Major Metabolites In Human Plasma Research Articles

Identification and quantification of daidzein-7-glucuronide-4′-sulfate, genistein-7-glucuronide-4′-sulfate and genistein-4′,7-diglucuronide as major metabolites in human plasma after administration of kinako

Much attention has been paid to the metabolism and disposition of isoflavones daidzein (Dein) and genistein (Gein) with regard to the prevention of several hormone-dependent diseases. Recent studies have reported that several conjugates as well as aglycones may be biologically active or may be activated within target cells. However, the disposition of Dein and Gein in plasma is still uncertain. This paper describes the identification and quantification of the highly polar metabolites, daidzein-7-glucuronide-4'-sulfate (D-7G-4'S), genistein-7-glucuronide-4'-sulfate (G-7G-4'S), daidzein-4',7-diglucuronide (D-4',7-diG), and genistein-4',7-diglucuronide (G-4',7-diG) in human plasma after dietary administration of kinako (baked soybean powder) to two healthy volunteers. The structure identification of these conjugated metabolites in plasma was performed in comparison to the LC-ESI-MS and 600 MHz (1)H-NMR spectral data of the chemically synthesized compounds. Furthermore, 16 isoflavone metabolites including D-7G-4'S, G-7G-4'S, D-4',7-diG, and G-4',7-diG in plasma were simultaneously measured by a high-performance liquid chromatography-UV-diode-array detector method combined with solid-phase extraction using an Oasis HLB cartridge. D-7G-4'S, G-7G-4'S and G-4',7-diG were found to be major metabolites of Dein and Gein in plasma, while intact aglycones were detected to be only ca. 2% in both subjects. The findings suggest that the conjugated metabolites could be the key compounds responsible for pharmacological and medicinal properties of isoflavones.

Chromatographic Behavior of Bupivacaine and Five of its Major Metabolites in Human Plasma, Utilizing Solid- Phase Extraction and Capillary Gas Chromatography

Bupivacaine and its five known metabolites were determined in underivatized form using capillary gas chromatography. Gas chromatography with mass spectrometry (GC-MS) and nitrogen-phosphorus detection was used for the determination of bupivacaine and its metabolites. A comparison between seven commercially available columns of differing polarity and from different manufacturers was made. Peak symmetries and retention factors varied significantly between the columns. The nonpolar columns (dimethylpolylsiloxane), Ultra1 and CP-Sil, gave good peak symmetries and an analysis time of 9 min with complete baseline separation for all of the analytes studied. The stability of the substances at different injector temperatures, 200-350 degrees C, was studied. The limit of detection was 0.13-0.16 pmol using GC-MS in positive chemical ionization mode. Different conditions for the extraction of analytes from plasma samples using solid-phase extraction (SPE) were investigated. Different sorbents (C(2), C(8), C(18), and ENV+, divinyl-polystyrene) were tested. The ENV+ phase gave recoveries of between 82-120% for all the analytes studied. Several attempts at varying the washing and elution steps in order to obtain a high recovery and clean extract were investigated.

Measurement of Total and Unbound Mycophenolic Acid and Its Glucuronide by LC Coupled with MS. Application to a Pharmacokinetic Study of Mycophenolate Mofetil in Patients with Autoimmune Diseases

Liquid chromatography coupled with mass spectrometry for the determination of total and unbound mycophenolic acid and its major metabolite in human plasma has been developed. Sample preparations were based on a fully automated solid-phase extraction process and ultrafiltration. Mass spectrometric data were acquired in a single-ion monitoring method. The analytes and nevirapine (internal standard) were well separated in an isocratic mode over 8 min. Validation study exhibited excellent linearity, with intra- and inter-day precision and accuracy of less than 12%. The assay was successfully applied to the pharmacokinetic study of mycophenolic acid in patients with autoimmune diseases.

Rapid and sensitive ultra-high-pressure liquid chromatography–tandem mass spectrometry analysis of the novel anticancer agent PR-104 and its major metabolites in human plasma: Application to a pharmacokinetic study

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC–MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC–MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50 mm × 2.1 mm, 1.8 μm) column and gradient of acetonitrile and 0.01% formic acid with a 6 min run time. The method had a linear range of 0.1–50 μM for PR-104, PR-104A and PR-104G, 0.05–5 μM for PR-104H, 0.025–2.5 μM for PR-104M and 0.01–1 μM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.

Quantification of Vincristine and its Major Metabolite in Human Plasma by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 x 150 mm) with a 5-mum particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3--> 271.7), vincristine (m/z 413.2--> 362.2), and M1 (m/z 397.3 --> 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 +/- 9.6% for intra-day, n = 5 each concentration; 90.9 +/- 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.

Immunochemical detection of flavonoid glycosides: Development, specificity, and application of novel monoclonal antibodies

Flavonoid-rich diets are expected to decrease the risk of cardiovascular diseases. The localization and target sites of flavonoids underlying the protective mechanism in vivo have not been fully investigated because the methods for detection of flavonoids have been limited to chemical analysis such as high-performance liquid chromatography. To further understand the actions of flavonoids in vivo, we developed a novel methodology that immunochemically evaluates flavonoids using specific antibodies. Quercetin-3-glucuronide (Q3GA), a major metabolite in human plasma, was coupled with keyhole limpet hemocyanin. Alternatively, the sugar moiety of quercetin-3-glucoside (Q3G) was succinylated and then coupled with a carrier protein. Using these two immunogens, we finally obtained two monoclonal antibodies, mAb14A2 and mAb11G6, from the immunogen using Q3GA and Q3G, respectively. Competitive enzyme-linked immunosorbent assay showed the unique difference in the specificity between the two similar antibodies: mAb14A2 recognized several quercetin-3-glycosides including Q3G and rutin but mAb11G6 was highly specific to the Q3G structure. The macrophage-derived foam cells in human atherosclerotic lesions were significantly stained with mAb14A2 but scarcely with mAb11G6. These results showed that the anti-flavonoid glycoside antibodies are useful tools for evaluating their localization in tissues and that the specificities strongly depend on the immunogen design for synthesizing the hapten–protein conjugates.

