Hydroxy Metabolites In Plasma Research Articles

Exploring CYP2B6 activity by measuring the presence ofnevirapine hydroxy metabolites in plasma.

Nevirapine is a reverse-transcriptase inhibitor widely used in combination therapy to treat HIV infection. Nevirapine is extensively metabolized in the liver and CYP2B6 is mainly responsible for oxidation of 3-hydroxynevirapine (3-OH NVP). This study aims to explore CYP2B6 activity by measuring 2-hydroxynevirapine (2-OH NVP) and 3-OH NVP in plasma and to identify factors associated with nevirapine pharmacokinetic parameters. A total of 112 patients were recruited and treated with nevirapine-based antiretroviral therapy. Plasma nevirapine and metabolite concentrations were assayed using high-performance liquid chromatography via liquid-liquid extraction. Thirty-nine (34.8%) of the patients had no 3-OH NVP detected in their plasma while 2-OH NVP was detected in all patients. Metabolite concentrations were low compared to nevirapine. Positive correlations were observed between nevirapine and its metabolites, 2-OH NVP (P < 0.01) and 3-OH NVP (P = 0.012). Nevirapine concentration was decreased when concomitantly administered with methadone. Univariate analysis showed that ALT level, AST level, and detection of 3-OH NVP were associated with nevirapine pharmacokinetic parameters. The variability of nevirapine pharmacokinetic parameters was caused by liver enzymes and the presence of 3-OH NVP metabolites. The presence of 3-OH NVP can probably be used to distinguished CYP2B6 activity and efficacy of nevirapine in patients with HIV infection.

Analysis of omeprazole, midazolam and hydroxy-metabolites in plasma using liquid chromatography coupled to tandem mass spectrometry

A method has been developed and validated for the quantitation of midazolam, alphahydroxy-midazolam, omeprazole, and hydroxyomeprazole from one 250 microL sample of human plasma using high performance liquid chromatography coupled to tandem mass spectrometry. The method was validated for a daily working range of 0.400-100 ng/mL, with limits of detection between 2 and 15 pg/mL. The inter-assay variation was less than 15% for all analytes at four control concentrations and the samples were stable for three freeze-thaw cycles under the analysis conditions and 24 h in the post-preparative analysis matrix. This method was used to analyze samples in support of clinical studies probing the activity of the cytochrome P-450 enzyme system.

Stereospecific analysis of flurbiprofen and its major metabolites in plasma and urine by chiral-phase liquid chromatography

Direct chiral-phase HPLC methods have been developed for the determination of flurbiprofen and its major metabolites, namely 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen, in biological fluids using a derivatized amylose chiral stationary phase (CSP; Chiral-pak AD). Quantification of all three analytes, both free and conjugated, in urine was carried out following liquid-liquid extraction using tandem ultraviolet (UV) and fluorescence detection. Determination of flurbiprofen and the 4′-hydroxy-metabolite in plasma utilized the same CSP but required modification in the mobile phase composition and sole use of fluorescence detection. The urine assay was linear (r>0.998) between 0.05–10 μg mL−1, 0.1–20 μg mL−1 and 0.01–2 μg mL−1 for the enantiomers of flurbiprofen, 4′-hydroxyflurbiprofen and 3′-hydroxy-4′-methoxyflurbiprofen respectively. The plasma assay was linear (r>0.997) between 0.1–6 μg mL−1 and 0.01–0.6 μg mL−1 for the enantiomers of flurbiprofen and 4′-hydroxyflurbiprofen respectively. Both assays, typically yielded within- and between-day imprecision and accuracy values less than 10% for the enantiomers of the different analytes. Initial volunteer studies suggest that the disposition of flurbiprofen displays modest enantioselectivity in humans.

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.

