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Background and aims It is well known that elevated cholesterol is associated with enhanced platelet aggregation and patients suffering from familial hypercholesterolemia (FH) have a high risk of thrombotic cardiovascular events. Although decreasing cholesterol level is associated with attenuation of platelet hyperactivity, there are currently no data on the effect of convertase subtilisin/kexin type 9 monoclonal antibodies (PCSK9ab) on platelet reactivity in FH. The aim of the study was to analyse the impact of different therapies including PCSK9ab on platelet aggregation in FH. Methods This study enrolled all 15 patients treated in the University Hospital Hradec Králové for FH. PCSK9ab have been administered in 12 of 15 patients while 8 patients were also undergoing lipid apheresis. Blood samples from all patients including pre- and post-apheresis period were tested for platelet aggregation triggered by 7 inducers, and the effect of 3 clinically used drugs (acetylsalicylic acid, ticagrelor and vorapaxar) was compared as well. Results Although apheresis decreased the reactivity of platelets in general, platelet responses were not different between non-apheresis patients treated with PCSK9ab and apheresis patients (post-apheresis values) with the exception of ristocetin. However, when compared to age-matched healthy population, FH patients had significantly lower platelet aggregation responses to 4 out of 7 used inducers and higher profit from 2 out of 3 used antiplatelet drugs even after exclusion of FH patients regularly receiving conventional antiplatelet treatment. Conclusion This study showed for the first time the suitability of PCSK9ab treatment for reduction of platelet reactivity in FH patients.

An ultra-high performance chromatography method with multichannel detection for fast, sensitive, and robust analysis of terbinafine and its three impurities β-terbinafine, (Z)-terbinafine, and 4-methylterbinafine in just 5.0 min has been developed.

The process of platelet aggregation is often influenced by several factors including sex and age. A literature review confirmed the existence of sex-related differences in platelet aggregation. Although 68 out of 78 papers found such differences, there are still some controversies regarding these differences, which can be due to multiple factors (age, trigger, concomitant disease, sample handling, etc.). These outcomes are discussed in line with novel results obtained from a local study, in which blood samples from a total of 53 overall healthy women and men with ages ranging from 20 to 66 years were collected. Aggregation was induced with seven different triggers (ristocetin, thrombin receptor activating peptide 6 [TRAP-6], arachidonic acid [AA], platelet-activating factor 16 [PAF-16], ADP, collagen, or thromboxane A2 analog U-46619) ex vivo. In addition, three FDA-approved antiplatelet drugs (vorapaxar, ticagrelor, or acetylsalicylic acid [ASA]) were also tested. In general, women had higher aggregation responses to some agonists (ADP, TRAP), as well as lower benefit from inhibitors (ASA, vorapaxar). The aggregatory responses to AA and TRAP decreased with age in both sexes, while responses to ADP, U-46619, and PAF were affected by age only in women. In conclusion, more studies are needed to decipher the biological importance of sex-related differences in platelet aggregation in part to enable personalized antiplatelet treatment.

Miniaturized LC has evolved at an exponential rate over the last 50 years. In the past decade, it has received considerable attention in the field of bioanalytical separation science and technology due to the need to measure different classes of biomolecules present in a variety of matrixes on a global scale to gain a deeper understanding of complex biological processes. This field has become a dominant area underpinning the molecular omics research (e.g., proteomics, metabolomics, lipidomics, and foodomics), allowing key insights into the function and mechanism of small to very large biomolecules on a molecular level. This Feature highlights the recent advances in molecular omics focusing on miniaturized LC technology combined with mass spectrometry-based platforms, with a particular emphasis on the strategies adopted and applications using new and sensitive nanoscale analytical methodologies.

Development of a chromatographic method in bioanalysis is a challenging and complex procedure with many pitfalls and often unexpected reversals that can require several months to accomplish. Even an experienced analytical team must contend many limitations mainly in connection with the strict requirements imposed on current clinical research. These restrictions typically persist throughout the whole development process, from clinical trial assignment, across optimization of extraction of biological materials and chromatographic separation, to validation and data interpretation. This paper describes questions and their possible answers raised during the pre-analytical phase such as use of modern sample preparation techniques in clinical methods, application of internal standards, as well as selection of stationary phases and detection techniques in the analytical phase. Validation problems and interpretation of results are demonstrated with three typical examples of characteristics to be considered, i.e. recovery, matrix effect, and limit of detection vs. lower limit of quantification.

