A rapid, sensitive and low-cost method for the determination of methamphetamine and its metabolites in urine was developed. The tested substances were isolated by SPE, dansylated and analyzed by HPLC with fluorescence detection. The method was validated, the limit of detection for methamphetamine was lower than 1 ng ml–1; the limit of quantitation was 3 ng ml–1.
- MeSH
- Amphetamine * analysis urine MeSH
- Chemistry Techniques, Analytical * methods instrumentation utilization MeSH
- Spectrometry, Fluorescence * methods utilization MeSH
- Indicators and Reagents MeSH
- Humans MeSH
- Methamphetamine * analogs & derivatives analysis urine MeSH
- Analytic Sample Preparation Methods MeSH
- Substance-Related Disorders MeSH
- Dansyl Compounds chemistry MeSH
- Chromatography, High Pressure Liquid * methods instrumentation utilization MeSH
- Check Tag
- Humans MeSH
V práci je popsána metoda vysokoúčinné kapalinové chromatografie, která byla zavedena pro účely fenotypizace enzymu CYP2D6 metoprololem jako substrátovou látkou. Metoprolol, ?-hydroxmetoprolol a nadolol (vnitřní standard) byly extrahovány ze séra dichlormethanem s přídavkem 1 mol/l NaOH. Látky byly separovány na koloně s reverzní fází SupercosilTM LC-18 (15 cm × 3 mm, 5 µm) mobilní fází o složení acetonitril : methanol : voda : triethylamin (15 : 5 : 80 : 0,1; pH 3,0) při průtoku 0,7 ml/min. Fluorescenční detekce (FL) probíhala při vlnové délce 230 nm (excitační) a 300 nm (emisní). Celková doba analýzy byla 12 min. Retenční časy byly pro ?-hydroxymetoprolol (h-MET) 2,04 minut, pro nadolol (IS) 3,02 minut a pro metoprolol (MET) 9,04 minut. Přesnosti v sérii a mezi sériemi vyjádřené jako relativní směrodatné odchylky byly nižší než 7,2 % a recovery se pohybovalo v rozmezí 98,2–103,0 %. Kalibrační křivka byla lineární v rozmezí 25–500 ng/ml (r = 0,999) pro obě látky. Detekční limit byl stanoven pro metoprolol a ?-hydroxymetoprolol na 5 ng/ml a kvantifikační limit na 25 ng/ml. Prezentovaná metoda může být vhodná pro analýzu metoprololu a ?-hydroxymetoprololu v séru u pacientů s hypertenzí, ICHS i jiných onemocnění.
High-performance liquid chromatography method has been developed with a view of its future use in the phenotyping of CYP2D6 enzyme by metoprolol as a probe drug. Metoprolol, ?-hydroxymetoprolol and the internal standard nadolol were extracted from serum with dichloromethane alkalinized with 1 mol/l NaOH. Chromatographic separations were performed on the reversed-phase column SupercosilTM LC-18 (15 cm × 3 mm, 5 µm) with the mobile phase containing acetonitril : methanol : water : triethylamine (15 : 5 : 80 : 0.1, pH 3.0) at a flow rate of 0.7 ml/min. Fluorescence detection (FL) was made at 230 nm (excitation) and 300 nm (emission). The total analysis time was 12 min. The retention time for ?-hydroxymetoprolol, nadolol and metoprolol were 2.04, 3.02 and 9.04 min, respectively. The intra-assay and inter-assay precisions (coefficients of variation) were less than 7.2 %, and recovery values were found to be within 98.2–103.0 %. The calibration curve of the method was linear over a concentration range of 25–500 ng/ml (r = 0.999) for both compounds. The limit of detection was 5 ng/ml and the limit of quantification was 25 ng/ml for both metoprolol and ?-hydroxymetoprolol. The reported method could be suitable for measurements of metoprolol and ?-hydroxymetoprolol in serum from patients with hypertension, IHD and other illnesses.
The determination of phenylalanine and tyrosine is presently the most reliable direct approach to the diagnosis of phenylketonuria. An HPLC method for the simultaneous measurement of phenylalanine and tyrosine in samples of dried blood spots and plasma has been developed and evaluated. We have used an inherent fluorescence of both phenylalanine and tyrosine. For the separation, a reverse-phase column LiChroCart 125-4, Purospher RP-18e, 5microm, was used. The mixture of ethanol and deionized water (5:95, v/v) was used as a mobile phase. Analytical performance of this method is satisfactory for both phenylalanine and tyrosine: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma and blood samples were between 92.0 and 102.9%. The limit of detection was 10.0 and 5.0micromol/L, respectively. The preliminary reference ranges of phenylalanine and tyrosine in a group of newborns are 69.3+/-13.1 and 42.7+/-12.9micromol/L, in a group of blood donors are 68.4+/-9.9 and 52.1+/-10.9micromol/L. The presented method is inexpensive and suitable for diagnosis of phenylketonuria.