Liquid chromatography–tandem mass spectrometric quantitation of cyclophosphamide and its hydroxy metabolite in plasma and tissue for determination of tissue distribution

Cyclophosphamide (CP) and its metabolite, hydroxycyclophosphamide (OH-CP) have been quantitated in mouse plasma and tissue by derivatization combined with liquid chromatography–tandem mass spectrometry (LC–MS–MS). The derivatization was conducted immediately upon sample collection, to trap the OH-CP metabolite intermediate prior to further conversion to phosphoramide mustard or other reaction products. This simple and straightforward derivatization procedure, combined with sample extraction via protein precipitation, allowed quantitation of CP and the oxime derivative of OH-CP in plasma for concentrations ranging from approximately 12.5–3333 ng/ml, and in spleen tissue for concentrations of 1250–50 000 ng/g. The short cycle time (2.5 min) of the LC–MS–MS method allowed high throughput analysis with minimal matrix interference. Mouse plasma levels were quantitated for doses of 40, 65 and 120 mg/kg; spleen concentrations were determined for mice dosed at 120 mg/kg. The CP and oxime plasma levels correlated well with dose amounts. The CP levels in the spleen and plasma were similar while the oxime levels in the spleen were significantly lower than the plasma.

Method for the analysis of perhexiline and its hydroxy metabolite in plasma using high-performance liquid chromatography with precolumn derivatization

A high-performance liquid chromatographic method for the analysis of perhexiline and its monohydroxy metabolite in plasma has been developed. After a simple extraction procedure, the analytes are derivatized over a 30-min period with trans-4-nitrocinnamoyl chloride. The derivatized products are monitored at 340 nm following separation on a 5-μm phenyl reversed-phase column under isocratic conditions. The limits of detection for perhexiline and its hydroxy metabolite are 0.03 and 0.02 mg/l, respectively. The between-day and within-day assay coefficients of variation for perhexiline and its hydroxy metabolite at concentrations of 0.2 and 1.0 mg/l were less than 10%. The method has proved robust and suitable for the routine monitoring of perhexiline and hydroxyperhexiline.

The effect of experimentally-induced renal failure on accumulation of bupropion and its major basic metabolites in plasma and brain of guinea pigs.

Dosage regimen adjustments because of poor renal function are often assumed to be unnecessary for extensively metabolized antidepressants. This assumption is being increasingly questioned in recognition of the role of active drug metabolites. The purpose of this study was to assess the steady-state accumulation of the new antidepressant bupropion and its three major basic metabolites in guinea pigs, with and without experimentally-induced renal failure. Two groups of guinea pigs were treated by intraperitoneal (IP) implantation of mini-osmotic pumps containing bupropion hydrochloride. Immediately after surgery, one group of animals received an injection of uranyl nitrate. After 4 days, all animals were sacrificed by decapitation following blood removal by cardiac puncture. Analysis of plasma and brain samples by high performance liquid chromatography (HPLC) for concentrations of bupropion (BUP) and its major basic metabolites, the erythro-amino alcohol (EB), the threo-amino alcohol (TB) and the hydroxy metabolite (HB) revealed greater accumulation of BUP, TB, and HB in plasma and brain of the animals with renal failure compared to controls. No difference was found between groups in the concentrations of the EB metabolite. As the guinea pig shows a BUP and metabolite plasma concentration profile similar to that seen in human studies, these results suggest that further studies of bupropion and its major metabolites are warranted in patients with impaired renal function to assess possible excessive drug and metabolite accumulation.