Elevated low-density lipoprotein (LDL) cholesterol levels lead to atherosclerosis and platelet hyperaggregability, both of which are known culprits of arterial thrombosis. Normalization of LDL cholesterol in familial hypercholesterolemia (FH) is not an easy task and frequently requires specific treatment, such as regularly performed lipid apheresis and/or novel drugs such as proprotein convertase subtilisin kexin 9 monoclonal antibodies (PCSK9Ab). Moreover, a high resistance rate to the first-line antiplatelet drug acetylsalicylic acid (ASA) stimulated research of novel antiplatelet drugs. 4-methylcatechol (4-MC), a known metabolite of several dietary flavonoids, may be a suitable candidate. The aim of this study was to analyse the antiplatelet effect of 4-MC in FH patients and to compare its impact on two FH treatment modalities via whole-blood impedance aggregometry. When compared to age-matched, generally healthy controls, the antiplatelet effect of 4-MC against collagen-induced aggregation was higher in FH patients. Apheresis itself improved the effect of 4-MC on platelet aggregation and blood from patients treated with this procedure and pretreated with 4-MC had lower platelet aggregability when compared to those solely treated with PCKS9Ab. Although this study had some inherent limitations, e.g., a low number of patients and possible impact of administered drugs, it confirmed the suitability of 4-MC as a promising antiplatelet agent and also demonstrated the effect of 4-MC in patients with a genetic metabolic disease for the first time.

Vitamin K is traditionally connected with blood coagulation, since it is needed for the posttranslational modification of 7 proteins involved in this cascade. However, it is also involved in the maturation of another 11 or 12 proteins that play different roles, encompassing in particular the modulation of the calcification of connective tissues. Since this process is physiologically needed in bones, but is pathological in arteries, a great deal of research has been devoted to finding a possible link between vitamin K and the prevention of osteoporosis and cardiovascular diseases. Unfortunately, the current knowledge does not allow us to make a decisive conclusion about such a link. One possible explanation for this is the diversity of the biological activity of vitamin K, which is not a single compound but a general term covering natural plant and animal forms of vitamin K (K1 and K2) as well as their synthetic congeners (K3 and K4). Vitamin K1 (phylloquinone) is found in several vegetables. Menaquinones (MK4-MK13, a series of compounds known as vitamin K2) are mostly of a bacterial origin and are introduced into the human diet mainly through fermented cheeses. Current knowledge about the kinetics of different forms of vitamin K, their detection, and their toxicity are discussed in this review.

This review summarizes the current knowledge on essential vitamins B1, B2, B3, and B5. These B-complex vitamins must be taken from diet, with the exception of vitamin B3, that can also be synthetized from amino acid tryptophan. All of these vitamins are water soluble, which determines their main properties, namely: they are partly lost when food is washed or boiled since they migrate to the water; the requirement of membrane transporters for their permeation into the cells; and their safety since any excess is rapidly eliminated via the kidney. The therapeutic use of B-complex vitamins is mostly limited to hypovitaminoses or similar conditions, but, as they are generally very safe, they have also been examined in other pathological conditions. Nicotinic acid, a form of vitamin B3, is the only exception because it is a known hypolipidemic agent in gram doses. The article also sums up: (i) the current methods for detection of the vitamins of the B-complex in biological fluids; (ii) the food and other sources of these vitamins including the effect of common processing and storage methods on their content; and (iii) their physiological function.

This review summarizes the current knowledge on essential vitamins B

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 Kinetex™ 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.

A polyphenol-rich diet has beneficial effects on cardiovascular health. However, dietary polyphenols generally have low bioavailability and reach low plasma concentrations. Small phenolic metabolites of these compounds formed by human microbiota are much more easily absorbable and could be responsible for this effect. One of these metabolites, 4-methylcatechol (4-MC), was suggested to be a potent anti-platelet compound. The effect of 4-MC was tested ex vivo in a group of 53 generally healthy donors using impedance blood aggregometry. The mechanism of action of this compound was also investigated by employing various aggregation inducers/inhibitors and a combination of aggregometry and enzyme linked immunosorbent assay (ELISA) methods. 4-MC was confirmed to be more potent than acetylsalicylic acid on both arachidonic acid and collagen-triggered platelet aggregation. Its clinically relevant effect was found even at a concentration of 10 μM. Mechanistic studies showed that 4-MC is able to block platelet aggregation caused by the stimulation of different pathways (receptors for the von Willebrand factor and platelet-activating factor, glycoprotein IIb/IIIa, protein kinase C, intracellular calcium elevation). The major mechanism was defined as interference with cyclooxygenase-thromboxane synthase coupling. This study confirmed the strong antiplatelet potential of 4-MC in a group of healthy donors and defined its mechanism of action.

Enhanced recovery after surgery (ERAS) is currently the modern perioperative method of care for improvement of post-surgery patient condition and for minimising various postoperative complications. A question of some negative impact of early postoperative parenteral nutrition on postoperative inflammatory response intensity has not clear-cut answer yet. This pilot project was focused on the possible influence of early parenteral nutrition on the intensity of inflammatory postoperative response to operating trauma in surgical patients. Elected as a model of these conditions were patients with colorectal cancer undergoing major surgery.45 patients (of whom 39 were analysed finally) operated for cancer of the large bowel were enrolled into the clinical, prospective, randomized, blinded, and monocentric trial - reference number 201811 S09P of the Ethics committee, University Hospital Hradec Kralove, Czech Republic. Patients were divided into two subgroups according to the type of nutrition: subgroup A - supplemented only with 10% glucose for supported mineral carrier; and subgroup B - supplemented with total parenteral nutrition. Samples of blood and urine were examined immediately after surgery, and on the first, second, and fourth days postoperatively. The inflammatory reaction was monitored by the serum or/and urine concentration of neopterin, tryptophan, and kynurenine, and their urinary ratios with creatinine. The results were analysed by multivariate analysis, and p-values ≤ 0.05 were considered statistically significant.The final total of 39 patients comprised 20 from subgroup A and 19 from subgroup B. The intensity of the inflammatory response detected by the selected inflammatory markers (serum and urine concentrations of neopterin, kynurenine, tryptophan, their serum ratios, and their urinary ratios to creatinine) did not demonstrate statistically significant differences after early administration of the two alternative types of parenteral nutrition.The results of the study demonstrated the same or a very similar impact on the intensity of postoperative inflammatory response, regardless of whether the patient received intravenous administration of a small simple sugar infusion or total parenteral nutrition during early postoperative care.

