Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 μmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.
- MeSH
- alkaloidy amarylkovitých krev farmakokinetika moč MeSH
- fenantridiny krev farmakokinetika moč MeSH
- krysa rodu rattus MeSH
- limita detekce MeSH
- pilotní projekty MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- žluč metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Boldine belongs to the group of aporphine alkaloids isolated from Boldo tree. In contrast with numerous reports on the pharmacological effects of boldine, the data about its pharmacokinetics and biotransformation are scarce. No validated bioanalytical method of sufficient sensitivity has so far been described in the literature which could be used for quantification of boldine in various body fluids collected in pharmacokinetic studies. This work presents, for the first time, the assay for boldine in the plasma, bile and urine of rats. It includes liquid-liquid extraction/back-extraction of boldine, its chromatographic separation and sensitive fluorescence detection. Separation was carried out on a pentafluorophenyl core-shell column (Kinetex PFP, 150×3mm, 2.6μm) in gradient elution mode with solvent system consisting of an acetonitrile-ammonium formate buffer (5mM, pH=3.8). Fluorimetric detection (λEX=320nm, λEM=370nm) was used for quantitative work. Validation according to the EMEA guideline proved the assay LLOQ (0.1μmolL(-1)), linearity over a broad range of 0.1-50μmolL(-1), precision (intra- and inter-day CVs less than 4.5% and 6.1%, respectively) and accuracy (relative errors between -5.8% and 4.8%). In a pilot pharmacokinetic experiment, the concentration-time profiles were described for boldine (single i.v. bolus 50mgkg(-1)) in plasma and bile and cumulative excretion in urine was investigated. The major metabolites identified by means of LC-MS(n) were boldine-O-glucuronide, boldine-O-sulphate and disulphate, boldine-O-glucuronide-O-sulphate and N-demethyl-boldine-O-sulphate.
- MeSH
- aporfiny analýza chemie farmakokinetika MeSH
- fluorescenční spektrometrie MeSH
- krysa rodu rattus MeSH
- metoda nejmenších čtverců MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stabilita léku MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- žluč chemie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
For the purpose of in vivo pharmacokinetic studies, an HPLC method was developed and validated for the quantification of N-(ω)-hydroxy-nor-L-arginine, L-arginine and N-(ω)-ethyl-L-arginine (internal standard) in rat plasma. Sample processing involved a solid-phase extraction on the Waters MCX cartridges and on-line pre-column derivatization of the analytes with o-phthaldialdehyde and 3-mercaptopropionic acid. Separation of the derivatives was carried out on a core-shell Kinetex C18 column in a gradient elution mode with a mobile phase consisting of methanol and water (pH=3.00 adjusted with formic acid). Fluorimetric detection with the excitation/emission wavelengths of 235/450 nm was used. The method was validated according to the FDA guidelines and applied to pilot pharmacokinetic experiments. An unknown metabolite was extracted from the plasma of Wistar rats after a single bolus of N-(ω)-hydroxy-nor-L-arginine (i.v. 10 mg kg(-1)). The metabolite was identified as nor-L-arginine using mass spectrometry. Validated method was successfully used for pilot pharmacokinetic experiment on rats.
- MeSH
- arginasa antagonisté a inhibitory MeSH
- arginin aplikace a dávkování analogy a deriváty krev farmakokinetika MeSH
- extrakce na pevné fázi metody MeSH
- krysa rodu rattus MeSH
- lineární modely MeSH
- pilotní projekty MeSH
- potkani Wistar MeSH
- reprodukovatelnost výsledků MeSH
- stabilita léku MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie přístrojové vybavení metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with gamma-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25-400 nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis.
