Cílem práce bylo zavést a validovat analytickou metodu, která by umožnila detekovat koncentrace nabumetonu a kyseliny 6-methoxy-2-naftyloctové (6-MNA) po jednorázovém podání terapeutické dávky léčiva. Byly porovnány dvě metody stanovení pomocí HPLC s UV a hmotnostní detekcí. Při úpravě vzorku bylo optimálních výsledků dosaženo pomocí extrakce na pevné fázi (SPE). Výtěžnost se pohybovala okolo 84 % pro nabumeton a 86–90 % pro 6-MNA. HPLC separace analytů byla prováděna na reverzní C18 koloně. Limit UV detekce pro 6-MNA byl 50 nM, pro nabumeton 0,1 µM. Limit MS detekce pro 6-MNA činil 1 µM, pro nabumeton 0,5 µM. Přesnost metody pro stanovení nabumetonu se pohybovala v rozmezí 4,2–14,4 % při UV detekci a 4,6–8,5 % při MS detekci. Přesnost metody pro 6-MNA byla 2,4–12,5% při UV detekci a 2,1–9,4 % při MS detekci. Správnost metody pro stanovení nabumetonu se pohybovala v rozmezí 93,4–109,6 % při UV detekci a 86,2–107,9 % při MS detekci. Správnost metody pro 6-MNA byla 87,8–107,4 % při UV detekci a 86,3–106,4 % při MS detekci. Vhodnost metody byla ověřená na vzorcích pro stanovení farmakokinetiky léčiva u 24 zdravých dobrovolníků.
The study aimed to establish and validate an analytical method for the determination of nabumetone and 6-methoxy-2-naphthylacetic acid (6-MNA) in human plasma after a single therapeutic dose of the drug. Two methods based on HPLC with UV and MS detection were compared. Optimal results in sample preparation were achieved using solid phase extraction. The recovery reached approximately 84% and 86–90% for nabumetone and 6-MNA, respectively. A reverse C18 column was used for HPLC separation of the analytes. The limit of UV detection was 50 nM and 0.1 µM for 6-MNA and nabumetone, respectively. The limit of MS detection was 1 µM and 0.5 µM for 6-MNA and nabumetone, respectively. Precision ranged between 4.2–14.4% and 4.6–8.5% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 2.4–12.5% and 2.1–9.4%. Accuracy ranged between 93.4–109.6% in UV detection and 86.2–107.9% using UV and MS detection for nabumetone, respectively. The respective values for 6-MNA were 87.8–107.4% and 86.3–106.4%. The method was subsequently applied to determine the pharmacokinetic parameters of nabumetone and 6-MNA in a group of 24 healthy volunteers.
Je zde popsána metoda pro specifické stanovení kyseliny močové v lidském séru. Byla testována účinnost různých deproteinačních činidel. Výsledky získané proměřením 300 vzorků sér pomocí vysoce účinné kapalinové chromatografie byly porovnány s výsledky získanými rutinní enzymatickou metodou. Vzorky sér byly deproteinovány pomocí kyseliny chloristé a supernatanty byly analyzovány vysoce účinnou kapalinovou chromatografií na reverzní fázi. Pro separaci byla užita kolona MAG 1, 4,6 x 150 mm, Biospher PSI 200, 5 µm. Mobilní fází byla směs 5% metanolu v 25 mmol/l dihydrogenfosforečnanu sodného (v/v), pH 4,75. Analytické parametry metody jsou vyhovující: variační koeficienty přesnosti v sérii a mezi sériemi se pohybovaly pod 5 %. Výtěžnost, určená metodou standardních přídavků, se pohybovala v rozmezí 90,0–103,4 %. Mez stavitelnosti byla 10,0 µmol/l (3,3 pmol/nástřik). Výsledky získané chromatografickou metodou dobře korelovaly s výsledky získanými rutinní enzymatickou metodou, avšak chromatografická metoda poskytovala vyšší hodnoty, a to v závislosti na dané skupině pacientů. Zde prezentovaná metoda je vhodná pro analýzu těch vzorků, u nichž klasická enzymatická metoda poskytuje nespolehlivé výsledky.