Determination of concentration of biomarkers of the activation of immune system, uric acid, and creatinine in the saliva can be useful tool for the diagnosis and monitoring of early manifestations of diseases such as malignant, inflammatory, and periodontal disorders. We have developed and validated a high-performance liquid chromatographic method coupled with fluorescence and diode array detection for the separation and quantification of neopterin, tryptophan, creatinine, uric acid, and kynurenine in the human saliva. A separation of these analytes was achieved within 9 min by using second-generation monolithic stationary phase and elution with phosphate buffer. The present method involves very simple sample preparation requiring small amount of sample matrix. The internal standard 3-nitro- l -tyrosine was used for a more precise quantification. The sensitivity of the present method was demonstrated with lower limits of quantification of 0.6 × 10−3 μmol/L for neopterin, 0.725 μmol/L for tryptophan, 0.12 μmol/L for creatinine, 0.18 μmol/L for uric acid, and 0.135 μmol/L for kynurenine. The method was validated with 67 real-life saliva samples collected from patients suffering from breast, ovarian, colorectal, and renal cancer, and 19 saliva samples from patients with periodontal diseases and allowed monitoring of inflammatory response.

Vitamin A is a group of vital micronutrients widely present in the human diet. Animal-based products are a rich source of the retinyl ester form of the vitamin, while vegetables and fruits contain carotenoids, most of which are provitamin A. Vitamin A plays a key role in the correct functioning of multiple physiological functions. The human organism can metabolize natural forms of vitamin A and provitamin A into biologically active forms (retinol, retinal, retinoic acid), which interact with multiple molecular targets, including nuclear receptors, opsin in the retina and, according to the latest research, also some enzymes. In this review, we aim to provide a complex view on the present knowledge about vitamin A ranging from its sources through its physiological functions to consequences of its deficiency and metabolic fate up to possible pharmacological administration and potential toxicity. Current analytical methods used for its detection in real samples are included as well.

Vitamin C (L-ascorbic acid) has been known as an antioxidant for most people. However, its physiological role is much larger and encompasses very different processes ranging from facilitation of iron absorption through involvement in hormones and carnitine synthesis for important roles in epigenetic processes. Contrarily, high doses act as a pro-oxidant than an anti-oxidant. This may also be the reason why plasma levels are meticulously regulated on the level of absorption and excretion in the kidney. Interestingly, most cells contain vitamin C in millimolar concentrations, which is much higher than its plasma concentrations, and compared to other vitamins. The role of vitamin C is well demonstrated by miscellaneous symptoms of its absence—scurvy. The only clinically well-documented indication for vitamin C is scurvy. The effects of vitamin C administration on cancer, cardiovascular diseases, and infections are rather minor or even debatable in the general population. Vitamin C is relatively safe, but caution should be given to the administration of high doses, which can cause overt side effects in some susceptible patients (e.g., oxalate renal stones). Lastly, analytical methods for its determination with advantages and pitfalls are also discussed in this review.

This review summarizes and critically evaluates the published approaches and recent trends in sample pre-treatment, as well as both separation and non-separation techniques used for the determination of uric acid (UA) in saliva. UA is the final product of purine nucleotide catabolism in humans. UA concentrations in biological fluids such as serum, plasma, and urine represent an important biomarker of diseases including gout, hyperuricemia, or disorders associated with oxidative stress. Previous studies reported correlation between UA concentrations detected in saliva and in the blood. The interest in UA has been increasing during the past 20 years from a single publication in 2000 to 34 papers in 2019 according to MEDLINE search using term “uric acid in saliva”. The evaluation of salivary UA levels can contribute to non-invasive diagnosis of many serious diseases. Increased salivary UA concentration is associated with cancer, HIV, gout, and hypertension. In contrast, low UA levels are associated with Alzheimer disease, progression of multiple sclerosis, and mild cognitive impairment.

A simple and fast ultra-high-performance liquid chromatography method with UV detection for the separation and quantification of omeprazole and the impurities of omeprazole and methylparaben (the internal standard) in six extemporaneous suspensions has been developed and fully validated. Isocratic elution was carried out using a Kinetex™ C18 column, 50 × 2.1 mm packed with 1.7 μm porous shell particles, and the mobile phase comprised a mixture of phosphate buffer and acetonitrile. The separation of all compounds was achieved within 2 min. This method was successfully applied for stability evaluation of the developed formulations, which are now being used in the therapy of acid-related disorders in paediatric patients.

