Investigations into Annona fruit consumption as a potential source of dietary higenamine intake in the context of sports drug testing.

Higenamine is prohibited in sports as a ß2 -agonist by the World Anti-Doping Agency. As a key component of a great variety of plants, including the Annonaceae family, one aim of this research project was to evaluate whether the ingestion of Annona fruit could lead to higenamine adverse analytical findings. Single-dose administration studies including three Annona species (i.e., Annona muricata, Annona cherimola, and Annona squamosa) were conducted, leading to higenamine findings below the established minimum reporting level (MRL) of 10 ng/mL in urine. In consideration of cmax values (7.8 ng/mL) observed for higenamine up to 24 h, a multidose administration study was also conducted, indicating cumulative effects, which can increase the risk of exceeding the applicable MRL doping after Annona fruit ingestion. In this study, however, the MRL was not exceeded at any time point. Further, the major urinary excretion of higenamine in its sulfo-conjugated form was corroborated, its stability in urine was assessed, and in the absence of reference material, higenamine sulfo-conjugates were synthesized and comprehensively characterized, suggesting the predominant presence of higenamine 7-sulfate. In addition, the option to include complementary biomarkers of diet-related higenamine intake into routine doping controls was investigated. A characteristic urinary pattern attributed to isococlaurine, reticuline, and a yet not fully characterized bismethylated higenamine glucuronide was observed after Annona ingestion but not after supplement use, providing a promising dataset of urinary biomarkers, which supports the discrimination between different sources of urinary higenamine detected in sports drug testing programs.

Modulation of oxidative stress/antioxidative defence in human serum treated by four different tyrosine kinase inhibitors.

Recent findings implied the significance of reactive oxygen species (ROS) as a part of tyrosine kinase inhibitors (TKIs) pharmacological activity. Evidences also suggested that toxic effects of TKIs were related to ROS production. The results regarding benefits of vitamin E usage alongside with prescribed TKIs therapy are ambiguous. We aimed to examine oxidative stress and antioxidative defense in human serum treated with four different TKIs and their possible interactions with hydrosoluble vitamin E analog (Trolox). An in-vitro experiment with serum pool as a substitute model was performed. Different parameters of oxidative stress and antioxidative defense were measured in serum pool with and without addition of TKIs (axitinib, crizotinib, nilotinib, and imatinib), before and after addition of Trolox. Z score statistic was used for calculation of Prooxidative and Antioxidative scores. The highest oxidative potential was recorded for samples incubated with imatinib and nilotinib, while the lowest damaging scores were observed for crizotinib and axitinib (nilotinib vs. imatinib, P < 0.05; axitinib vs. imatinib, P < 0.01; crizotinib vs. imatinib, P < 0.001). The best capability for antioxidative protection was seen in samples with nilotinib, then with imatinib, while the lowest level was obtained in samples with crizotinib and axitinib (imatinib and axitinib vs. nilotinib, P < 0.05 for both; crizotinib vs. nilotinib, P < 0.01; axitinib vs. imatinib, P < 0.05, crizotinib vs. imatinib, P < 0.01). Our results demonstrated the opposite effects of Trolox in combination with imatinib and nilotinib. Usage of antioxidant in combination with TKIs should be carefully evaluated in each specific case.

Synthesis of a human long-term oxymetholone metabolite.

A long-term metabolite of the doping agent oxymetholone (OXM-M2, 17ß-hydroxymethyl-2,17α-methyl-18-norandrost-13-en-3-one) which has been identified by GC-MS/MS was synthesized from commercially available materials. Two efficient synthetic routes to access both C-17 epimers of tentative metabolites were developed. The identity and molecular configuration of the in vivo metabolite: 17ß-hydroxymethyl-2α,17α-methyl-18-norandrost-13-en-3-one was confirmed by single crystal X-ray diffraction.

Quality by Design oriented development of hydrophilic interaction liquid chromatography method for the analysis of amitriptyline and its impurities.