Dose dependent disposition of sulphadimidine and of its N4‐acetyl and hydroxy metabolites in plasma and milk of dairy cows

The disposition of sulphadimidine (SDM) and of its N4-acetyl (N4-SDM) and two hydroxy metabolites, 6-hydroxymethyl-(SCH2OH) and 5-hydroxyasulphadimidine (SOH), was studied in plasma and milk of dairy cows following intramuscular or intravenous administration of sulphadimididine-33.3% at doses of 10, 45, 50, and 100 mg/kg. The main metabolite in plasma as well as in milk was SCH2OH. The metabolite percentages, the final plasma elimination half-lives, and the time of peak SDM concentrations in milk are presented for different dosages. The concentrations of SDM and its metabolites in milk ran parallel to those in plasma beyond 4 hours p.i. The metabolite concentrations in plasma and milk were lower than those of the parent SDM. Sulphate and glucuronide metabolites could not be detected in milk. At high doses (45 mg/kg or more) and SDM plasma concentrations exceeding 20 micrograms/ml, a capacity limited metabolism of SDM to SCH2OH was noticed, viz. a steady state concentration of SCH2OH and a biphasic elimination pattern for SDM and SCH2OH in plasma and milk. The mean ultrafiltrate ratios of the milk to plasma concentrations with respect to SDM, SCH2OH, SOH, and N4-SDM were: 0.69, 0.22, 020, and 0.63, respectively. The total amount of SDM and its metabolites recovered from the milk after milking twice daily over the whole experimental time was less than 2% of the applied dose. A bioassay method allowed of detecting qualitatively SDM concentrations exceeding 0.2 micrograms/ml in plasma or milk. Withholding times for edible tissues and milk are suggested.

Determination of Clobazam, n-desmethylclobazam and their hydroxy metabolites in plasma and urine by high-performance liquid chromatography

Owing to the pharmacological and clinical importance of the determination of plasma and urine levels of the hydroxy metabolites of clobazam and N-desmethylclobazam in healthy volunteers and in epileptic patients, a high-performance liquid chromatographic (HPLC) method was developed that permits the determination of all these compounds in the same plasma or urine sample. The method involved ether extraction at pH 13 with diazepam as internal standard for the measurement of clobazam and N-desmethylcobazam, followed by ether extraction at pH 9 with nitrazepam as internal standard for the measurement of the hydroxy derivatives. The limit of detection was about 1020 ng/ml for each of these compounds. Applications to patients were limited by chromatographic interferences between the hydroxy metabolites and some medications currently associated with clobazam in the treatment of epilepsy. The only interference in clobazam and N-desmethylclobazam analysis was from N-desmethyldiazepam. Despite these inconveniences, this HPLC procedure appears to be the only available method for the simultaneous quantification of clobazam and its three main metabolites.

Pharmacokinetics of anthracyclines in dogs: evidence for structure-related body distribution and reduction to their hydroxy metabolites.

The pharmacokinetic disposition of the anthracyclines, adriamycin (doxorubicin), daunorubicin, 4'-epi-adriamycin, carminomycin, and 4-demethoxy-daunorubicin, and the formation of their reduced C13 hydroxy metabolites were studied in dogs. The presence of a C14hydroxy group (adriamycin and 4'epi-adriamycin) drastically reduces the appearance of the C13 hydroxy metabolites in plasma. Substitution of the C4-H with C4-OH and C4-OCH3, in this rank order, decreases the area under the plasma concentration-time curves of the parent compounds and their C13 hydroxy metabolites.

Simultaneous assay of triazolam and its main hydroxy metabolite in plasma and urine by capillary gas chromatography

A gas—liquid chromatographic method for the simultaneous determination of triazolam and its major hydroxy metabolite (1-hydroxymethyltriazolam) in human plasma and urine is described. After addition of two internal standards to the biological fluid, extraction at pH 9, acid washing, back-extraction, and derivatization, the analysis was performed on a wall-coated superior capacity open-tubular (WSCOT) CP-Sil 5 capillary column with electron-capture detection. The detection limit was 0.1–0.2 ng/ml; reproducibility was about 6–7% for plasma concentrations below 1 ng/ml. No interference from other possible minor hydroxy metabolites of triazolam was found. Gas chromatography coupled with mass spectrometry validated the chromatographic results. The method was successfully applied to plasma specimens collected from healthy human volunteers following a single intravenous administration of 1 mg of triazolam or 1-hydroxymethyltriazolam.

Simultaneous Determination of Brotizolam and Its Main Hydroxymetabolite in Plasma by Capillary Gas Chromatography with Double Internal Standardization

Abstract A gas-liquid chromatographic (GLC) method for the simultaneous determination of brotizolam (I) and its major hydroxymetabolite (1-hydroxymethylbrotizolam) (II) in human plasma is described here. After extraction at pH 13, acid washing, alkaline back extraction and derivatization with a double internal standardization, the analysis was performed on a WSCOT CPsil 5 capillary column, with detection by electron capture. Sensitivity limit was 0.2 ng/ml; reproducibility was about 6–8% for plasma concentrations below 1 ng/ml. No interference of other possible minor hydroxymetabolites of brotizolam was found. Mass fragmentography was used for the validation of hydroxymetabolite derivazation. This method was applied to plasma specimens collected from dogs, after single IV administration of I and II and from healthy volunteers, after single oral administration of 0.5 mg brotizolam. Preliminary pharmacokinetic results are presented here.

Determination of midazolam and its major hydroxy metabolite in plasma by gas liquid chromatography. Application to a pharmacokinetic study

AbstractA sensitive gas liquid chromatographic (GLC) assay was developed for plasma determinations of 8‐chloro‐6‐(2′ ‐fluorophenyl)‐1‐methyl‐4H‐imidazo[1,5 α][1,4]benzodiazepine (compound I) and its hydroxymethylimidazo metabolite (compound II). The internal standards used were 8‐chloro‐6‐(2′ ‐chlorophenyl)‐1 ‐methyl‐4H‐imidazo[1,5 α][1,4]benzodiazepine (compound VI) and 7‐chloro‐5‐(2′ ‐fluorophenyl)‐1, 3‐dihydro‐1‐(hydroxyethyl)‐2H‐1,4‐benzodiazepin‐2‐one (compound VII) for compounds I and II, respectively. Following extraction, and silylation for compound II, compounds I and II were analyzed by GLC using a glass column packed with 5% OV‐101 on Gas‐Chrom Q, and a 63Ni electron‐capture detector. The GLC method was validated by a CI‐GC/MS technique. The detection limit of the assay is approximately 4–5 ng/ml for compound I and 3 ng/ml for compound II. The method was used in comparative pharmacokinetic studies of the distribution of the two compounds in arterial and venous blood.

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

PROCEEDINGS OF THE AUSTRALASIAN SOCIETY OF CLINICAL AND EXPERIMENTAL PHARMACOLOGISTS

Determination of therapeutic plasma concentrations of tetrabenazine and an active metabolite by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the determination of tetrabenazine and a hydroxy metabolite in plasma is described. Tetrabenazine and the hydroxy metabolite are quantified as their dehydro derivatives using fluorescence detection. This method has been applied to the analysis of plasma samples from patients with Huntington's chorea and has been found to be sensitive, reliable and specific for tetrabenazine and the hydroxy metabolite. The plasma concentrations of tetrabenazine found in patients were lower than could be detected using previously published methods.

Simultaneous determination of pentoxifylline and its hydroxy metabolite in plasma by high-performance liquid chromatography

Simultaneous determination of pentoxifylline and its hydroxy metabolite in plasma by high-performance liquid chromatography

Liquid Chromatography in Pharmaceutical Analysis X: Determination of Chlorzoxazone and Hydroxy Metabolite in Plasma

A method for the high-pressure liquid chromatographic determination of chlorzoxazone and its hydroxy metabolite in human plasma samples is presented. The separation of the compounds is achieved on an octadecylsilane column with a mobile phase of absolute methanol-distilled water (40:60) at a flow rate of 2.0ml/min (3100psig). The chromatographic separation is achieved within 10min. The overall analysis time is about 45 min, which includes extraction of the drug and metabolite from plasma followed by high-pressure liquid chromatographic separation and quantification. The accuracy of the procedure is in the 1-5% range.