To compare preoperative intestinal trefoil factor 3 (TFF3), allograft inflammatory factor-1 (AIF-1) and calgizzarin (S100-A11) serum levels in patients with endometrial cancer, endometrial hyperplasia and in healthy female controls. Serum levels of TFF3, S100- A11 and AIP-1 were analyzed in 98 consecutive patients with histologically verified endometrial cancer, in 43 patients with endometrial hyperplasia diagnosed during hysteroscopy and 24 controls with benign disease. Results were correlated with urinary neopterin/creatinine ratio, serum kynurenine, tryptophan, retinol, alpha-tocopherol, vitamin D, citrulline, C-reactive protein, interleukin-6 and clinical characteristics. S100-A11, and AIF-1 levels were higher in endometrial hyperplasia patients than in controls, and also significantly higher in endometrial cancer than in patients with endometrial hyperplasia. Serum concentrations of TFF3 and S100-A11 were associated with tumor stage and lymph node status. TFF3 exhibited positive correlation with age, IL-6, vitamin D, kynurenine, urinary neopterin/creatinine ratio and kynurenine/tryptophan ratio. S100-A11, as well as AIF-1 correlated positively with Il-6 and TFF3. TFF3, S100-A11 and AIF-1 represent potential biomarkers in patients with endometrial cancer. TFF3 and S100-A11 increase with tumor stage and lymph node involvement, reflecting higher tumor mass that is also associated with increased concentration of biomarkers of immune dysfunction.

Introduction At present, there are no strong predictors, nor a useful scoring system, that clearly identifies patients at risk for anastomotic leakage. Aim This study aimed to investigate a new method that assesses this risk by monitoring levels of neopterin, tryptophan, and kynurenine, in bodily fluids. Material and methods This prospective study included patients who underwent elective rectal resection for carcinoma. The basic condition for inclusion was rectal anastomosis using the double-stapling technique. Preoperative levels of neopterin, tryptophan, kynurenine, and their ratios, were assessed with blood and urine samples. These levels were then monitored for 6 postoperative days in venous blood, urine, and abdominal drainage fluid. Results A total of 42 patients were enrolled in the study. Thirty-six patients underwent a laparoscopic resection and 6 patients had an open procedure. No differences were found among neopterin, tryptophan, and kynurenine serum levels. However, the groups were observed to have significant differences in the urinary neopterin/creatinine ratio: the preoperative neopterin/creatinine ratio was 139.5 μmol/mol in the group with leakage, vs 114.8 μmol/mol in the group without complications, p = 0.037. The same results were observed during the postoperative period, p = 0.012. Additionally, the group with complications had a higher mean value of neopterin in drainage fluid, p = 0.048. Conclusions Our study demonstrated that high preoperative levels of urinary neopterin could be interpreted as a risk for anastomotic leakage. Moreover, pathological levels of neopterin in urine and abdominal drainage fluid could be useful for early identification of anastomotic leakage during the postoperative period prior to its clinical development.

The efficient sample preparation is fundamental in the bioanalysis. Application of solid-phase extraction represents effective sample clean-up and allows final compatibility with selected analytical output. Routine practise desires more efficient and greener approaches. Various μ-solid-phase extraction pipette tips and disposable pipette extraction tips are commercially available. Also, many researchers use home-made tips filled with their own sorbents to improve the efficiency and selectivity of their applications. This review details application of extraction pipette tips in clinical and forensic toxicology with a close focus on the most abused groups of substances. We also cover practical aspects of use of microextraction in pipette tips, their applications for treatment of biological samples of human origin, and sorbents applied for this process. Different approaches to microextraction, their advantages and disadvantages are discussed that enable informed decision which extraction pipette tips can be applied in clinical practice.

The aim of the present pilot study was to investigate the concentrations of neopterin, kynurenine and tryptophan in wound secretion in patients undergoing surgery for breast cancer or malignant melanoma. Twenty-two patients, 16 females and 6 males, undergoing surgery for breast cancer (n=15) or malignant melanoma (n=7) were evaluated. Neopterin, kynurenine and tryptophan were determined using a high-performance liquid chromatography method. When the concentrations in wound secretions from the primary breast tumor and the axilla were compared, the neopterin/tryptophan ratio was significantly higher in the tumor wound secretions (0.92±0.41 vs. 0.61±0.14 mmol/mol; p=0.049), but no significant differences were observed in neopterin (49.2±28.6 vs. 31.5±11.1 nmol/L), tryptophan (52.9±13.0 vs. 51.2±13.3 μmol/L) and kynurenine concentrations (5.97±7.49 vs. 5.34±6.25 μmol/L) and kynurenine/tryptophan ratio (108.1±107.7 vs. 103.5±106.7 mmol/mol). No marked differences were noted in neopterin, tryptophan and kynurenine concentrations and kynurenine/tryptophan and neopterin/tryptophan ratios in sequential samples from the axilla of breast cancer patients obtained on days 1 and 2. In conclusion, present data demonstrate that the measurement of neopterin, kynurenine and tryptophan can be used to monitor local immune response after cancer surgery.