- MeSH
- Adult MeSH
- Phenylalanine blood MeSH
- Fluorescence MeSH
- Middle Aged MeSH
- Humans MeSH
- Tyrosine blood MeSH
- Chromatography, High Pressure Liquid methods instrumentation MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: New high-performance liquid chromatography (HPLC) method was developed for the determination of vitamin K1 and two forms of vitamin K2 (MK-4 and MK-7) in human serum, and the levels of vitamin K were determined in 350 samples of postmenopausal women. METHODS: Vitamin K was determined by HPLC with fluorescence detection after postcolumn zinc reduction. The detection was performed at 246 nm (excitation) and 430 nm (emission). The internal standard and 2 mL of ethanol were added to 500 μL of serum. The mixture was extracted with 4 mL of hexane, and solid phase extraction was then used. RESULTS: The HLPC method was fully validated. The intra- and interday accuracy and precision were evaluated on two QC samples by multiple analysis, and CV were less than 10%. The limit of quantification for MK-4 was found at 0.04 ng/mL, for K1 0.03 ng/mL, and for MK-7 0.03 ng/mL. The mean recoveries of the corresponding compounds were 98%-110%. Serum levels of MK-4, K1 , and MK-7 in postmenopausal women with osteoporosis were 0.890 ± 0.291 ng/mL, 0.433 ± 0.394 ng/mL, and 1.002 ± 1.020 ng/mL, respectively (mean ± SD). Serum levels of MK-4, K1 , and MK-7 in postmenopausal women without osteoporosis were 0.825 ± 0.266 ng/mL, 0.493 ± 0.399 ng/mL, and 1.186 ± 1.076 ng/mL, respectively (mean ± SD). CONCLUSION: New HPLC method for the determination of vitamins K1 , MK-4, and MK-7 in serum was evaluated and validated. This method is highly specific and sensitive with the low limit of quantification.
- MeSH
- Time Factors MeSH
- Fluorescence * MeSH
- Humans MeSH
- Postmenopause blood MeSH
- Vitamin K 1 blood MeSH
- Vitamin K 2 blood classification MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Check Tag
- Humans MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
Práca je zameraná na hodnotenie kvality vzoriek slovenských medov na základe obsahu minoritných sacharidov. Vzorky zahrňovali jednodruhové a viacdruhové medy. Obsah sacharidov bolo stanovený pomocou HPLC metódy v kombinácií s fluorescenčnou detekciou. Z dôvodu zvýšenia citlivosti detekcie, bola použitá derivatizácia cukrov s dansylhydrazínom. Na HPLC separáciu analytov bola použitá stacionárna fáza typu C18 a zmes acetonitrilu a kyseliny octovej ako mobilná fáza. Vo vzorkách bola zistená prítomnosť sacharózy, maltózy, turanózy a tiež hlavných zložiek glukózy a fruktózy. Medze detekcie boli v rozsahu 15–100 μg.ml–1 a naznačujú, že metóda je vhodná na stanovenie nízkych koncentrácií sacharidov. Pre vzorky slovenských medov a vzorky z iných geografických oblastí (Francúzsko, Chorvátsko) boli zístené rozdiely v obsahu maltózy, turanózy a tiež sacharózy. Pomocou viacrozmernej štatistickej analýzy boli na základe sacharidového profilu slovenské medy rozlíšené od ostatných testovaných medov.
The work is focused on the quality evaluation of Slovak honey samples on the basis of minor saccharide contents. The samples included unifloral and multifloral types of honeys. The saccharide contents were determined by HPLC with fluorescence detection. Derivatization of sugars with dansylhydrazine was used to increase detection sensitivity. The C18 type stationary phase was used for the separation of analytes and the mixture of acetonitrile and acetic acid was used as the mobile phase. The determined carbohydrates were saccharose, maltose, turanose, and also the main sugars glucose and fructose. Limits of detection were in the range from 15 to 100 μg.ml–1 and suggest that low quantities of all studied compounds can be detected. Differences in saccharide contents between Slovak samples and samples from different geographical regions (France, Croatia) for maltose, turanose and also for saccharose were obtained. Using multivariate statistical treatment, Slovak honeys can be distinguished from other tested honeys using the saccharide profiles.