A sensitive method for the specific determination of uric acid in human serum is described. The effectiveness of various protein precipitants was tested. Results from measurements by high-performance liquid chromatography on 300 serum samples were compared with an enzymatic method. Serum samples were deproteinized with perchloric acid and analyzed by reversed-phase high-performance liquid chromatography. For the separation, a reverse phase column MAG 1, 4.6 x 150 mm, Biospher PSI 200 C18, 5 µm, was used. The mobile phase consisted of 5% methanol in 25- mmol/L sodium dihydrogenphosphate (v/v), pH 4.75. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 5%. Quantitative recoveries of spiked serum samples were between 90.0 – 103.4%. The limit of quantification was 10.0 µmol/L (3.3 pmol/inject). Results obtained by chromatographic method correlated well with an enzymatic method, but gave at average higher values depending on a group of patients. Presented method is useful for the analysis of samples where the classical enzymatic method do not give reliable results.
- Keywords
- porovnání metod,
- MeSH
- Electrochemical Techniques methods instrumentation MeSH
- Financing, Organized MeSH
- Uric Acid analysis blood MeSH
- Reproducibility of Results MeSH
- Spectrophotometry, Ultraviolet methods instrumentation MeSH
- Urate Oxidase diagnostic use MeSH
- Chromatography, High Pressure Liquid methods instrumentation MeSH
Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25-hydroxyvitamin and 1,25-dihydroxyvitamin D(3). The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25-hydroxyvitamin D(3) in human plasma. A method for the measurement of 25-hydroxyvitamin D(3) using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125-4, Purospher RP-18e, 5 microm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra- and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0-103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25-hydroxyvitamin D(3) in a group of blood donors is 62 +/- 26 nmol/L.
- MeSH
- Adult MeSH
- Solid Phase Extraction methods MeSH
- Calcifediol blood MeSH
- Humans MeSH
- Reference Values MeSH
- Sensitivity and Specificity MeSH
- Ultraviolet Rays MeSH
- Chromatography, High Pressure Liquid methods instrumentation MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.
- MeSH
- Antioxidants chemistry MeSH
- Calibration MeSH
- Ascorbic Acid chemistry blood MeSH
- Humans MeSH
- Reference Standards MeSH
- Reproducibility of Results MeSH
- Solvents MeSH
- Sensitivity and Specificity MeSH
- Spectrophotometry, Ultraviolet methods instrumentation MeSH
- Chromatography, High Pressure Liquid methods instrumentation MeSH
- Check Tag
- Humans MeSH
- Publication type
- Case Reports MeSH
Efavirenz is an antiretroviral drug used in the treatment of HIV-positive patients. A simple, fast and sensitive high-performance liquid chromatography (HPLC) method was developed in order to determine efavirenz in three types of samples provided from pharmacokinetic studies. The analysis took 5min and was performed using a C18 analytical column (Discovery HS C18, 150×4.6mm, particle size of 5μm) in isocratic mode with a mobile phase containing acetonitrile and water (65:35, v/v), a flow rate of 1.6mLmin(-1), a sample volume of 10μL and UV detection at 245nm. Three different sample matrices (Opti-MEM medium, Krebs perfusion liquid and tissue lysate) and their treatment (dilution, SPE) were considered. The validated method was applied for the analysis of 805 real samples arising from in vitro transcellular transport assays and in vivo organ perfusion experiments in order to evaluate the interaction of efavirenz with ATP-dependent drug efflux transporters. The lack of interaction of efavirenz with ABCB1, ABCG2 and ABCC2 transporters as well as technical aspects of this analysis, including the adhesion of efavirenz to the plastic materials and the stability of the drug during different tissue lysis approaches are discussed.
