Quantitative Real-Time Polymerase Chain Reaction, better known as qPCR, is the most sensitive and specific technique we have for the detection of nucleic acids. Even though it has been around for more than 30 years and is preferred in research applications, it has yet to win broad acceptance in routine practice. This requires a means to unambiguously assess the performance of specific qPCR analyses. Here we present methods to determine the limit of detection (LoD) and the limit of quantification (LoQ) as applicable to qPCR. These are based on standard statistical methods as recommended by regulatory bodies adapted to qPCR and complemented with a novel approach to estimate the precision of LoD.

• Klíčová slova
• Data analysis, GenEx software, Limit of detection, Limit of quantification, LoD, LoQ, MIQE, Quality control, Real-time PCR, Replicates, Standardization, qPCR,
• Publikační typ
• časopisecké články MeSH

Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.

• Klíčová slova
• limit of detection, Prototheca bovis, milk, real-time PCR,
• MeSH
• DNA chemie   izolace a purifikace   MeSH
• farmy MeSH
• klonování DNA MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• limita detekce MeSH
• mikrobiologie životního prostředí MeSH
• mlékárenství MeSH
• mléko mikrobiologie   MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• plazmidy genetika   MeSH
• Prototheca genetika   růst a vývoj   izolace a purifikace   MeSH
• senzitivita a specificita MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• DNA MeSH

The quantification of gold nanoparticles (AuNP) in environmental samples at ultratrace concentrations can be accurately performed by sophisticated and pricey analytical methods. This paper aims to challenge the analytical potential and advantages of cheaper and equally reliable alternatives that couple the well-established extraction procedures with common spectrometric methods. We discuss several combinations of techniques that are suitable for separation/preconcentration and quantification of AuNP in complex and challenging aqueous matrices, such as tap, river, lake, brook, mineral, and sea waters, as well as wastewaters. Cloud point extraction (CPE) has been successfully combined with electrothermal atomic absorption spectrometry (ETAAS), inductively coupled plasma mass spectrometry (ICP-MS), chemiluminescence (CL), and total reflection X-ray fluorescence spectrometry (TXRF). The major advantage of this approach is the ability to quantify AuNP of different sizes and coatings in a sample with a volume in the order of milliliters. Small volumes of sample (5 mL), dispersive solvent (50 µL), and extraction agent (70 µL) were reported also for surfactant-assisted dispersive liquid-liquid microextraction (SA-DLLME) coupled with electrothermal vaporization inductively coupled plasma mass spectrometry (ETV-ICP-MS). The limits of detection (LOD) achieved using different combinations of methods as well as enrichment factors (EF) varied greatly, being 0.004-200 ng L-1 and 8-250, respectively.

• Klíčová slova
• environmental waters, extraction techniques, gold nanoparticles, quantification, separation, spectrometric methods,
• MeSH
• kovové nanočástice * MeSH
• odpadní voda MeSH
• povrchově aktivní látky MeSH
• rozpouštědla MeSH
• zlato * MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• odpadní voda MeSH
• povrchově aktivní látky MeSH
• rozpouštědla MeSH
• zlato * MeSH

Apidaecins represent an important group of antimicrobial peptides occurring in honey bee hemolymph, where they play an important role as key components of humoral immunity. The present study demonstrates the development of a highly sensitive assay for apidaecin 1 isoforms quantification in the hemolymph or body parts from honey bee individuals. The analytical protocol comprises apidaecins 1 purification and enrichment steps by weak cation-exchange chromatography (WCX) in laboratory-made WCX-Tip microcolumns combined with a desalting step on a reversed-phase sorbent (C8) carried in StageTips. Apidaecin-enriched fraction was analyzed by a reversed-phase based nanoliquid chromatography (C4) separation coupled with high-resolution mass spectrometry. The method performance was validated in its specificity, linearity (0-5pmol), recovery (∼45%), precision (<10% at 0.1pmol), limit of detection (∼50fmol), limit of quantification (0.1pmol) and sample stability. The method was successfully applied to analyze the content of apidaecin 1 isoforms in the following samples: hemolymph - 13.0ng/μL (95% confidence interval of 7.5-18.6ng/μL), thoraxes - 36.2ng/unit (95% CI of 18.9-53.6ng/unit) and heads - 12.9ng/unit (95% CI of 9.1-16.7ng/unit). Freshly emerged bees had apidaecin 1 isoforms levels below the limit of detection. Thus it was possible to use them as a competitive matrix for calibration standards to prevent losses of highly basic apidaecins. This new protocol for apidaecin 1 isoforms quantification represents a promising tool to study the role of apidaecins in honey bee immunity and can be considered as a proof-of-concept for the development of sensitive quantification methods for basic antimicrobial peptides in various organisms.

