Metabolites In Plasma Of Patients Research Articles

Concentrations of the DNA methyltransferase inhibitor 5-fluoro-2′-deoxycytidine (FdCyd) and its cytotoxic metabolites in plasma of patients treated with FdCyd and tetrahydrouridine (THU)

Although the DNA methyltransferase inhibitor 5-fluoro-2'-deoxycytidine (FdCyd), is being evaluated clinically, it must be combined with the cytidine deaminase inhibitor tetrahydrouridine (THU) to prevent rapid metabolism of FdCyd to the pharmacologically active, yet unwanted, metabolites 5-fluoro-2'-deoxyuridine (FdUrd), 5-fluorouracil (FU), and 5-fluorouridine (FUrd). We assessed plasma concentrations of FdCyd and metabolites in patients receiving FdCyd and THU. We validated an LC-MS/MS assay, developed for a preclinical study, to quantitate FdCyd and metabolites in human plasma. Patients were treated with five daily, 3-h infusions of FdCyd at doses of 5-80 mg/m(2) with 350 mg/m(2) THU. Plasma was obtained during, and before the end of infusions on days 1 and 5. The lower limits of quantitation for FU, FdUrd, FUrd, FC and FdCyd were 1, 1.5, 10, 3, and 10 ng/ml, respectively. Plasma FdCyd increased with dose, from 19-96 ng/ml at 5 mg/m(2) to 1,600-1,728 ng/ml at 80 mg/m(2). FdUrd was undetectable in patients treated with FdCyd doses <20 mg/m(2), and increased from 2.3 ng/ml at 20 mg/m(2) to 3.5-5.7 ng/ml at 80 mg/m(2). FU increased from 1.2-5.5 ng/ml at 5 mg/m(2) to 6.0-12 ng/ml at 80 mg/m(2). By co-administering FdCyd with THU, FdCyd plasma concentrations were achieved that are known to inhibit DNA methylation in vitro. The accompanying plasma FU and FdUrd concentrations are <10% those observed after therapeutic infusions of FU or FdUrd, while FdCyd levels are well above those required to inhibit methylation in vitro. Therefore, inhibition of DNA methylation with FdCyd and THU appears feasible.

Simultaneous determination of clomipramine and its desmethyl and hydroxy metabolites in plasma of patients by high-performance liquid chromatography after solid-phase extraction

Clomipramine (CMI) is a typical tricyclic antidepressant with a wide clinical spectrum, being used in major depressive, panic and obsessive-compulsive disorders. The relationship between clinical response and plasma levels of clomipramine and its N-desmethylated ( N-desmethylclomipramine, DMCMI) and hydroxy-metabolites remains unclear. In particular, limited information is available on the correlation with clinical response in patients with obsessive-compulsive disorder (OCD). This study describes a new sensitive method to simultaneously determine CMI and its major N-desmethylated and hydroxy-metabolites present in human plasma by HPLC with a UV detector. After a solid-phase extraction from plasma (Isolute C2 columns) the separation of the compounds was performed on a Lichrospher CN column (250×4 mm, 5 μm with a 2-cm pre-column) by an eluent consisting of 10 m M K 2HPO 4–acetonitrile–methanol (35:25:40 v/v/v) at a flow of 1.5 ml/min. UV detector was set at 214 nm. The lower limit of quantification for all the analytes was at least 5 ng/ml. The coefficients of variation ranged between 2.0 and 4.9% with recovery rates between 97.0 and 100.3%. Linear regression analyses showed correlation coefficients between 0.98 and 0.99. This method is simple, fast and reliable with good specificity and sensitivity. Solid phase extraction is efficient and rapid, allowing the extraction of several plasma samples on the same day and may therefore be usefully and realistically applied in the clinical context. We thus investigated the relevance of plasma levels of CMI and its metabolites as a predictor of clinical outcome in a group of 15 patients with OCD.

Assay of 6-mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection

A reversed-phase high-performance liquid chromatography (HPLC) method was developed to determine 6-mercaptopurine (MP) and seven of its metabolites (6-thioguanine, 6-thioxanthine, 6-mercaptopurine riboside, 6-thioguanosine, 6-thioxanthine riboside, 6-methylmercaptopurine and 6-methylmercaptopurine riboside) simultaneously in human plasma. A volume of 100 μl of plasma was used. Protein was removed from the sample by a simple and easy ultrafiltration step and ultrafiltrate was directly injected onto the HPLC system. Analytes were detected and confirmed with a diode-array detector before quantitation at 295 and 330 nm. The limit of detection for the analytes ranged from 20 to 50 n M. For the majority of patients receiving a 1 g/m 2 MP intravenous infusion, MP and all metabolites except 6-thioguanine and 6-methylmercaptopurine riboside were present. This method serves as useful tool to characterize pharmacokinetics and pharmacodynamics of MP in oncology patients, and the small volume of plasma lends itself to pediatric studies.

