In our previous study we proposed a screening approach using ultra-high performance supercritical fluid chromatography for the determination of 10 pharmaceutical quality control mixtures. Most of resulting methods offered baseline separation of all analytes. However, some of these methods had to be further optimized to ensure their successful validation and applicability to impurity control in drug substance and drug products. Several challenges occurred during the optimization including: (i) the necessity of the resolution of active pharmaceutical ingredient and following impurity equaling at least 3, which was especially difficult to achieve for mixtures of structurally close compounds, (ii) unrepeatable elution of compounds eluting close to the dead volume or at the end of the gradient elution, and (iii) shifts in retention times due to the column aging and effects of additive. The most frequent optimization adjustments involved changes in gradient program. Other adjustments such as the substitution of Viridis UPC2 HSS C18 SB column with a slightly different Acquity UPLC HSS C18 SB column, the addition of acetonitrile in the modifier, and the column coupling also led to beneficial changes in selectivity. Subsequently, validation of all 10 methods was carried out to prove the applicability of ultra-high performance supercritical fluid chromatography methods for the impurity control in pharmaceuticals. Parameters recommended by ICH guidelines Q2 and Q3 including specificity, linearity, range, lower and upper limit of quantification, limit of detection, accuracy, and precision were examined. In addition, intermediate precision and the accuracy profiles were determined for selected methods. Overall, only two impurities did not meet the validation criteria due to low resolution and low sensitivity, respectively. Only identification threshold and not reporting threshold was met for this impurity.

• Klíčová slova
• Impurity control, Method validation, Pharmaceutical compounds, Quality control, Ultra-high performance supercritical fluid chromatography,
• Publikační typ
• časopisecké články MeSH

A complete analytical procedure combining optimized tea infusion preparation and validated UHPLC-MS/MS method was developed for routine quantification of eight naturally occurring catechin derivatives in various tea samples. The preparation of tea infusions was optimized in terms of temperature, time and water-to-tea ratio in green, white and black teas. The catechins were analyzed using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry in a run of only 4 min including equilibration of the system. The UHPLC-MS/MS method was fully validated in terms of inter/intra-day precision, accuracy, linearity (r(2)>0.9991), range (50-5000 ng/ml), LOD (1.5-7.5 ng/ml) and LOQ (5-25 ng/ml). Validation of the method included also the determination of the matrix effects that were evaluated in both flavored and unflavored green, white and black teas. Dilution of the resulting tea infusions appeared to be crucial for the matrix effects and also for subsequent catechin quantification in real tea samples in order to fit into the linear range of the UHPLC-MS/MS method. This complete procedure for catechin quantification was finally applied to real sample analysis represented by 70 commercial tea samples.

• Klíčová slova
• Catechins, Matrix effects, Method validation, Tea, UHPLC–MS/MS,
• MeSH
• analýza potravin metody   MeSH
• čaj chemie   MeSH
• katechin chemie   MeSH
• limita detekce MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie metody   MeSH
• teplota MeSH
• voda chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• čaj MeSH
• katechin MeSH
• voda MeSH

AIM: This study proceeds to rigorously examine and validate the Low Vision Quality-of-Life Questionnaire (LVQOL) on a Greek population of ophthalmic patients employing Rasch measurement techniques. METHODS: It is a prospective observational study of 150 cataract patients and 150 patients with other ophthalmic diseases, all followed longitudinally for a period of two months pending surgical or other corrective therapy, after which they were administered the LVQOL for a second time. RESULTS: The original 25-item LVQOL demonstrated high reliability and validity, excellent measurement precision and ordered response category thresholds. A small number of items carry an acceptable level of measurement error while three items had some differential functioning for gender, Age and underlying disorder that did not exceed the established thresholds. CONCLUSIONS: This validation study is the first to employ Rasch measurement to examine the validity of the LVQOL and it supports its use with no changes to the original structure. The LVQOL can be employed in a large range of ophthalmic diseases and reliably assess improvements in quality-of-life following phacoemulsification surgery or any other intervention.