• Klíčová slova
• Antimicrobial peptide, Apidaecin, Honey bee, Mass spectrometry, Nanoflow liquid chromatography, Quantification,
• MeSH
• hmotnostní spektrometrie metody   MeSH
• kalibrace MeSH
• kationické antimikrobiální peptidy izolace a purifikace   MeSH
• kationty chemie   MeSH
• limita detekce MeSH
• protein - isoformy izolace a purifikace   MeSH
• včely chemie   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• apidaecin MeSH Prohlížeč
• kationické antimikrobiální peptidy MeSH
• kationty MeSH
• protein - isoformy MeSH

The potential use of ethanol as an internal standard (IS) for GC-MS analysis was studied. The paper describes the analysis of spirit drinks and other alcoholic products which consist of a mixture of water, ethanol, and volatile compounds. In the suggested method, ethanol was employed as an IS for the common procedure of volatile compounds quantification. A number of standard solutions of nine compounds with different concentrations was prepared in a water-ethanol matrix and measured with GC-MS in the SIM mode. Two possible approaches were suggested to avoid detector saturation during ethanol detection. The first one consisted in using less abundant m/z 47 as quantifiers. These ions mainly correspond to unfragmented heavy ethanol molecules containing one 13 C isotope. The second method consisted in reduction of the voltage of MS electron multiplier. The experiment also included the preparation and subsequent dilution of the standard solution and ethanol with water, which determined the linearity of the modified MS response relative to the ethanol content. Analysis of the obtained results revealed that volatile compounds can be successfully accurately determined with GC-MS by employing ethanol as an IS. Application of the suggested method is not limited to the reported volatile compounds and alcoholic products.

• Klíčová slova
• GC-MS, alcoholic products, ethanol, internal standard, quantification, volatile compounds,
• Publikační typ
• časopisecké články MeSH

Internal normalization (IN) serves as a quantitative tool in gas chromatography. Nevertheless, its utilization in liquid chromatography is not widely employed, as several requirements need to be taken into account. However, IN can be used in case of relative amounts estimation when the absolute concentration is not the crucial factor. This suits very well in pharmaceutical analysis when the relative amount of active pharmaceutical ingredient (API) impurities is to be estimated with a limited knowledge of statistics, such as t-test and linear regression. The determination of three prasugrel impurities in the real sample by means of IN and the comparison of these results with external standard calibration was presented. The IN method was validated by test of population means and variances agreement and the agreement of external calibration and IN was performed by Student t-test. The influence of impurities concentration above and below is also discussed as well as the validation parameters, LOD and LOQ. It was found that the results achieved by external calibration and IN are statistically the same and, therefore, IN is a proper method for relative amount estimation of API impurities.