Determination of Antipyrine and Metabolites in Plasma of a Patient With Mild Renal Failure

A method has been developed for the simultaneous determination of antipyrine and its three major metabolites in plasma of patients with renal failure. Plasma samples (500 microl) were hydrolyzed with beta-glucuronidase/aryl sulphatase. The compounds, after addition of sodium chloride, were extracted with chloroform:ethanol (90:10, v/v) in acidic medium. Chromatographic conditions comprise a C18 column, a mobile phase with 30% methanol and 70% 0.25N sodium acetate buffer (pH 5.0), a total run time of 10 minutes, and ultraviolet absorbance detection at 254 nm. Confidence limits showed 0.5 to 40.0 microg/ml(-1) linearity (r2 = 0.999); 0.1 microg/ml(-1) HMA, 0.05 microg/ml(-1) antipyrine and NORA, and 0.5 microg/ml(-1) OHA sensitivity and absolute recovery >95%. Interprecision and intraprecision expressed as coefficient of variation were <10% for all compounds investigated. The assay shows to be suitable for pharmacokinetics and drug metabolism studies after administration of a single oral dose of 500 mg of antipyrine to a patient with hypertension and chronic renal failure (CL(CR) = 34.17 ml/min(-1); 1.73 m(-2)).

Microdialysis-HPLC for plasma levodopa and metabolites monitoring in parkinsonian patients

We used in vitro microdialysis-HPLC to determine L-3,4-dihydroxyphenylalanine (L-DOPA) and its metabolites in plasma of patients with advanced Parkinson disease. Blood samples and clinical evaluations were obtained 0, 30, 60, 90, 120, and 150 min after oral administration of carbidopa/L-DOPA (25/100 mg, 12.5/125 mg, and 50/200 mg). In vitro recoveries for L-DOPA and metabolites ranged from 22% to 36%. Linear correlation was found between metabolite concentrations in the dialysate and in the surrounding medium. There was a significant positive correlation between L-DOPA dose and plasma concentration of L-DOPA and homovanillic acid (P < 0.04). Clinical response was maximum 60 min after L-DOPA administration. Threshold L-DOPA plasma concentration averaged 7.74 +/- 3.3 mumol/L. Motor effect is longer with the highest L-DOPA peak concentration (P < 0.01). Microdialysis-HPLC is readily applicable, reproducible, and allows monitoring of plasma L-DOPA and metabolites in parkinsonian patients.

Analysis of l-[methyl- 11C]methionine and metabolites in human plasma by an automated solid-phase extraction and a high-performance liquid chromatographic procedure

A fully automated method for separation of l-[methyl- 11C]Methionine from metabolites in patient plasma was developed. l-[methyl- 11C]Methionine was isolated from plasma by solid-phase extraction (SPE). The radioactivity retained on the SPE column was eluted and injected onto the HPLC system for separation of in vivo formed l-[methyl- 11C]methionine radiolabeled metabolites. The yield through the isolation procedure and HPLC analysis was greater than 95% with a precision better than 5% (R.S.D.). The calculated rate of l-[methyl- 11C]methionine transport into tumor tissue was markedly different with and without compensation for radiolabeled metabolises in patient plasma.

Concentrations of vitamin D-binding protein and vitamin D metabolites in plasma of patients with liver cirrhosis.

Plasma levels of vitamin D-binding protein (DBP) and vitamin D metabolites in patients with decompensated and compensated liver cirrhosis were assayed. Plasma levels of DBP in the decompensated group were significantly lower than those in the compensated group, but both were lower than the normal range. The plasma levels of 25-hydroxyvitamin D (25-OH-D) and 1 alpha,25-dihydroxyvitamin D [1,25(OH)2D] in the compensated group were within the respective normal ranges, whereas both values in the decompensated group were significantly lower than those in the compensated group. Most of 25-OH-D (higher than 96%) was confirmed to be circulated as a bound form with DBP in the plasma of not only the compensated but also the decompensated group. When vitamin D2 was given to the decompensated group, a significant increase of 1,25(OH)2D levels in the plasma could not be observed while 25-OH-D levels were increased. On the other hand, the administration of 1 alpha-hydroxyvitamin D3 (1 alpha-OH-D3) to the decompensated group caused a significant increase in the plasma levels of 1,25(OH)2D. Therefore, we suggest that the administration of 1 alpha-OH-D3 is useful for the treatment of bone disease induced by liver cirrhosis.

The assay of fentanyl and its metabolites in plasma of patients using gas chromatography with alkali flame ionisation detection and gas chromatography—mass spectrometry

Fentanyl was determined using gas chromatography (GC) and alkali flame ionisation detection (AFID), in the plasma of patients who had received a high single dose (up to 60 μg/kg body weight). The relative standard deviation is 6% for 11 ng/ml while the calculated detection limit is 3.3 ng of fentanyl per 1 ml of plasma. The concentration of fentanyl in patients ranged from 40 to 3 ng/ml of plasma in the first hour after administration. In the plasma of patients treated with fentanyl two metabolites could be detected and identified using GC—AFID and GC—MS.

Determination of hexamethylmelamine and metabolites in plasma or serum by gas—liquid chromatography with a nitrogen-sensitive detector

A gas chromatographic method for the quantitative determination of hexamethylmelamine (HMM) and five of its metabolites in plasma (or serum) is described. After adjustment of the pH of the plasma sample to about 9.5, the compounds are extracted with chloroform containing 5% of isopropanol. Amyl alcohol is added to the extract, which is then evaporated until a small volume remains. An aliquot of this solution is injected into a gas chromatograph equipped with a nitrogen—phosphorus flame ionisation detector. Separation of the methylmelamines is achieved with a 10% Carbowax 20M–2% KOH column. By programming the oven temperature unnecessarily long retention times are avoided. Using 1 ml of plasma, concentrations as low as 5 ng/ml of HMM and its metabolites can be quantitated. The method has been applied to the determination of HMM and metabolites in plasma of patients who received oral doses of HMM.