This paper presents integration of Quality by Design concept in the development of hydrophilic interactions liquid chromatographic methods for analysis of amitriptyline and its impurities (A, B, C, and F). This is the first time that HILIC method for amitriptyline and its impurities is developed. Using QbD concept, it is possible to design a robust method and incorporate quality directly into its development. QbD concept in combination of Design of Experiments methodology (DoE) enables creation of well-defined design space. In this study, for method optimization a Box-Behnken design was used to test the effect of acetonitrile content, buffer concentration and pH of water phase on critical system responses such as retention factor of impurity A, resolution between impurity B and impurity C, amitriptyline peak asymmetry factor and retention time of last eluted impurity F. The defined mathematical models and Monte Carlo simulations were used to identify the design space. For robustness testing, fractional factorial design was applied. Optimal chromatographic conditions were the analytical column ZORBAX NH2 (250 mm x 4.6 mm, 5 µm particle size); mobile phase consisted of acetonitrile-water phase (60 mM ammonium acetate, pH adjusted to 4.5 with glacial acetic acid) (92.5:7.5 v/v); column temperature 30 °C, mobile phase flow rate 1 mL min-1, wavelength of detection 254 nm. Finally, method was fully validated and applicability of the method in tablet analysis was confirmed.

Assessment of Quality of Life, Anxiety and Depressive Symptoms in Serbian Children with Celiac Disease and their Parents.

OBJECTIVES: To evaluate the level of health-related quality of life (QoL) and presence of anxiety and depressive symptoms in Serbian children with celiac disease from the perspective of patients and their parents. METHODS: This cross-sectional study investigated the group of children and adolescents with celiac disease aged 5-18 y, and at least one parent of each patient with celiac disease. The patients and their parents were recruited at the Institute of Mother and Child Health of Serbia and the University Children's Hospital in Belgrade. The instruments used in this study were child-self and parent-proxy versions of the Pediatric Quality of Life Inventory (PedsQL), Screen for Child Anxiety Related Emotional Disorder (SCARED) and Short Mood and Feelings Questionnaire (MFQ). Additional information was collected from the medical records of each patient. RESULTS: According to the PedsQL questionnaire, the quality of life was similarly assessed by both parents and their children (p > 0.05), as well as the presence of depressive symptoms according to MFQ questionnaire. However, a statistically significant difference was observed in the total score of the SCARED questionnaire for children and parents [total score (p < 0.05), panic-somatic disturbance (p < 0.01) and social anxiety (p < 0.01)]. CONCLUSIONS: The patients and their parents in Serbia have similarly assessed the quality of life of children with celiac disease, but the differences in the scores of SCARED questionnaire indicate that it is necessary to include both children and parents in the assessment of QOL.

Analytical Quality by Design-based development and validation of ultra pressure liquid chromatography/MS/MS method for glycopeptide antibiotics determination in human plasma.

AIM: An ultra pressure liquid chromatography (UPLC)/MS/MS method for vancomycin and teicoplanin determination in human plasma was developed in accordance with analytical quality by design (AQbD) concept and fully validated. MATERIALS & METHODS: Chromatographic separation was performed on ACQUITY UPLC C18 charge surface hybrid (CSH) column (2.1 mm × 50 mm, 1.7 µm particle size) in gradient mode and the mobile phase consisted of 0.1% formic acid in water and pure acetonitrile. The experimental design methodology was used for the definition of optimal chromatographic and protein precipitation conditions. RESULTS: The linearity ranges were 0.05-10 µg ml-1 for vancomycin and 0.5-200 µg ml-1 for total teicoplanin. The relative standard deviations for precision estimation were below 15% and the accuracy was within 85-115% for all quality control levels. CONCLUSION: The method was utilized for glycopeptide antibiotics bioanalysis.

Urinary excretion studies of meldonium after multidose parenteral application.

Meldonium is a drug exhibiting cardioprotective and anti-ischemic effects. Due to its potential performance-enhancing benefit in sports, meldonium was added to the World Anti-Doping Agency list of prohibited substances in 2016. Since then, a high number of adverse analytical findings reported on meldonium has questioned meldonium`s detection time in urine. Hence, the objective of the current study was to characterize the pharmacokinetic urinary excretion pattern of meldonium when administered as multiple intravenous injections. Three injections of 250 mg meldonium were given over a time period of five days to six healthy volunteers and urine samples were collected for eight months after the last injection of the drug. For the quantification of meldonium in urine, a liquid chromatography-tandem mass spectrometry method was fully validated according to the World Anti-Doping Agency guidelines in terms of specificity, matrix interferences, intra- and inter-day precision, accuracy, carry-over, robustness, linearity, limit of detection, and limit of quantification. The assay was successfully applied to the pharmacokinetic study. A three-compartment model was found to best describe the pharmacokinetics of meldonium with average alpha, beta, and gamma half-lives of 1.4 h, 9.4 h, and 655 h, respectively. The detection time in urine varied between 94 and 162 days.

