isotope-dilution method
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Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatography connected to electrospray tandem mass spectrometry (UHPLC-ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24h. The standard isotope dilution method was used for quantitative determination. The method's lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15nmol/L and 0.3 to 45nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method's accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of compounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies.
- MeSH
- biochemická analýza krve metody MeSH
- chemické techniky analytické metody MeSH
- indikátorové diluční techniky MeSH
- izotopy MeSH
- lidé MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie * MeSH
- tryptofan krev MeSH
- vysokoúčinná kapalinová chromatografie * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- MeSH
- mléko MeSH
- progesteron analýza MeSH
- radioizotopy jodu MeSH
- skot MeSH
- těhotenství MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- zvířata MeSH
Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of μg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.
- MeSH
- biomasa MeSH
- chromatografie kapalinová metody MeSH
- cystein analýza MeSH
- glutathion analýza MeSH
- limita detekce MeSH
- mikrocystiny analýza chemie MeSH
- radioisotopová diluční technika MeSH
- reprodukovatelnost výsledků MeSH
- sinice chemie MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Therapeutic drug monitoring is recommended for the optimal management of patients with several malignant diseases. The aim of this study was to develop and validate an isotope dilution direct injection mass spectrometry method for the high throughput determination of tyrosine kinase inhibitors in plasma from leukemic and cancer patients. METHODS: The plasma for analysis was deproteinated by methanol and the centrifuged supernatant was directly injected to mass spectrometer without separation step. Multiple reaction monitoring modes on a hybrid triple quadrupole - linear ion trap mass spectrometer (5500 QTRAP) were used for the detection and quantification of imatinib, nilotinib, lapatinib, and dasatinib. RESULTS: We developed a fast method with analysis time of 55 s and 19s in multiple injection setting. The method was successfully validated and applied to the patient plasma samples. In order to overcome insufficient sensitivity of dasatinib, multiple reaction monitoring cube mode in linear ion trap (MRM(3)) was successfully applied. The limits of quantification were in the range 1.0-5.5 ng/ml. Imprecisions were lower than 6.9% and the accuracy of the quality control samples ranged between 99.0 and 107.9%. CONCLUSIONS: Isotope dilution direct injection mass spectrometry method allows high-throughput therapeutic drug monitoring of tyrosine kinase inhibitors in plasma. The method offers low-cost analyses as a result of its speed and the exclusion of separation step and can be advantageously used in routine clinical practice. The method can be applied on various drugs and biochemical markers with the use of triple quadrupole instruments.
- MeSH
- biochemická analýza krve ekonomika metody MeSH
- časové faktory MeSH
- hmotnostní spektrometrie ekonomika metody MeSH
- inhibitory proteinkinas krev MeSH
- injekce MeSH
- izotopy MeSH
- lidé MeSH
- metody pro přípravu analytických vzorků MeSH
- reprodukovatelnost výsledků MeSH
- tyrosinkinasy antagonisté a inhibitory MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A stable isotope dilution assay using D3-mevalonic acid was developed and applied to the study of mevalonic aciduria. The method also appears to be suitable for the evaluation of different therapeutic regimens in patients with hypercholesterolemia. Mevalonic acid was isolated by liquid partition chromatography and quantified as the underivatized lactone by means of ammonia chemical ionization selected ion monitoring capillary gas chromatography-mass spectrometry. In heterozygotes there was significantly greater urinary excretion of mevalonic acid, while the range of enzymatic activity of mevalonate kinase showed an overlap with that of controls. The analysis of amniotic fluids of two pregnancies at risk for mevalonic aciduria showed a 3277-fold elevation as compared to controls in the first case, diagnostic of an affected fetus, and a normal value in the second one. Mevalonic acid concentration was much increased in tissues of the affected and aborted fetus. Concentrations ranged from 840 to 1120 mumol/kg in various tissues and were as high as 1810 mumol/kg in brain. Concentrations in control fetal tissues were approximately 1 mumol/kg.
- MeSH
- buněčné linie MeSH
- cholesterol biosyntéza MeSH
- dítě MeSH
- dospělí MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem * MeSH
- fosfotransferasy metabolismus MeSH
- heterozygot * MeSH
- indikátorové diluční techniky MeSH
- kojenec MeSH
- kyselina mevalonová analýza krev metabolismus moč MeSH
- lidé MeSH
- nemoci plodu * diagnóza MeSH
- novorozenec MeSH
- plod chemie MeSH
- plodová voda chemie MeSH
- plynová chromatografie s hmotnostně spektrometrickou detekcí metody MeSH
- prenatální diagnóza * MeSH
- senzitivita a specificita MeSH
- těhotenství MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
INTRODUCTION: Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. OBJECTIVE: To develop a rapid and reliable method for quantification of ingenol in various plant extracts. METHODOLOGY: Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. RESULTS: The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. CONCLUSION: The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.