- MeSH
- antirevmatika farmakokinetika krev terapeutické užití MeSH
- biologická dostupnost MeSH
- biotransformace MeSH
- erytrocyty metabolismus MeSH
- financování organizované MeSH
- fluorescence MeSH
- hydrolýza MeSH
- juvenilní artritida farmakoterapie krev MeSH
- kalibrace MeSH
- kyselina polyglutamová agonisté farmakokinetika krev MeSH
- lidé MeSH
- methotrexát analogy a deriváty farmakokinetika krev terapeutické užití MeSH
- monitorování léčiv metody normy MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie normy MeSH
- Check Tag
- lidé MeSH
BACKGROUND: Clinical studies of low-dose oral methotrexate (MTX) in the treatment of psoriasis and rheumatoid arthritis document a large interpatient variability in the pharmacokinetics of MTX, including its polyglutamates (MTXPGs) in erythrocytes (RBC). This can be a factor contributing to the variability of therapeutic and toxic effects. AIM: This pilot trial aimed to investigate the MTXPG concentrations in RBC as well as their relation to therapeutic and adverse effects during the initial 4 months of pharmacokinetically guided therapy with a divided-dose schedule (three doses of MTX separated by 12-h intervals once a week). SUBJECTS AND METHODS: Sixteen psoriatic patients (4 men and 12 women; mean age, 53 years; range, 28-69 years) with moderate-to-severe chronic plaque psoriasis [mean Psoriasis Area and Severity Index (PASI) = 24; range, 9-42] were enrolled in the study. Concentrations of plasma MTX and that of MTXPGs in RBC were assayed using liquid chromatography methods. The area under the concentration-time curve of plasma MTX in the interval 0-8 h post-dose (AUC(0-8 h)) was measured after a test bolus dose of 10 mg, and the starting weekly dose was individualized in order to achieve the target AUC(0-8 h) of 1800 nmol.h/L. The PASI, biochemistry, and haematology tests and MTXPGs levels in RBC were evaluated at baseline and at 4-week intervals. RESULTS: The AUC(0-8 h )achieved 1360 +/- 425 nmol.h/L (mean +/- SD: range, 778-2400 nmol.h/L). The mean (range) of individualized doses was 14.5 mg/week (7.5-22.5 mg). The mean (SD) steady-state concentration of total MTXPGs observed between days 85 to 110 reached 113 (34.6) nmol/L (range, 66.1-174 nmol/L). The PASI decreased from 24.0 +/- 8.0 (mean +/- SD) at baseline to 8.0 +/- 6.1 at day 110 (P < 0.001). Thirteen patients (87%) achieved a greater than 50% improvement in baseline PASI, and seven (47%) experienced a greater than 75% improvement. There was no relationship between the percent improvement from baseline PASI and the steady-state concentration of MTXPGs in RBC. All patients tolerated MTX well. Throughout the study period, there was a continuous increasing trend in the geometric mean values of the mean corpuscular volume from 92.6 to 96.4 fL (P < 0.001) and of plasma homocysteine from 9.5 to 12.3 micromol/L (P < 0.005). The geometric mean serum alanine aminotransferase (ALT) activity slightly increased from 0.49 to 0.80 microkat/L (P < 0.05). However, only two patients had the ALT activity transiently elevated above twice the upper limit of normal. CONCLUSION: Results of this pilot trial show that the steady-state levels of MTXPGs in RBC vary less than threefold between patients and did not correlate with the change in PASI observed after 4 months of therapy with an individualised weekly dose of MTX. Whether pharmacokinetically guided dosing can improve the results of psoriasis therapy with MTX should be prospectively tested in large controlled studies.
- MeSH
- aplikace orální MeSH
- dermatologické látky aplikace a dávkování farmakokinetika krev MeSH
- dospělí MeSH
- erytrocyty metabolismus MeSH
- financování organizované MeSH
- lidé středního věku MeSH
- lidé MeSH
- methotrexát aplikace a dávkování farmakokinetika krev MeSH
- nežádoucí účinky léčiv MeSH
- pilotní projekty MeSH
- psoriáza farmakoterapie krev MeSH
- rozvrh dávkování léků MeSH
- senioři MeSH
- stupeň závažnosti nemoci MeSH
- výsledek terapie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- klinické zkoušky MeSH