Determination of olopatadine in human tears by hydrophilic interaction liquid chromatography-MS/MS method.

AIM: The objective of the study was development of hydrophilic interaction liquid chromatography-ESI/MS/MS method for the determination of olopatadine in tear matrix. MATERIALS & METHODS: Separation was performed on Acquity BEH amide column (2.1 × 100 mm, 1.7 µm). The mobile phase was consisted of 0.1% formic acid in water and acetonitrile. Mianserin hydrochloride was implemented as an internal standard. The artificial tear fluid was used as matrix. The tear samples were collected using Schirmer test strips. For the optimization of ultra pressure liquid chromatography conditions, Box-Benhken design was utilized. RESULTS: The optimal values of the ion source and collision cell parameters were found. Quantification was performed in multiple reaction monitoring mode. The optimized method was fully validated. CONCLUSION: The proposed method was utilized for monitoring of olopatadine in human tear.

Quality by Design in the development of hydrophilic interaction liquid chromatography method with gradient elution for the analysis of olanzapine.

This paper deals with the development of hydrophilic interaction liquid chromatography (HILIC) method with gradient elution, in accordance with Analytical Quality by Design (AQbD) methodology, for the first time. The method is developed for olanzapine and its seven related substances. Following step by step AQbD methodology, firstly as critical process parameters (CPPs) temperature, starting content of aqueous phase and duration of linear gradient are recognized, and as critical quality attributes (CQAs) separation criterion S of critical pairs of substances are investigated. Rechtschaffen design is used for the creation of models that describe the dependence between CPPs and CQAs. The design space that is obtained at the end is used for choosing the optimal conditions (set point). The method is fully validated at the end to verify the adequacy of the chosen optimal conditions and applied to real samples.

Application of Analytical Quality by Design concept for bilastine and its degradation impurities determination by hydrophilic interaction liquid chromatographic method.

This paper deals with the development of hydrophilic interaction liquid chromatographic (HILIC) method for the analysis of bilastine and its degradation impurities following Analytical Quality by Design approach. It is the first time that the method for bilastine and its impurities is proposed. The main objective was to identify the conditions where an adequate separation in minimal analysis duration could be achieved within a robust region. Critical process parameters which have the most influence on method performance were defined as acetonitrile content in the mobile phase, pH of the aqueous phase and ammonium acetate concentration in the aqueous phase. Box-Behnken design was applied for establishing a relationship between critical process parameters and critical quality attributes. The defined mathematical models and Monte Carlo simulations were used to identify the design space. Fractional factorial design was applied for experimental robustness testing and the method is validated to verify the adequacy of selected optimal conditions: the analytical column Luna(®) HILIC (100mm×4.6mm, 5µm particle size); mobile phase consisted of acetonitrile-aqueous phase (50mM ammonium acetate, pH adjusted to 5.3 with glacial acetic acid) (90.5:9.5, v/v); column temperature 30°C, mobile phase flow rate 1mLmin(-1), wavelength of detection 275nm.

Hydrophilic interaction liquid chromatography in analysis of granisetron HCl and its related substances. Retention mechanisms and method development.

In this paper separation of granisetron and its two related substances in HILIC mode is presented. Separation was done on silica column derivatized with sulfoalkylbetaine groups (ZIC-HILIC). Firstly, retention mechanisms were assessed whereby retention factors of substances were followed in wide range of acetonitrile content (80-97%), at constant concentration of aqueous buffer (10mM) as well as at constant pH value of 3.0. Further, in order to developed optimal HILIC method, Design of Experiments (DoE) methodology was applied. For optimization full factorial design 3(2) was employed. Influence of acetonitrile content and ammonium acetate concentration were investigated while pH of the water phase was kept at 3.3. Adequacy of obtained mathematical models was confirmed by ANOVA. Optimization goals (α>1.15 and minimal run time) were accomplished with 94.7% of acetonitrile in mobile phase and 70 mM of ammonium acetate in water phase. Optimal point was in the middle of defined Design Space. In the next phase, robustness was experimetally tested by Rechtschaffen design. The investigated factors and their levels were: acetonitrile content (±1%), ammonium acetate molarity in water phase (±2 mM), pH value of water phase (±0.2) and column temperature (±4 °C). The validation scope included selectivity, linearity, accuracy and precision as well as determination of limit of detection (LOD) and limit of quantification (LOQ) for the related substances. Additionally, the validation acceptance criteria were met in all cases. Finally, the proposed method could be successfully utilized for estimation of granisetron HCl and its related substances in tablets and parenteral dosage forms, as well as for monitoring degradation under various stress conditions.