• Klíčová slova
• LVQOL, Rasch validation, cataract, phacoemulsification, quality of life,
• MeSH
• dospělí MeSH
• extrakce katarakty * MeSH
• kvalita života * MeSH
• lidé středního věku MeSH
• lidé MeSH
• longitudinální studie MeSH
• oční nemoci chirurgie   MeSH
• prospektivní studie MeSH
• průzkumy a dotazníky normy   MeSH
• reprodukovatelnost výsledků MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH
• validační studie MeSH
• Geografické názvy
• Řecko MeSH

Antibody reagents are the key components of multiparametric flow cytometry analysis. Their quality performance is an absolute requirement for reproducible flow cytometry experiments. While there is an enormous body of antibody reagents available, there is still a lack of consensus about which criteria should be evaluated to select antibody reagents with the proper performance, how to validate antibody reagents for flow cytometry, and how to interpret the validation results. The achievements of cytometry moved the field to a higher number of measured parameters, large data sets, and computational data analysis approaches. These advancements pose an increased demand for antibody reagent performance quality. This review summarizes the codevelopment of cytometry, antibody development, and validation strategies. It discusses the diverse issues of the specificity, cross-reactivity, epitope, titration, and reproducibility features of antibody reagents, and this review discusses the validation principles and methods that are currently available and those that are emerging. We argue that significant efforts should be invested by antibody users, developers, manufacturers, and publishers to increase the quality and reproducibility of published studies. More validation data should be presented by all stakeholders; however, the data should be presented in sufficient experimental detail to foster reproducibility, and community effort shall lead to the public availability of large data sets that can serve as a benchmark for antibody performance. © 2019 International Society for Advancement of Cytometry.

• Klíčová slova
• flow cytometry, monoclonal antibody, validation,
• MeSH
• indikátory a reagencie MeSH
• protilátky * MeSH
• průtoková cytometrie MeSH
• reprodukovatelnost výsledků MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• indikátory a reagencie MeSH
• protilátky * MeSH

The present study describes a rapid and effective capillary electrophoresis (CE) method for the enantioseparation of pindolol using single-isomer octa(6-O-sulfo)-γ-cyclodextrin. The complexation parameters were determined under neutral and high pH conditions to identify optimal separation conditions using a theoretical model. Baseline separation of pindolol enantiomers was achieved within 6 min in a sodium/MOPS buffer, pH 7.2, with a selector concentration of 6 mM. The method was validated according to the ICH guidelines using imidazole as an internal standard. Low limits of detection and quantification were found, specifically 1.2 μg/mL and 4 μg/mL (0.6 μg/mL and 2 μg/mL per enantiomer), respectively. The calibration curves showed good linearity, with a coefficient of determination R2 ≥ 0.999 over a 5 - 55 μg/mL concentration range and over a 50 - 300 μg/mL concentration range of the racemic mixture. The relative standard deviations (%RSD) of intra-day and inter-day precision were lower than 8% at LOQ level, lower than 3% at 50 μg/mL level and lower than 1.5% at 300 μg/mL level. Accuracy ranged from 95 to 103% (106% at LOQ level). The proposed method was successfully tested on a medical formulation of Visken® Sandoz intravenous solution and Visken® Teofarma pills for oral use.

• Klíčová slova
• Capillary electrophoresis, Enantioseparation, Method validation, Pindolol, Single-isomer cyclodextrin, β-blockers,
• MeSH
• časové faktory MeSH
• elektroforéza kapilární metody   MeSH
• gama-cyklodextriny chemie   MeSH
• kalibrace MeSH
• koncentrace vodíkových iontů MeSH
• limita detekce MeSH
• pindolol izolace a purifikace   MeSH
• pufry MeSH
• reprodukovatelnost výsledků MeSH
• software * MeSH
• stereoizomerie MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• gama-cyklodextriny MeSH
• gamma-cyclodextrin MeSH Prohlížeč
• pindolol MeSH
• pufry MeSH

At present, no standardized tool to measure therapeutic outcomes of a central European traditional healing system called Ethicotherapy exists. Ethicotherapy focuses on the relationship between physical health and moral health. The aim was to develop and validate the Ethicotherapy quality of life questionnaire (EQLQ) using a sequential exploratory mixed-method study design. An EQLQ with good psychometric properties was developed. An exploratory factor analysis identified eight domains consisting of 30 items. Future research investigating a relationship between EQLQ, health, and physical illness in different populations and cultural settings is recommended.