In the present study, we determined complex indices of inflammatory activity and compared the performance of these indices as prognostic biomarkers in a cohort of breast cancer patients. All proposed composite biomarkers could be evaluated in 418 out of 474 patients in the cohort with complete data on peripheral blood cell count, urinary neopterin, albumin and C-reactive protein. Neutrophil-to-lymphocyte ratio, lymphocyte-to-monocyte ratio, platelet-to-lymphocyte ratio, systemic inflammatory index, Glasgow prognostic index, modified Glasgow prognostic index, prognostic nutritional index and C-reactive protein/albumin ratio were calculated and further complex indices were proposed. Although a number of the investigated indices were significantly associated with survival in the univariate analysis, only age and stage, but none of the laboratory biomarkers or composite biomarkers, were significant predictors of survival in the whole group in the multivariate analysis. In patients evaluated before the start of the treatment, age, stage and urinary neopterin were significant predictors of survival. These results underscore the importance of neopterin as a prognostic biomarker in breast cancer.

The aim of the present study was to investigate the association of peripheral-blood cell count (PBC)-derived ratios, other biomarkers of inflammation and biomarkers of tumor growth with outcome in a cohort of patients presenting for the next line of therapy after the failure of prior systemic treatment. The data of 51 patients with advanced/metastatic colorectal carcinoma treated with cetuximab in the second or higher line of therapy were retrospectively analyzed. The median duration of cetuximab therapy and the median survival were 5.1 and 12.1 months, respectively. C-reactive protein (CRP), but not urinary neopterin correlated significantly with PBC-derived ratios. Both CRP and urinary neopterin correlated positively with carcinoembryonic antigen (CEA) concentrations and biomarkers of liver dysfunction. Although a number of parameters predicted overall survival in univariate analysis, only hemoglobin, CEA change and serum bilirubin were independent predictors of survival. In conclusion, in patients with metastatic colorectal carcinoma and predominantly liver metastases, the outcome of therapy in the advanced line setting was associated with initial hemoglobin level, a decrease of CEA concentration and initial presence of liver dysfunction. Urinary neopterin did not correlate with PBC-derived ratios, in contrast to CRP, but both urinary neopterin and serum CRP concentrations correlated with laboratory parameters of liver dysfunction.

We report a patient who presented with synchronous second primary human epidermal growth factor receptor (HER)-2-positive breast cancer and rectal cancer that both required simultaneous neoadjuvant therapy. A modified regimen combining anti-HER-2 monoclonal antibody trastuzumab with chemotherapy and external beam radiation was selected. An organ-preserving surgical procedure was possible both in the breast and the rectum. Citrulline decreased rapidly after the start of the treatment, and then gradually returned to pre-treatment levels after the completion of chemoradiation. Urinary neopterin concentrations exhibited a fluctuating course. Both serum neopterin and C-reactive protein concentrations were more or less stable during the initial administration of trastuzumab, paclitaxel and carboplatin and then increased steeply during chemoradiation and subsequently declined to pre-treatment levels during the weekly trastuzumab administration. Changes were observed in the serum retinol concentrations. A decline in lymphocyte counts was accompanied by marked changes in peripheral blood cell count-derived ratios. The present case report demonstrates a successful combination of two neoadjuvant regimens in a patient with two synchronous different second primary tumors. Data from this case also illustrate the use of biomarkers for monitoring of intensive therapeutic regimens in medical and radiation oncology.

The importance of vitamin A and its various forms and metabolites for the human body, as well as their methods of analysis, has often been described previously. The excretion of retinol into the urine has been observed in diseases accompanied with kidney damage such as diabetes mellitus, cancer, and serious infections. This review summarizes all available information concerning the importance of the determination of retinol in urine. Analytical aspects such as sample collection, sample preparation, and analysis are discussed in detail, and the main emphasis is focused on the enormous potential of retinol as an early biomarker for the detection of renal failure.

Over the last five decades, many methods to analyze thiamine (vitamin B1) and its phosphorylated forms in urine, whole blood, serum, plasma and erythrocytes have been proposed. Some of the methods are presently used in routine practice, but analytical problems regarding reproducibility, standardization, lack of automation, time consuming procedures for pretreatment and analysis are often discussed. With modern approaches to bioanalysis in clinical research of vitamins, whole processes can be automated, making analysis less time consuming, with reduced consumption of solvents and samples. This review critically discusses various analytical techniques, their advantages and disadvantages that are used for determination of thiamine and its derivatives in clinical practice, with emphasis on accurate, reliable and fast analytical procedures.