Theoretical Models and QSRR in Retention Modeling of Eight Aminopyridines.

In this article, retention modeling of eight aminopyridines (synthesized and characterized at the Faculty of Pharmacy) in reversed-phase high performance liquid chromatography (RP-HPLC) was performed. No data related to their retention in the RP-HPLC system were found. Knowing that, it was recognized as very important to describe their retention behavior. The influences of pH of the mobile phase and the organic modifier content on the retention factors were investigated. Two theoretical models for the dependence of retention factor of organic modifier content were tested. Then, the most reliable and accurate prediction of log k was created, testing multiple linear regression model-quantitative structure-retention relationships (MLR-QSRR) and support vector regression machine-quantitative structure-retention relationships (SVM-QSRR). Initially, 400 descriptors were calculated, but four of them (POM, log D, M-SZX/RZX and m-RPCG) were included in the models. SVM-QSRR performed significantly better than the MLR model. Apart from aminopyridines, four structurally similar substances (indapamide, gliclazide, sulfamethoxazole and furosemide) were followed in the same chromatographic system. They were used as external validation set for the QSRR model (it performed well within its applicability domain, which was defined using a bounding box approach). After having described retention of eight aminopyridines with both theoretical and QSRR models, further investigations in this field can be conducted.

Quality by Design approach in the development of hydrophilic interaction liquid chromatographic method for the analysis of iohexol and its impurities.

This study presents the development of hydrophilic interaction liquid chromatographic method for the analysis of iohexol, its endo-isomer and three impurities following Quality by Design (QbD) approach. The main objective of the method was to identify the conditions where adequate separation quality in minimal analysis duration could be achieved within a robust region that guarantees the stability of method performance. The relationship between critical process parameters (acetonitrile content in the mobile phase, pH of the water phase and ammonium acetate concentration in the water phase) and critical quality attributes is created applying design of experiments methodology. The defined mathematical models and Monte Carlo simulation are used to evaluate the risk of uncertainty in models prediction and incertitude in adjusting the process parameters and to identify the design space. The borders of the design space are experimentally verified and confirmed that the quality of the method is preserved in this region. Moreover, Plackett-Burman design is applied for experimental robustness testing and method is fully validated to verify the adequacy of selected optimal conditions: the analytical column ZIC HILIC (100 mm × 4.6 mm, 5 µm particle size); mobile phase consisted of acetonitrile-water phase (72 mM ammonium acetate, pH adjusted to 6.5 with glacial acetic acid) (86.7:13.3) v/v; column temperature 25 °C, mobile phase flow rate 1 mL min(-1), wavelength of detection 254 nm.

Chromatographic analysis of olopatadine in hydrophilic interaction liquid chromatography.

In this paper, chromatographic analysis of active substance olopatadine hydrochloride, which is used in eye drops as antihistaminic agent, and its impurity E isomer by hydrophilic interaction liquid chromatography (HILIC) and application of design of experiments (DoE) methodology are presented. In addition, benzalkonium chloride is very often used as a preservative in eye drops. Therefore, the evaluation of its chromatographic behavior in HILIC was carried out as well. In order to estimate chromatographic behavior and set optimal chromatographic conditions, DoE methodology was applied. After the selection of important chromatographic factors, Box-Behnken design was utilized, and on the basis of the obtained models factor effects were examined. Then, multi-objective robust optimization is performed aiming to obtain chromatographic conditions that comply with several quality criteria simultaneously: adequate and robust separation of critical peak pair and maximum retention of the first eluting peak. The optimal conditions are identified by using grid point search methodology. The experimental verification confirmed the adequacy of the defined optimal conditions. Finally, under optimal chromatographic conditions, the method was validated and applicability of the proposed method was confirmed.

Retention mechanism assessment and method development for the analysis of iohexol and its related compounds in hydrophilic interaction liquid chromatography.

