than a peripheral venous approach. The management of these complications is difficult and may increase the morbidity and even mortality of critically sick patients. This is why there is such emphasis on the prevention of these serious complications. Strict antiseptic procedures are an absolute must when handling such catheters. To prevent catheter sepsis, as well as any contamination and colonization of a central venous catheter, it is essential to insert such a catheter under aseptic conditions; it calls for handling in a sterile manner and the same applies to all tubing and other connecting systems and to the preparation of infusion liquids and drugs. Moreover, the site of insertion has to be correctly selected and the catheter left in place only for the absolutely necessary time. Most effective in the prevention of catheter infections are the so-called maximum barrier measures applied to the insertion of CVCs.

[Microbiological findings in children with an indwelling central venous catheter]. / Mikrobiologické nálezy u detí so zavedeným centrálnym zilovým katétrom.

INTRODUCTION: Besides their obvious advantages for the patient, central venous catheters (CVC) also carry the risk of possible infectious complications. The purpose of our investigation was to carry out a microbiological evaluation of a 5-year set of paediatric patients with indwelling CVCs. PATIENTS AND METHODS: In the group were 218 CVCs inserted to 165 children over a period of 5 years. There were 26 multi-lumen catheters (11.927 %) and 192 single-lumen catheters (88.073 %). The mean indwelling period was 10.1 days per 1 CVC. Blood for microbiology was removed by a physician from the CVC after disinfecting its opening under standard sterile conditions into a commercial sampling vessel HEMOD (Imuna, Sarisske Michalany, Slovak Republic) or into a vessel of an automated haemoculture system BactecPeds PLUS/F (Becton Dickinson and Comp., Spark,MA, USA). When removing the tip of the CVC we disinfected, before removing the CVC, the area around the insertion with isopropyl or ethyl alcohol. We released the fixed CVC and 1 minute after disinfection we pulled out the CVC and cut off the end or rather the tip of the catheter (approx. 1-3 cm of the tip) into a sterile test tube. To establish the diagnosis of infectious complications we used the 1995 Sirges-Serra classification and the CDC criteria. RESULTS: In 5 years (1995-1999) we had 71 infectious complications. There were 31 contaminated catheters, 27 cases of catheter sepsis and 11 cases of catheter bacteraemia. With 147 catheters (67.43%) there were no infectious complications. Dominant microbes were Staphylococcus epidermidis (32 cases - 11 from haemocultures and 21 from CVCs) and Candida spp. (30 cases, 17 from haemocultures and 13 from CVCs). Among the microbiological agents of catheter sepsis predominated Gram-negative bacteria. Out of the whole analysed group 41 children (24.8 %) died. CVC as the cause of death was demonstrated in 6 children (3.636 % of patients with CVC). CONCLUSIONS: Microbiological findings in our group are in line with literary data. To reduce the incidence of infectious complications it is important to limit sampling from CVC to a minimum. Insertion of CVCs under strict sterile conditions and aseptic handling of all entries into the central bloodstream reduces to a minimum the risk of infectious complications.