A new, rapid and effective ultra-high-performance liquid chromatography method with mass spectrometry detection is described for the separation and quantification of 8-hydroxy-2-deoxyguanosine, 8-hydroxyguanosine and creatinine in human urine. The present study uses an isotope-labelled internal standard ([15N]5-8-hydroxy-2-deoxyguanosine), a BIO core-shell stationary phase and an isocratic elution of methanol and water. Sample preparation of human urine was performed by solid-phase extraction (SPE) on Oasis HLB cartridges with methanol/water 50:50 (v/v) elution. Extraction recoveries ranged from 98.1% to 109.2%. Biological extracts showed high short-term stability. Several aspects of this procedure make it suitable for both clinical and research purposes: a short elution time of less than 3.2 min, an intra-day precision of 2.5-8.9%, an inter-day precision of 3.4-8.7% and low limits of quantification (27.7 nM for 8-hydroxyguanosine, 6.0 nM for 8-hydroxy-2-deoxyguanosine). Finally, simultaneous analysis of DNA and RNA oxidative stress biomarkers is a useful tool for monitoring disease progression in neurodegenerative disorders and cancer. Graphical abstract UHPLC-MS/MS analysis of DNA and RNA oxidative stress biomarkers.

Determination of urinary retinol, which is a new promising early biomarker of renal damage typically expressed in the clinical environment as retinol/creatinine ratio, is currently difficult to accomplish. We have developed and validated the new ultra-high-performance liquid chromatography method with UV and mass spectrometry detection for the separation and quantification of retinol and creatinine in human urine in a single run. The separation of these two substances with completely different physicochemical properties was achieved using a column packed with fluorinated stationary phase and acetonitrile and aqueous ammonium formate buffer as the mobile phases. The separation was completed within 4 min. Our new method involves very fast and simple sample preparation requiring small amount of sample matrix and solvents. Deuterium labeled internal standard was used for the more precise quantification. The method was tested with real-life samples using urine collected from patients suffering from breast, colorectal, head, and neck cancer.

The immune response crucially determines the survival of patients with malignant tumors including breast carcinoma. The aim of the present study was to evaluate retrospectively an association of peripheral blood cell count (PBC)-derived ratios and urinary neopterin concentration with prognosis in breast cancer patients. Urinary neopterin, neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to-lymphocyte ratio (PLR) were retrospectively analyzed in a cohort of 474 breast cancer patients. NLR and PLR correlated positively with each other and negatively with LMR, but no correlation between neopterin concentrations and PBC-derived ratios was observed. Increased urinary neopterin concentration was a significant predictor of poor survival in patients with active disease, but PLR, NLR or LMR were not significantly associated with survival in multivariate analysis. In conclusion, increased urinary neopterin was a significant predictor of poor survival in patients with breast cancer and active disease.

The aim of the present study was to investigate serial changes in urinary neopterin and the populations of peripheral blood lymphocytes in breast cancer patients treated with primary chemotherapy. Thirty patients with breast cancer, including 20 patients undergoing primary chemotherapy treated with the combination of doxorubicin and cyclophosphamide with sequential administration of paclitaxel-based therapy (cohort A), and 10 patients presenting for first-line therapy of metastatic disease and treated with the combination of bevacizumab, taxane and carboplatin (cohort M) were studied. Healthy female blood donors served as control group. Significant differences were observed in baseline relative and absolute counts of peripheral blood lymphocyte subpopulations between both patient cohorts and controls. Significant changes of peripheral blood lymphocyte subpopulations were also observed during the course of chemotherapy. However, no significant changes of urinary neopterin concentrations and no correlation between urinary neopterin and peripheral blood lymphocyte counts were observed. In conclusion, present data demonstrate the presence of significant differences in peripheral blood leukocyte phenotype in breast cancer patients even before the start of primary chemotherapy. Significant changes were observed during the course of chemotherapy.

Vitamin E comprises eight related compounds: α-, β-, γ-, δ-tocopherols and α-, β-, γ-, δ-tocotrienols. In the past, α-tocopherol has been the isomer that was studied most, and its anti-inflammatory and anti-proliferative effects have been described. Therefore, many prevention trials have investigated the effect of α-tocopherol on human health. Current research studies have also defined the important roles of other tocopherols, such as anti-inflammatory, anti-proliferative and cancer preventative effects. Knowledge of the individual tocopherols could help to understand their roles in various metabolic pathways. This review summarizes the recent trends in sample pretreatment, liquid chromatography and selected applications of the determination of tocopherols in various biological materials. The relationship between tocopherol isomers and serious diseases is also described. Graphical Abstract Article structure.

The aim of the present study was to evaluate the correlations of peripheral blood cell count (PBC)-derived ratios with neopterin concentration and biomarkers of atherosclerosis risk in patients with history of breast cancer. Neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to-lymphocyte ratio (PLR) were calculated in three cohorts of patients with a history of breast cancer and in controls. Significant differences were observed between PBC-derived ratios obtained from automated and manual counts. NLR and PLR were significantly higher and LMR was significantly lower in patients. NLR and PLR correlated positively with each other and negatively with LMR. NLR exhibited a significant correlation with age, glucose and C-reactive protein (CRP) concentrations, whereas LMR correlated negatively with CRP. With the exception of a correlation between LMR and urinary or serum neopterin concentrations in controls, no other correlation between neopterin concentrations and PBC-derived ratios was observed. NLR ≥3 was a significant predictor of poor survival, but neither urinary neopterin ≥205 μmol/mol creatinine, NLR ≥150 nor LMR ≥4.25 was significantly associated with survival. In conclusion, no consistent correlation was observed between urinary and serum neopterin concentrations and any of the PBC-derived ratios. In a cohort of breast cancer patients, a higher NLR predicted poor survival.