• Klíčová slova
• Internal normalization, Pharmaceutical substances, Quantification, Validation,
• MeSH
• farmaceutická chemie normy   MeSH
• kalibrace MeSH
• koncentrace vodíkových iontů MeSH
• léčivé přípravky chemie   MeSH
• limita detekce MeSH
• prasugrel hydrochlorid analýza   MeSH
• reprodukovatelnost výsledků MeSH
• vysokoúčinná kapalinová chromatografie metody   normy   MeSH
• Publikační typ
• časopisecké články MeSH
• validační studie MeSH
• Názvy látek
• léčivé přípravky MeSH
• prasugrel hydrochlorid MeSH

Clavulanic acid is a molecule with antimicrobial effect used in several livestock species treatment. Its inclusion in the treatment of infectious diseases of broilers requires determination of pharmacokinetic and pharmacodynamic parameters in order to determine the appropriate dosage for broilers and ensure safety of chicken products for human health. The present study describes the optimisation of analytical LC-MS/MS method for identification and quantification of clavulanic acid in broiler chicken plasma and meat. The limit of detection and the limit of quantification for the developed method were 3.09 μg·L-1 and 10.21 μg·L-1 for plasma and 2.57 μg·kg-1 and 8.47 μg·kg-1 for meat. The recoveries of the developed plasma and tissue extraction procedure were > 105.7% and > 95.6%, respectively. The achieved coefficient of variation of within-run precision ranged from 2.8 to 10.9% for plasma and from 6.5 to 8.5% for meat. The pharmacokinetic experiment was performed in 112 Ross broiler chickens assigned into time interval groups ranging from 10 min to 24 h in accredited animal facilities. Administered dose of clavulanic acid was 2.5 mg·kg-1 according to the manufacturer's recommendations. The pharmacokinetic parameters obtained from the experiment are as follows: Cmax = 1.82 ± 0.91 mg·L-1, Tmax = 0.25 h, T1/2 = 0.87 h, Kel = 0.80 ± 0.04 h-1, AUC0-∞ = 2.17 mg·h ·L-1.

• Klíčová slova
• Antibiotics, Clavulanic acid, Pharmacokinetics, Tandem mass spectrometry,
• MeSH
• hmotnostní spektrometrie metody   MeSH
• inhibitory beta-laktamasy krev   metabolismus   farmakokinetika   MeSH
• kur domácí MeSH
• kyselina klavulanová krev   metabolismus   farmakokinetika   MeSH
• limita detekce MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• inhibitory beta-laktamasy MeSH
• kyselina klavulanová MeSH

Two new impurities were described and determined using gradient HPLC method with UV detection in retigabine (RET). Using LC-HRMS, NMR and IR analysis the impurities were identified as RET-dimer I: diethyl {4,4'-diamino-6,6'-bis[(4-fluorobenzyl)amino]biphenyl-3,3'-diyl}biscarbamate and RET-dimer II: ethyl {2-amino-5-[{2-amino-4-[(4-fluorobenzyl) amino] phenyl} (ethoxycarbonyl) amino]-4-[(4-fluorobenzyl)amino] phenyl}carbamate. Reference standards of these impurities were synthesized followed by semipreparative HPLC purification. The mechanism of the formation of these impurities is also discussed. An HPLC method was optimized in order to separate, selectively detect and quantify all process-related impurities and degradation products of RET. The presented method, which was validated in terms of linearity, limit of detection (LOD), limit of quantification (LOQ) and selectivity is very quick (less than 11min including re-equilibration time) and therefore highly suitable for routine analysis of RET related substances as well as stability studies.

• Klíčová slova
• HPLC, Identification, Process-related impurities, Retigabine, Synthesis,
• MeSH
• fenylendiaminy chemie   MeSH
• karbamáty chemie   MeSH
• kontaminace léku MeSH
• limita detekce MeSH
• referenční standardy MeSH
• stabilita léku MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ezogabine MeSH Prohlížeč
• fenylendiaminy MeSH
• karbamáty MeSH