- MeSH
- Chemistry Techniques, Analytical methods instrumentation utilization MeSH
- Spectrometry, Fluorescence methods instrumentation utilization MeSH
- Data Interpretation, Statistical MeSH
- Honey analysis MeSH
- Carbohydrates analysis MeSH
- Chromatography, High Pressure Liquid methods instrumentation utilization MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Slovakia MeSH
The determination of α-keto acids derived from amino acids is currently the most reliable approach for the diagnosis of some congenital metabolic diseases. An HPLC method for the simultaneous measurement of selected α-keto acids in dried blood samples has been developed and evaluated. Blood spot samples from a group of healthy blood donors were collected onto #903 Specimen Collection Paper. Prior the separation, the α-keto acids were derivatized with 1,2-diamino-4,5-dimethoxybenzene to the corresponding 3-substituted-6,7-dimethoxy-2(1 H)-quinoxalinol derivatives. For the separation, a reverse-phase column LichroCart 125-4, Purospher RP-18e, 5 μm, was used. The mixture of 25% ACN in deionized water (mobile phase A) and 100% ACN (mobile phase B) were used for a gradient elution of α-keto acids derivatives. Analytical performance of this method is satisfactory for all α-keto acids. The intra-assay and inter-assay coefficients were below 10% and recoveries were close to 100%. We have developed relatively simple, rapid, selective and sufficiently sensitive HPLC method with fluorescence detection for the determination of selected α-keto acids in dried blood samples. The presented method is suitable for clinical testing purposes.
- MeSH
- Amino Acids * MeSH
- Indicators and Reagents MeSH
- Keto Acids * MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
We describe a relatively simple method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human whole blood. We have used an HPLC with coulometric electrochemical detection for the simultaneous measurement of GSH and GSSG. Diluted and filtered trichloroacetic acid extracts were injected directly into the HPLC system and were eluted isocratically on a Polaris 5u C18-A, 250 × 4.6 mm analytical column. Glutathione in samples extracted with trichloroacetic acid and diluted with 1.0 mM hydrochloric acid was stable at 4 °C for at least 8 h. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked whole blood samples were at intervals 91.6–97.6% for GSH and 85.0–104.4% for GSSG. The linear range is 5.0–2000.0 µmol/l, with a detection limit of 2.1 µmol/l (signal-to-noise ratio = 3) for GSH and 2.0–250.0 µmol/l, with a detection limit of 0.9 µmol/l for GSSG.
A rapid procedure based on direct extraction and RP-HPLC separation of pregabalin and its possible impurities with fluorescence detection has been developed. The separation conditions and parameters of derivatization reaction for postcolumn derivatization of pregabalin with o-phtaldialdehyde/2-mercaptoethanol were studied. Purospher STAR RP-8e column with isocratic elution was employed. Fluorescence detection was performed at excitation and emission wavelength of 345 nm and 450 nm, respectively. The proposed method has an advantage of a simple sample pre-treatment and a quick and very sensitive HPLC method. The applicability of developed method was successfully verified during analysis of commercial samples of tablets of Lyrica (Pfizer, USA).
- MeSH
- Spectrometry, Fluorescence MeSH
- gamma-Aminobutyric Acid analogs & derivatives analysis chemistry MeSH
- Calibration MeSH
- Drug Contamination MeSH
- Limit of Detection MeSH
- o-Phthalaldehyde MeSH
- Sensitivity and Specificity MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The biosynthetic pathway of the clinically important antibiotic lincomycin is not known in details. The precise knowledge of the lincomycin biosynthesis is a prerequisite for generation of improved derivatives by means of combinatorial genetics. Methods allowing determination of the key intermediates are very important tools of the pathway investigation. Two new high-performance liquid chromatography methods with fluorescence detection for determination of lincomycin precursors in fermentation broth of Streptomyces lincolnensis and its lincomycin nonproducing mutants were developed. The first one enables simultaneous analysis of methylthiolincosamide (MTL) and N-demethyllincomycin (NDL), whereas the second one is suitable for 4-propyl-L-proline (PPL) assay. Both methods are based on the pre-column derivatization: MTL and NDL with 4-chloro-7-nitrobenzofurazan; PPL with o-phthaldialdehyde. The methods were validated with lower limit of quantification values of 2.50, 3.75, and 3.75 microg ml(-1) for MTL, NDL, and PPL, respectively. The inter- and intra-day accuracies and precisions were all within 12%. Stability of oxidized and derivatized analytes was investigated.
- MeSH
- Amides analysis MeSH
- Fermentation MeSH
- Financing, Organized MeSH
- Fluorescence MeSH
- Lincomycin analogs & derivatives biosynthesis MeSH
- Molecular Structure MeSH
- Proline analogs & derivatives analysis MeSH
- Reproducibility of Results MeSH
- Streptomyces metabolism MeSH
- Sulfhydryl Compounds analysis MeSH
- Chromatography, High Pressure Liquid methods MeSH