Vancomycin is a glycopeptide antibiotic used in the therapy of severe bacterial infection. The monitoring of vancomycin levels is recommended because of its narrow therapeutic index and toxicity. This measurement is especially appropriate in patients with unstable renal functions, who receive high doses of vancomycin or present serious bacterial infections accompanied by important sequestration of liquids when it could be difficult to achieve the optimal therapeutic dose. Most of the methods for vancomycin determination in routine practice are immunoassays. However, chromatography-based techniques in combination with UV or mass spectrometry detection provide results with greater accuracy and precision also in complicated biological matrices. This review provides a detailed overview of modern approaches for the chromatographic separation of vancomycin in various biological samples and useful sample preparation procedures for vancomycin determination in various biological fluids.

Aims: Rheohaemapheresis treatment influences rheological markers and most likely improves metabolism in affected retinal areas, resulting not only in absorption of soft drusen but also reduction or complete disappearance of drusenoid retinal pigment epithelium detachments. However, the character of the treatment process has raised suspicion that there is a decrease not only in cholesterol but also in antioxidants, such as vitamin E and vitamin A. Methods: Twenty-three patients with the progressive dry form of age-related macular degeneration were each treated with 8 procedures of rheohaemapheresis. We measured levels of vitamin E (α-tocopherol), the vitamin E/cholesterol ratio in serum and lipoproteins (VLDL, LDL, HDL). Vitamin E in erythrocyte membrane and serum vitamin A (retinol) were also measured. These parameters were determined before and after rheohaemapheresis. Erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase and serum malondialdehyde were analysed as markers of antioxidant activity and lipid peroxidation, respectively. Results: In serum, the VLDL and LDL fraction ratios of vitamin E/cholesterol increased significantly. Additionally, the HDL fraction ratio showed an increase but this was not statistically significant. The patients showed no clinical signs of vitamin E deficiency, and their serum concentrations of vitamin E did not differ from normal values. The results show that rheohaemapheresis in addition to causing a significant reduction in atherogenic LDL cholesterol, may have favourable additive anti-atherogenic effects due to a relative increase in the content of vitamin E in the lipoprotein fractions.

Vitamin D plays an important role in calcium metabolism and affects other metabolic pathways. Despite the intense interest in vitamin D, no comprehensive overview addressing the analysis of vitamin D in milk has been published. Historically, immunoassay techniques have been mainly used for the routine quantification of vitamin D and its metabolites. However, the greater accuracy and precision of chromatography makes it one of the most important methods in the analysis of vitamin D. The determination of vitamin D and its metabolites by LC-MS is the gold standard for its assessment. LC-MS has unique advantages for vitamin D determination and quantification due to its high sensitivity and specificity. In this review, the current status of vitamin D and its metabolites analysis in milk, human and bovine, including sample pre-treatment and chromatography analysis, are critically discussed and summarised.

A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes.

With the advent of immunotherapy the topic of biomarkers of immune response is of high interest. Along with the expression of programmed death ligand 1 (PD-L1) or tumor infiltrating lymphocytes (TIL), biomarkers of macrophage activation could be of interest. Neopterin is a biomarker of immune activation increased in different disorders associated with immune activation, including cancer. Neopterin synthesis is induced by interferon-γ that also induces indoleamine 2,3-dioxygenase (IDO), an enzyme catalyzing catabolism of tryptophan to kynurenine. Increased urinary or serum concentrations of neopterin have been associated with poor prognosis across a spectrum of malignant disorders of different primary location. Neopterin concentration in peripheral blood as well as in the tumor microenvironment correlates with phenotypic and functional changes of lymphocytes, indicating immune dysfunction. Increased neopterin concentrations are also accompanied by increased rate of conversion of tryptophan to kynurenine. Increasing neopterin concentrations also accompany side effects of anticancer treatment and could predict subsequent complications. Although almost four decades have elapsed since the discovery of increased neopterin concentrations in cancer patients, the full potential of neopterin as a biomarker in this setting has not been so far realized.

A high-throughput miniaturized liquid-liquid extraction procedure followed by a simple ultra-high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core-shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λex = 325, 295 nm and λem = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 μM, α-tocopherol 0.38 μM, β-tocopherol 0.18 μM, γ-tocopherol 0.14 μM, and δ-tocopherol 0.01 μM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.