We carried out a calibration of FLOTAC for ciliates Troglodytella abrassarti and Neobalantidium coli based on the selection of a most appropriate flotation solutions, and we also tested its accuracy (i.e., number of detected stages out of known added number of stages to fecal samples) and sensitivity for trophozoites of both ciliates in chimpanzee feces and N. coli cysts in pig feces, compared the detection threshold of FLOTAC with MIF-based sedimentation, and, subsequently, tested the losses of ciliate stages during sample preparation. Nine flotation solutions were evaluated, and ZnSO4 solution (specific gravity [s.g.] 1.2) showed to be the most suitable for trophozoite detection, while Sheather's solution (s.g. 1.33) was selected as most suitable for cysts. The FLOTAC sensitivity in detection of both stages varied: for trophozoites, we found all samples were positive when the intensity of infection 10 trophozoites per gram and higher, whereas for cysts the sensitivity was lower. The accuracy of FLOTAC negatively correlated with infection intensity, and the merthiolate-iodine-formaldehyde sedimentation-based quantification had a lower detection threshold. We demonstrated additional losses of stages of T. abrassarti and N. coli due to their retention in the sediment, which is probably a major reason for discrepancies in the numbers of countable ciliates between both methods. In conclusion, the FLOTAC should not be considered as a gold standard for quantification of intestinal ciliates in primates; instead, we recommend the modified MIF method.

• MeSH
• Ciliophora izolace a purifikace   MeSH
• feces parazitologie   MeSH
• infekce prvoky kmene Ciliophora diagnóza   parazitologie   veterinární   MeSH
• kalibrace MeSH
• limita detekce MeSH
• nemoci lidoopů diagnóza   parazitologie   MeSH
• nemoci prasat diagnóza   parazitologie   MeSH
• Pan troglodytes parazitologie   MeSH
• prasata MeSH
• roztoky chemie   klasifikace   MeSH
• senzitivita a specificita MeSH
• specifická hustota MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• roztoky MeSH

Hippocampal dysfunction is known to be associated with several neurological and neuropsychiatric disorders such as Alzheimer's disease, epilepsy, schizophrenia and depression; therefore, there has been significant clinical interest in studying hippocampal neurochemistry. However, the hippocampus is a challenging region to study using (1) H MRS, hence the use of MRS for clinical research in this region has been limited. Our goal was therefore to investigate the feasibility of obtaining high-quality hippocampal spectra that allow reliable quantification of a neurochemical profile and to establish inter-session reproducibility of hippocampal MRS, including reproducibility of voxel placement, spectral quality and neurochemical concentrations. Ten healthy volunteers were scanned in two consecutive sessions using a standard clinical 3 T MR scanner. Neurochemical profiles were obtained with a short-echo (T(E) = 28 ms) semi-LASER localization sequence from a relatively small (~4 mL) voxel that covered about 62% of the hippocampal volume as calculated from segmentation of T1 -weighted images. Voxel composition was highly reproducible between sessions, with test-retest coefficients of variation (CVs) of 3.5% and 7.5% for gray and white matter volume fraction, respectively. Excellent signal-to-noise ratio (~54 based on the N-acetylaspartate (NAA) methyl peak in non-apodized spectra) and linewidths (~9 Hz for water) were achieved reproducibly in all subjects. The spectral quality allowed quantification of NAA, total choline, total creatine, myo-inositol and glutamate with high scan-rescan reproducibility (CV ≤ 6%) and quantification precision (Cramér-Rao lower bound, CRLB < 9%). Four other metabolites, including glutathione and glucose, were quantified with scan-rescan CV below 20%. Therefore, the highly optimized, short-echo semi-LASER sequence together with FASTMAP shimming substantially improved the reproducibility and number of quantifiable metabolites relative to prior reports. In addition, the between-session variation in metabolite concentrations, as well as CRLB, was lower than the between-subject variation of the concentrations for most metabolites, indicating that the method has the sensitivity to detect inter-individual differences in the healthy brain.

• Klíčová slova
• 3 T, MRS, coefficient of variation, human hippocampus, metabolites, quantification precision, reproducibility, segmentation,
• MeSH
• algoritmy MeSH
• biopolymery metabolismus   MeSH
• dospělí MeSH
• hipokampus anatomie a histologie   metabolismus   MeSH
• lidé MeSH
• molekulární zobrazování metody   MeSH
• protonová magnetická rezonanční spektroskopie metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• studie proveditelnosti MeSH
• tkáňová distribuce MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• biopolymery MeSH