- MeSH
- ATP Binding Cassette Transporter, Subfamily G, Member 2 metabolism MeSH
- Acetonitriles chemistry MeSH
- Benzoxazines chemistry metabolism MeSH
- Biological Transport physiology MeSH
- Cell Line MeSH
- Madin Darby Canine Kidney Cells MeSH
- Rats MeSH
- ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism MeSH
- Perfusion MeSH
- Placenta chemistry MeSH
- Rats, Wistar MeSH
- Multidrug Resistance-Associated Proteins metabolism MeSH
- Dogs MeSH
- Reproducibility of Results MeSH
- Drug Stability MeSH
- Pregnancy MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Dogs MeSH
- Pregnancy MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
A simple, sensitive and quick HPLC method was developed for the determination of ketoprofen in cell culture media (EMEM, DMEM, RPMI). Separation was performed using a gradient on the C18 column with a mobile phase of acetonitrile and miliQ water acidified by 0.1 % (v/v) formic acid. The method was validated for parameters including linearity, accuracy, precision, limit of quantitation and limit of detection, as well as robustness. The response was found linear over the range of 3-100 μg/mL as demonstrated by the acquired value of correlation coefficient R2=0.9997. The described method is applicable for determination of various pharmacokinetic aspects of ketoprofen in vitro.
Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.
- MeSH
- Acetonitriles chemistry MeSH
- Chromatography, Reverse-Phase MeSH
- Adult MeSH
- Smoking metabolism MeSH
- Ascorbic Acid analysis MeSH
- Uric Acid analysis MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Statistics, Nonparametric MeSH
- Reproducibility of Results MeSH
- Spectrophotometry, Ultraviolet MeSH
- Semen chemistry MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Adolescent MeSH
- Young Adult MeSH
- Male MeSH
- Publication type
- Journal Article MeSH
An HPLC method with UV and electrospray ionization - mass spectrometry (ESI-MS) detection was developed for the separation and determination of obeticholic acid (OBE) and its related compounds in development process and quality control. OBE and its related compounds were classified into three major group based on the mass spectra profiles: (A) those containing a hydroxyl group at position 3 and 7, (B) those containing a hydroxyl group and/or carbonyl group at position 3, hydrogen, ethyl or ethylidene group at position 6 and a hydroxyl group and/or carbonyl group at position 7, and (C) those containing carbonyl groups at position 3 and 7. ESI-MS ionization of OBE and its related compounds often produced intense adduct ions [M+H+98]+ and/or [M+H+196]+ that were identified as the adduct ions of phosphoric acid ([M+H+H3PO4]+ and [M+H+2H3PO4]+) originating from the mobile phase. The separation on HPLC system was accomplished using stationary phase based on XSelect CSH C18 (3.0×150mm×2.5μm) and a linear gradient elution using acetonitrile and 0.05% of o-phosphoric acid. The condition of chromatographic system was set as follows: flow rate 0.7mL/min, temperature 45°C and UV detection at 192nm. The separation of the 19 compounds was finished in less than 18min (including equilibration time). The HPLC/UV method was partially validated according to International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines in terms of repeatability, selectivity, linearity and limit of quantification and detection.
- MeSH
- Chemical Fractionation instrumentation methods MeSH
- Chemistry, Pharmaceutical methods MeSH
- Spectrometry, Mass, Electrospray Ionization instrumentation methods MeSH
- Drug Contamination prevention & control MeSH
- Chenodeoxycholic Acid analogs & derivatives analysis chemistry MeSH
- Limit of Detection MeSH
- Drug Compounding instrumentation methods MeSH
- Reproducibility of Results MeSH
- Quality Control * MeSH
- Sensitivity and Specificity MeSH
- Spectrophotometry, Ultraviolet instrumentation methods MeSH
- Chromatography, High Pressure Liquid instrumentation methods MeSH
- Publication type
- Journal Article MeSH
- Validation Study MeSH