BACKGROUND: The calibration of Jaffe method for serum creatinine using one serum-based standard complemented with artificial matrix compensating standard's Jaffe-interfering substances allows two-point calibration with results well comparable with enzymatic methods. METHOD: Spectrophotometry was used. RESULTS: Jaffe procedures compensating serum/plasma intereferents by subtracting a constant amount of creatinine poorly overcompensate creatinine in children. Two-point calibration with a pair of primary serum standards certified by the reference measurement procedure (isotope-dilution, mass spectrometry) or with a pair of secondary standards linked to primary materials could provide results well agreeable with enzymatic determination. Such a calibration comprises an absorbance offset corresponding to other-than-creatinine Jaffe-interfering chromogens present in standards in the calibration line, while a two-point calibration combining one standard with physiological saline/water always grossly distorts calibration line. We calculated/prepared artificial serum matrices capable of compensating Jaffe-interfering chromogens in serum standards. The combination of even one standard with its artificial matrix also enables two-point calibration with practically the same results as with a pair of primary standards. CONCLUSIONS: A two-point calibration of Jaffe method for serum creatinine combining only one serum standard with creatinine solution matching standard's allow matrix-present interferents present results well comparable with enzymatic determination, providing the standard is attested/linked to the reference measurement procedure.
- MeSH
- artefakty MeSH
- bilirubin metabolismus MeSH
- biochemická analýza krve metody normy MeSH
- časové faktory MeSH
- dítě MeSH
- dospělí MeSH
- financování organizované MeSH
- kalibrace MeSH
- kinetika MeSH
- kreatinin krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- oxidace-redukce MeSH
- referenční standardy MeSH
- senioři MeSH
- tartaráty metabolismus MeSH
- Check Tag
- dítě MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
A rapid and precise method for the identification and quantification of cysteinyl leukotrienes (leukotriene C(4), leukotriene D(4) and leukotriene E(4)), essential markers of bronchial asthma, in exhaled breath condensate was developed. The protocol consists of immunoaffinity separation and a detection step, liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the selected reaction monitoring mode was used for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized with a high precision (≤ 7.7%, determined as RSD), an acceptable accuracy (90.4-93.7%, determined as recovery), a low limit of detection (≤ 2 pg/ml EBC) and a low limit of quantification (≤ 10 pg/ml EBC). It was compared to other simple, clinically appropriate combinations of pre-treatment methods (solid phase extraction and lyophilization) with LC/MS. Finally, the method (a combination of immunoaffinity separation with LC-MS) was successfully tested in a clinical study where a significant difference was found in the concentration levels of cysteinyl leukotrienes between patients with occupational bronchial asthma and healthy subjects.
- MeSH
- bronchiální astma diagnóza MeSH
- chromatografie kapalinová metody MeSH
- cystein analýza MeSH
- dechové testy metody MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací metody MeSH
- imunoanalýza metody MeSH
- leukotrieny analýza MeSH
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- vydechnutí MeSH
- Check Tag
- lidé MeSH
- mladiství MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The understanding of fluid fluxes in pediatric cardiac critical care is crucial to effective management. Knowledge of variations in total body water in this situation would aid this understanding, but most available methods are unsuitable for routine use. Recently, estimation of total body water by a tetrapolar bioelectric impedance has been validated in older children and adolescents. We undertook a study to validate the method in the taxing conditions of pediatric cardiac critical care. A prospective comparative study was done in 16 children whose ages ranged from 6 days to 10 years (mean 23 months) after a variety of cardiac operations. Total body water was estimated by a standard isotope dilution method (deuterium oxide) and by bioelectric impedance by means of a Holtain body composition analyzer. Individual estimations of total body water were made on two successive days on each patient at varying intervals after a cardiac operation, bioelectric impedance being measured hourly during 4 hourly urine collections for the deuterium oxide method. Thirty-two simultaneous values of total body water (by isotope and by impedance) were collected. Population-specific regression relationship was established by plotting total body water (isotope) against height2/bioelectric impedance. From this data plot r = 0.911, giving this equation: total body water = 0.158 +/- 0.662 x (height2/bioelectric impedance). Levels of agreement of -1.771 to +1.725 were observed, with a standard error of measurement of 16% across the range. The data suggest that bioelectric impedance is a satisfactory and reliable method of estimating total body water in children requiring cardiac critical care. The standard error of 16% suggests that the method may be more useful for measuring trends than absolute values, but the technique should be a valuable noninvasive tool both for continuous monitoring of total body water and in longitudinal research studies of rapid fluid flux and in the assessment of capillary leak.
- MeSH
- časové faktory MeSH
- deuterium MeSH
- dítě MeSH
- elektrická vodivost * MeSH
- kardiochirurgické výkony * MeSH
- kojenec MeSH
- kritický stav MeSH
- lidé MeSH
- novorozenec MeSH
- oxid deuteria MeSH
- předškolní dítě MeSH
- radioisotopová diluční technika MeSH
- tělesná voda * MeSH
- voda MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- práce podpořená grantem MeSH
- srovnávací studie MeSH