Hydrophilic interaction liquid chromatography (HILIC) has emerged in recent years as a valuable alternative to reversed-phase liquid chromatography in the analysis of polar compounds. Research in HILIC is divided into two directions: the assessment of the retention mechanism and retention behavior, and the development of HILIC methods. In this work, four polar neutral analytes (iohexol and its related compounds A, B, and C) were analyzed on two silica and two diol columns in HILIC mode with the aim to investigate thoroughly the retention mechanisms and retention behavior of polar neutral compounds on these four columns. The adsorption and partition contribution to the overall HILIC retention mechanism was investigated by fitting the retention data to linear (adsorption and partition) and nonlinear (mixed-retention and quadratic) theoretical models. On the other hand, the establishment of empirical second-order polynomial retention models on the basis of D-optimal design made possible the estimation of the simultaneous influence of several mobile-phase-related factors. Furthermore, these models were also used as the basis for the application of indirect modeling of the selectivity factor and a grid point search approach in order to achieve the optimal separation of analytes. After the optimization goals had been set, the grids were searched and the optimal conditions were identified. Finally, the optimized method was subjected to validation.

Chemometrical tools in the study of the retention behavior of azole antifungals.

Certain chemometrical tools allow an efficient way to provide valuable data to evaluate the retention behavior of analytes in liquid chromatography. In this study of the retention behavior of azole antifungals, the experimental design was applied in combination with artificial neural networks (ANNs). Three potentially significant factors (methanol content, pH of the mobile phase and column temperature) were incorporated in the plan of experiments, defined by central composite design. As the system outputs, the retention factors of all six investigated substances (fluconazole, ketoconazole, bifonazole, clotrimazole, econazole and miconazole) were determined. The pattern for the analyzed behavior of the system was created by employing ANNs. The final, optimized topology of the highly predictive network was 3-8-6. Twelve experiments were used in a training set, whereas a back-propagation algorithm was optimal for network training. The ability of the defined network to predict the retention of the investigated azoles was confirmed by correlations higher than 0.9912 for all analytes. The presented approach allowed the adequate prediction of the retention behavior of azoles, in addition to the extraction of important information for a better understanding of the analyzed system.

Stepwise optimization approach for improving LC-MS/MS analysis of zwitterionic antiepileptic drugs with implementation of experimental design.

In this article, a step-by-step optimization procedure for improving analyte response with implementation of experimental design is described. Zwitterionic antiepileptics, namely vigabatrin, pregabalin and gabapentin, were chosen as model compounds to undergo chloroformate-mediated derivatization followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. Application of a planned stepwise optimization procedure allowed responses of analytes, expressed as areas and signal-to-noise ratios, to be improved, enabling achievement of lower limit of detection values. Results from the current study demonstrate that optimization of parameters such as scan time, geometry of ion source, sheath and auxiliary gas pressure, capillary temperature, collision pressure and mobile phase composition can have a positive impact on sensitivity of LC-MS/MS methods. Optimization of LC and MS parameters led to a total increment of 53.9%, 83.3% and 95.7% in areas of derivatized vigabatrin, pregabalin and gabapentin, respectively, while for signal-to-noise values, an improvement of 140.0%, 93.6% and 124.0% was achieved, compared to autotune settings. After defining the final optimal conditions, a time-segmented method was validated for the determination of mentioned drugs in plasma. The method proved to be accurate and precise with excellent linearity for the tested concentration range (40.0 ng ml(-1)-10.0 × 10(3) ng ml(-1)).

Robust optimization of psychotropic drug mixture separation in hydrophilic interaction liquid chromatography.

This paper presents multiobjective optimization of complex mixtures separation in hydrophilic interaction liquid chromatography (HILIC). The selected model mixture consisted of five psychotropic drugs: clozapine, thioridazine, sulpiride, pheniramine and lamotrigine. Three factors related to the mobile phase composition (acetonitrile content, pH of the water phase and concentration of ammonium acetate) were optimized in order to achieve the following goals: maximal separation quality, minimal total analysis duration and robustness of an optimum. The consideration of robustness in early phases of the method development provides reliable methods with low risk for failure in validation phase. The simultaneous optimization of all goals was achieved by multiple threshold approach combined with grid point search. The identified optimal separation conditions (acetonitrile content 83%, pH of the water phase 3.5 and ammonium acetate content in water phase 14 mM) were experimentally verified.