• Klíčová slova
• Development, Ethicotherapy, Psychometric properties, Questionnaire, Validation,
• MeSH
• faktorová analýza statistická MeSH
• kvalita života * MeSH
• lidé MeSH
• průzkumy a dotazníky * normy   MeSH
• psychometrie MeSH
• reprodukovatelnost výsledků MeSH
• výzkumný projekt * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

This paper presents a new method for the determination of the volume, surface area and depth of skin defects. The method is based on the description of a spatial defect using a pyramid (made, for example, from injection needles), which is placed over the defect. The projection of the pyramid on to the defect is photographed using a digital camera and subsequently compared with the projection of the same pyramid on to a sheet of grid paper. The defect is mathematically reconstructed on a computer, and an optimal body shape describing the defect is found, using a number of simplifications and assumptions. The method was then validated using a plaster mold of a real foot with 19 defects simulating real wounds. These plaster wounds were molded using alginate hydrocolloid, and the volume, surface area and depth were measured and compared with the results of the pyramid projection by means of regression analysis.This method correlates in all variables with correlation coefficients higher than 0.9. It can be concluded that the projection pyramid method correlates well with the reference mold method and can be used with good results for a whole range of variables.

• MeSH
• fotografování metody   MeSH
• kůže patologie   MeSH
• lidé MeSH
• počítačové zpracování signálu * MeSH
• rány a poranění patologie   MeSH
• reprodukovatelnost výsledků MeSH
• software MeSH
• vředy na noze (od hlezna dolů) patologie   MeSH
• zobrazování trojrozměrné * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

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.

• Klíčová slova
• Alternative validation, Bile acids, LC-MS/MS,
• MeSH
• chromatografie kapalinová metody   MeSH
• lidé MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Aderamastat (FP-025) is a small molecule, selective matrix metalloproteinase (MMP)-12 inhibitor, under development for respiratory conditions which may include chronic inflammatory airway diseases and pulmonary fibrosis. To support evaluation of the pharmacokinetic parameters of Aderamastat in humans, we developed and validated a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analytical method for the quantification of Aderamastat in human plasma. This assay was validated in compliance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations (GLP) and European Medicines Agency (EMA) guidelines. K2EDTA human plasma samples were spiked with internal standard, processed by liquid-liquid extraction, and analyzed using reversed-phase HPLC with Turbo Ion Spray® MS/MS detection. Separation was done using a chromatographic gradient on 5 µm C6-Phenyl 110 Å, 50*2 mm analytical column at a temperature of 35 °C. The LC-MS/MS bioanalytical method, developed by QPS Taiwan to determine the concentration of Aderamastat in K2EDTA human plasma, was successfully validated with respect to linearity, sensitivity, accuracy, precision, dilution, selectivity, hemolyzed plasma, lipemic plasma, batch size, recovery, matrix effect, and carry-over. These data indicate that the method for determination of Aderamastat concentrations in human K2EDTA plasma can be used in pharmacokinetics studies and subsequent clinical trials with Aderamastat. Authors declare that, this novel data is not published and not under consideration for publication by another journal than this journal. All data will be made available on request.

• Klíčová slova
• Aderamastat, Bioanalysis Assay, FP-025, LC-MS/MS, Matrix Metalloproteinase (MMP)-12 inhibitor, Validation,
• MeSH
• adamantan analogy a deriváty   krev   farmakokinetika   chemie   MeSH
• chromatografie kapalinová metody   MeSH
• EDTA * chemie   krev   farmakokinetika   MeSH
• kapalinová chromatografie-hmotnostní spektrometrie MeSH
• lidé MeSH
• limita detekce MeSH
• lineární modely MeSH
• reprodukovatelnost výsledků MeSH
• stabilita léku MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adamantan MeSH
• EDTA * MeSH

Here, we present a practical overview of four commonly used validation methods for DNA methylation assessment: methylation specific restriction endonucleases (MSRE) analysis, pyrosequencing, methylation specific high-resolution DNA melting (MS-HRM) and quantitative methylation specific polymerase chain reaction (qMSP). Using these methods, we measured DNA methylation levels of three loci in human genome among which one was highly methylated, one intermediately methylated and one unmethylated. We compared the methods in terms of primer design demands, methods' feasibility, accuracy, time and money consumption, and usability for clinical diagnostics. Pyrosequencing and MS-HRM proved to be the most convenient methods. Using pyrosequencing, it is possible to analyze every CpG in a chosen region. The price of the instrument may represent the main limitation of this methodology. MS-HRM is a simple PCR-based method. The measurement was quick, cheap and very accurate. MSRE analysis is based on a methylation specific digestion of DNA. It does not require a bisulfite conversion of DNA as the other methods. MSRE analysis was very easy to perform, however, it was not suitable for intermediately methylated regions and it was also quite expensive. qMSP is a qPCR-based method that uses primers designed specifically for methylated and unmethylated alleles of a chosen region. This was the least accurate method and also the primer design and optimization of PCR conditions were highly demanding.

• Klíčová slova
• DNA methylation, MS-HRM, MSRE, Pyrosequencing, Validation methods, qMSP,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH