This review is a Part II of the series aiming to provide comprehensive overview of currently used antiviral drugs and to show modern approaches to their analysis. While in the Part I antivirals against herpes viruses and antivirals against respiratory viruses were addressed, this part concerns antivirals against hepatitis viruses (B and C) and human immunodeficiency virus (HIV). Many novel antivirals against hepatitis C virus (HCV) and HIV have been introduced into the clinical practice over the last decade. The recent broadening portfolio of these groups of antivirals is reflected in increasing number of developed analytical methods required to meet the needs of clinical terrain. Part II summarizes the mechanisms of action of antivirals against hepatitis B virus (HBV), HCV, and HIV, their use in clinical practice, and analytical methods for individual classes. It also provides expert opinion on state of art in the field of bioanalysis of these drugs. Analytical methods reflect novelty of these chemical structures and use by far the most current approaches, such as simple and high-throughput sample preparation and fast separation, often by means of UHPLC-MS/MS. Proper method validation based on requirements of bioanalytical guidelines is an inherent part of the developed methods.
- MeSH
- antivirové látky analýza farmakologie terapeutické užití MeSH
- biologické faktory analýza metabolismus MeSH
- hepatitida farmakoterapie metabolismus MeSH
- HIV infekce farmakoterapie metabolismus MeSH
- HIV-1 účinky léků metabolismus MeSH
- lidé MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
This review article is the first in the series providing an overview of currently used antiviral drugs and presenting contemporary approaches to their analysis. Large number of available antivirals and their structural variability makes this task very challenging. Trying to cover this topic comprehensively while maintaining reasonable size of the article, the review is presented in two parts. For the purpose of the overall review, antivirals were divided into four groups: (i) antivirals against herpes viruses, (ii) antivirals against respiratory viruses, (iii) antivirals against hepatitis viruses, and (iv) antivirals against HIV. Part one is devoted to the groups (i) and (ii) and also concerns the key features of the bioanalytical method. The mechanisms of action of antivirals against respiratory and herpes viruses and their use in clinical practice are briefly outlined, and the analytical methods for selected representatives of each class are described in more detail. The methods developed for the determination of drugs from these classes mostly include conventional procedures. In contrast, current trends such as UHPLC are used rarely and proper method validation based on requirements of bioanalytical guidelines can be often considered insufficient.
- MeSH
- antivirové látky analýza terapeutické užití MeSH
- biologické faktory analýza MeSH
- Herpesviridae účinky léků metabolismus MeSH
- herpetické infekce farmakoterapie metabolismus MeSH
- infekce dýchací soustavy farmakoterapie metabolismus MeSH
- lidé MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- virové nemoci farmakoterapie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Estrogen steroids, represented by estradiol and its related substances, include both structurally very close and simultaneously different analogs. Their separation still remains an analytical challenge. Subcritical fluid chromatography (SbFC) on sub-2-micron particles was found to be an appropriate tool to obtain fast and efficient separation of nine target analytes. Among the four tested stationary phases charged hybrid modified with PFP (pentafluorophenyl) moiety was found to be the most convenient providing the fastest separation within 1.6 min using quick gradient elution with carbon dioxide and methanol as an organic modifier. However, complete separation was obtained also on other tested phases including bare hybrid stationary phase, hybrid stationary phase modified with 2-EP (2-ethylpyridine) and also C18, which is less typical in SbFC. The baseline separation on the latter columns was achieved by means of a temperature increase, a change in organic modifier type and gradient time increase respectively. Quantitative performance was evaluated at optimized conditions and method validation was accomplished. Excellent repeatability of both retention times (RSD<0.15%) and peak areas (RSD<1%) was observed. The method was linear in the range of 1.0-1000.0 μg/ml for all steroids with the lowest calibration point being an LOQ, except for Δ-derivatives, that provided better sensitivity and thus LOQ of 0.5 μg/ml. The sensitivity was sufficient for the analysis of real samples although it was still five times lower compared to UHPLC-UV experiments.
The development of bioanalytical methods has become more and more challenging over the past years due to very demanding requirements in terms of method reliability, sensitivity, speed of analysis and sample throughput. LC-MS/MS has established itself as a method of choice for routine analysis of biological materials. A development of such method consists of several steps including sample preparation and clean-up step, efficient chromatographic separation, sensitive and selective detection of analytes in complex matrices, a choice of convenient data processing and calibration approach and finally method validation. Each of these steps has its own constraints and challenges, which are discussed in detail in this review. Novel and modern approaches in sample preparation, chromatography and detection are especially emphasized. Attention is paid to proper calibration approach and matrix effects that can seriously affect method accuracy and precision.
- MeSH
- časové faktory MeSH
- chromatografie kapalinová ekonomika přístrojové vybavení metody MeSH
- hmotnostní spektrometrie ekonomika přístrojové vybavení metody MeSH
- lidé MeSH
- validační studie jako téma MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Karcinom prsu patří mezi nejčastější malignitu u žen a podílí se i na vysoké invalidizaci pacientek. Důležitá je prevence a screening. V článku je popsána diagnostika, léčba a následné komplikace. Byl porovnán soubor pacientek od 1. 1. 2008 do 30. 9. 2011 podle vyhlášky č. 284/1995 Sb. a č. 359/2009 Sb.
Breast cancer belongs to the most frequent malignancies in women and participates in a high invalidism of the patients. Prevention and screening are very important. The contribution describes the diagnostics, therapy and subsequent complications. The group of patients in the period of January 1st 2008 to September 30th 2011 was analyzed according to the Regulation No. 284/1995 Sb. and No. 359/2009 Sb.
- MeSH
- chirurgie operační metody MeSH
- diagnostické zobrazování metody MeSH
- dospělí MeSH
- klasifikace MeSH
- lidé středního věku MeSH
- lidé MeSH
- lymfedém MeSH
- mamografie MeSH
- mastektomie MeSH
- nádory prsu * diagnóza prevence a kontrola terapie MeSH
- plošný screening MeSH
- posudkové řízení ve zdravotní péči normy MeSH
- posuzování zdravotní způsobilosti * MeSH
- rizikové faktory MeSH
- sentinelová uzlina MeSH
- staging nádorů MeSH
- statistika jako téma MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- přehledy MeSH
In this study, the selectivity, retention properties, peak shape and loading capacity for bases were practically evaluated using two UHPLC mixed-mode hybrid CSH stationary phases modified by C18 or Phenyl group. The data were compared with the data obtained on other UHPLC hybrid stationary phases (BEH C18, BEH C8, BEH Phenyl and BEH Shield RP18) at both basic and acidic conditions using conventional HPLC buffers (50mM ammonium formate/acetate) as well as low ionic-strength additives such as, e.g. 0.1-0.01% formic/acetic acid and 1mM solution of ammonium formate/acetate, which are widely used in LC-MS applications. Ten pharmaceutically important compounds encompassing acids, bases and neutral were included into the study. Due to properties of CSH sorbent (which possess positively charged surface besides RP group), much improved peak shapes and weaker retention was obtained for bases even at very low concentration of acidic additives. Such conditions are ideally suited for LC-MS analysis of bases, where typical RP chromatographic separation (retention and good selectivity at basic pH) and LS-MS conditions (efficient ionization at acidic pH) are not in agreement. On the other hand, acids were more strongly retained and for some compounds the peak shape was influenced negatively due to ion-exchange mechanism. Further, the behavior of acidic, basic and neutral solutes is discussed using various additives at both basic and acidic pH for all above stated columns. The robustness of retention times after pH change from basic to acidic was also evaluated. The new CSH stationary phases represent an interesting selectivity tool preferably for separation of basic compounds.
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Entecavir is a deoxyguanosine nucleotide antiviral agent with the activity against hepatitis B virus (HBV). The agent possesses a polar structure, which is predetermined for hydrophilic interaction chromatography (HILIC). Novel, fast and sensitive HILIC-UHPLC method developed in this study included separation from matrix component on BEH Amide stationary phase by isocratic elution using binary mobile phase composed of acetonitrile/5mM ammonium acetate pH 4.0 (75:25) at flow-rate 0.3 ml/min. Analysis under RP-UHPLC conditions was also possible on BEH C18 stationary phase with mostly aqueous binary mobile phase composed of (4:96) acetonitrile/0.01% formic acid. The comparison of sensitivity of the two UHPLC-MS/MS methods both using selected reaction monitoring (SRM) for quantitation revealed only slightly higher sensitivity for HILIC determination, however much better method linearity, repeatability and accuracy. HILIC separation mode provided also more convenient conditions for straightforward coupling with solid phase extraction (SPE). Entecavir was extracted on Oasis HLB cartridge (1 ml, 30 mg) and eluted by 75% acetonitrile in water, which is actually the HILIC mobile phase used in this study. Therefore the evaporation/reconstitution step was omitted, which substantially accelerated the sample preparation step. The method was validated using stable isotopically labeled internal standard entecavir-C(2)(13) N(15), which is the most appropriate internal standard. Validation results demonstrated good method accuracy (with < 5% error, and 26% at LOQ), recovery (87-114%), precision (<4% RSD), selectivity and sensitivity (LOQ=100 pg/ml). The matrix effects determined by both post-column infusion method as well as post-extraction addition method were negligible (<15%).
- MeSH
- extrakce na pevné fázi MeSH
- guanin analogy a deriváty farmakokinetika moč MeSH
- hydrofobní a hydrofilní interakce MeSH
- krysa rodu rattus MeSH
- limita detekce MeSH
- lineární modely MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Invariant natural killer T (iNKT) cells, CD1d restricted T cells, are involved in the immune responses against various infection agents. Here we describe their behavior during reactivation of human herpes simplex virus (HSV). iNKT cells exhibit only discrete changes, which however, reached statistically significant level due to the relatively large patient group. Higher percentage of iNKT cells express NKG2D. iNKT cells down-regulate NKG2A in a subset of patients. Finally, iNKT cells enhance their capacity to produce TNF-α. Our data suggests that iNKT cells are involved in the immune response against HSV and contribute mainly to its early, innate phase.
- MeSH
- aktivace lymfocytů MeSH
- aktivace viru MeSH
- antigeny CD1d genetika imunologie MeSH
- dospělí MeSH
- exprese genu MeSH
- herpes simplex imunologie patologie virologie MeSH
- lektinové receptory NK-buněk - podrodina K genetika imunologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- NKT buňky imunologie patologie MeSH
- přirozená imunita * MeSH
- senioři MeSH
- Simplexvirus fyziologie MeSH
- TNF-alfa genetika imunologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