Management of the therapy of life-threatening bacterial infection is extremely based on an optimal antibiotic treatment. Achieving the correct vancomycin dosage in blood and target tissues can be complicated in special situations, e.g., where large fluid sequestration and/or acute renal failure occur. A UHPLC–MS/MS method operating in electrospray (ESI) positive ion mode was applied for the determination of vancomycin in serum, urine and peritoneal/pleural effusion. Sample pretreatment was composed of dilution and simple protein precipitation where only a small volume (50 μL) of serum, urine or peritoneal/pleural effusion was required. The separation of vancomycin was performed on a Meteoric Core C18 BIO column (100 × 4.6 mm, 2.7 μm) by gradient elution with 0.1% formic acid in water and acetonitrile. The total time of analysis was 4.5 min. The method was found to be linear in the range of 2–60 μM (or 0.5–10 μM) for serum, 0.27–10 μM (or 2–60 μM) for peritoneal/pleural effusion and 25–300 μM for urine, which was adequate for the determination of vancomycin in patient samples. The intra- and inter-day precision was below 8% RSD, and accuracy was from 89 to 104%. The UHPLC/MS–MS method offers a fast and reliable approach to determine vancomycin concentrations in three different human body fluid samples (serum, urine and peritoneal/pleural effusion) with a simple sample pretreatment that was the same for all selected specimens. This method should be applicable to large sample series in clinical (pharmacokinetic/pharmacodynamic) studies.

A sensitive capillary electrophoretic method featuring spectrophotometric detection using a commercial Z-cell was devised for the assay of 8-hydroxy-2'-deoxyguanosine (8OHdG) in human urine. Solid-phase extraction (SPE) based on hydrophilic-lipophilic-balanced RP sorbent was utilized for urine sample pretreatment and analyte preconcentration. The separation was carried out in conventional fused-silica capillaries employing a Z-cell with hydrodynamic sample injection (at 50 mbar for 12 s). The BGE (pH* 9.2, adjusted with 1 M NaOH) contained 0.15 M boric acid and 10% v/v ACN. The detection wavelength was 282 nm. The calibration curve for 8OHdG (measured in spiked urine) was linear in the range 10-1000 ng/mL; R(2) = 0.9993. The LOD was 3 ng/mL (11 nmol/L) of 8OHdG. Determination of the 8OHdG urinary levels was possible even in healthy individuals.

Neopterin is a biomarker of host response to neoplasia. In the present study, urinary neopterin was determined during the course of (chemo)radiation in ten patients with gynecological tumors (nine patients with cervical carcinoma and one patient with carcinoma of the vulva). Baseline urinary neopterin concentrations were, generally, above the normal range. Neopterin concentrations were relatively stable during the first 5 weeks of combined (chemo)radiation. Marked peaks of neopterin concentrations reflected the emergence of complications. Neopterin could represent a useful biomarker for the assessment of the condition of the patients during this aggressive therapy.

The aim of the present study was to evaluate the prevalence of the risk factor of atherosclerosis in patients with history of breast cancer. C-reactive protein, lipoprotein (a), cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein cholesterol, triglycerides, homocysteine, fibrinogen, glucose, magnesium, uric acid, and urinary albumin were determined by routine methods in 61 patients with history of breast cancer and 74 control subjects. Urinary neopterin and creatinine concentrations were measured by high-performance liquid chromatography, and intima-media thickness was determined sonographically. Breast cancer patients were significantly older and also had higher systolic blood pressure, glycosylated hemoglobin, lipoprotein (a), serum neopterin, and intima-media thickness. Serum HDL cholesterol and magnesium concentrations were significantly higher in controls compared with patients. Differential patterns of associations between the risk factors of atherosclerosis were observed in patients with history of breast cancer compared with controls. In conclusion, the prevalence of risk factors of atherosclerosis is high in patients with history of breast cancer. Differential associations between risk factors suggest possible differences in the pathogenesis of atherosclerosis in breast cancer patients and controls.

Biomarkers, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , are important indicators of the vitamin D general status and are monitored in several pathophysiological disorders, such as osteoporosis, diabetes, heart disease, etc. A novel ultra-HPLC with MS/MS methodology for the analysis of 25-hydroxyvitamin D derivatives coupled with a very simple and highly rapid sample preparation step was developed. Analytical parameters obtained showed linearity (R(2) ) above 0.999 for both vitamins with accuracies between 95.8 and 102%. The LODs were as low as 0.22 and 0.67 nmol/L for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , respectively. Intra-assay precision (%RSD) was lower than 4.5%, and inter-assay precision (%RSD) was lower than 6.5%. The feasibility of the developed methodology to be applied in clinical routine analysis has been proved by its application in blood samples from non-agenarian patients, patients with familial hypercholesterolemia and patients suffering from age-related macular degeneration.

Microplates are routinely used in Radio- or Immuno-assays. Recently, microplates have found use not only in analytical but also in the pre-analytical phase in bioanalyses (sample storage, sample preparation). New connection of this technology to liquid chromatography could be economical, fast and simple solution for many routine laboratories handling large sequences of biological samples. This review summarises the application of microplates in bioanalytical laboratories. Different types of sorbents, materials and shapes of microplates are discussed, and the main advantages and disadvantages of microplates used in clinical research are presented.