An improved HPLC method for rapid quantitation of diazepam and its major metabolites in human plasma

A rapid and specific HPLC method was developed and validated for simultaneous determination of diazepam and its main active metabolites, desmethyldiazepam, oxazepam and temazepam in human plasma. Plasma samples were extracted using toluene. HPLC system included a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using 10 mM phosphate buffer (pH 2.5)–methanol–acetonitrile (63:10:27, v/v) as mobile phase running at 2 mL min −1. UV detector ( λ = 230 nm) was used. The calibration curves were linear in the concentration range of 2–800 ng mL −1 for diazepam and 2–200 ng mL −1 for the three metabolites ( r 2 > 0.99). The lower limit of quantification was 2 ng mL −1 for all analytes. Within and between-day precisions in the measurement of QC samples were in the range of 1.8–18.0% for all analytes. The developed procedure was used to assess the pharmacokinetics of diazepam and its main metabolites following single dose administration of 10 mg diazepam orally to healthy subjects.

Enantioselective analysis of mirtazapine and its two major metabolites in human plasma by liquid chromatography–mass spectrometry after three-phase liquid-phase microextraction

A three-phase liquid-phase microextraction (LPME) method using porous polypropylene hollow fibre membrane with a sealed end was developed for the extraction of mirtazapine (MRT) and its two major metabolites, 8-hydroxymirtazapine (8-OHM) and demethylmirtazapine (DMR), from human plasma. The analytes were extracted from 1.0 mL of plasma, previously diluted and alkalinized with 3.0 mL 0.5 mol L −1 pH 8 phosphate buffer solution and supplemented with 15% sodium chloride (NaCl), using n-hexyl ether as organic solvent and 0.01 moL L −1 acetic acid solution as the acceptor phase. Haloperidol was used as internal standard. The chromatographic analyses were carried out on a chiral column, using acetonitrile–methanol–ethanol (98:1:1, v/v/v) plus 0.2% diethylamine as mobile phase, at a flow rate of 1.0 mL min −1. Multi-reaction monitoring (MRM) detection was performed by mass spectrometry (MS–MS) using a triple-stage quadrupole and electrospray ionization interface operating in the positive ion mode. The mean recoveries were in 18.3–45.5% range with linear responses over the 1.25–125 ng mL −1 concentration range for all enantiomers evaluated. The quantification limit (LOQ) was 1.25 ng mL −1. Within-day and between-day assay precision and accuracy (2.5, 50 and 100 ng mL −1) showed relative standard deviation and the relative error lower than 11.9% for all enantiomers evaluated. Finally, the method was successfully used for the determination of mirtazapine and its metabolite enantiomers in plasma samples obtained after single drug administration of mirtazapine to a healthy volunteer.

High-performance liquid chromatographic method for the determination of moclobemide and its two major metabolites in human plasma

A selective, sensitive, and simple high-performance liquid chromatographic (HPLC) method was developed for the determination of moclobemide and its two major metabolites, Ro 12-5637 and Ro 12-8095, in human plasma. Sample preparation (0.5 ml of plasma) involved solid-phase extraction (SPE) using Speedisk ® H 2O-Philic DVB columns. Separations were performed on a Waters XTerra™ RP18 column (5 μm, 150 mm × 4.6 mm). The mobile phase consisted of 10 mM KH 2PO 4 with 1% triethylamine (pH 3.9) and acetonitrile (83:17, v/v), and a flow-rate was 1.2 ml/min. The total run time was 13 min. UV detection was performed at 240 nm. Mean absolute recoveries were ≥90% and the limit of quantification (LOQ) for all analytes was 0.02 mg/l. Calibration curves were linear ( r > 0.995) over a wide range of the analyte concentrations in plasma; thus, the method is suitable for different clinical studies when large variations in the drug/metabolites concentrations are observed. During a 5-day assay validation procedure the accuracy and precision were tested and proven (relative errors (RE) ≤ 13%; intra-day coefficient of variation (CV) ≤ 7%; inter-day CV ≤ 13%). Many drugs frequently used in the target patient population were evaluated for potential interference in order method selectivity to be ensured. The assay has been used in a clinical pharmacokinetic study to assess steady-state pharmacokinetics of moclobemide and two metabolites in depressive patients on mono- and combined therapy.

PHARMACOKINETICS AND METABOLISM OF THE FLAVONOID SCUTELLARIN IN HUMANS AFTER A SINGLE ORAL ADMINISTRATION

Scutellarin is widely used in treating various cardiovascular diseases. Few data are available regarding its metabolism and pharmacokinetics in humans. The objectives of this study were to develop methods to identify major metabolites of scutellarin in human urine and plasma and to determine simultaneously the parent drug and its major metabolites in human plasma for pharmacokinetic studies. Four metabolites were detected in urine samples by liquid chromatography coupled with electrospray multi-stage mass spectrometry (MS), but only one of them was found in plasma. Its structure was confirmed as scutellarein 6-O-beta-D-glucuronide by MS, NMR, and UV absorbance spectra. The plasma concentrations of scutellarin and the major metabolite were simultaneously determined using liquid chromatography-tandem MS. After a single p.o. administration of 60 mg of scutellarin to 20 healthy subjects, the plasma concentrations of scutellarin were very low, and its plasma concentration-time curve was also anomalous. Plasma concentration of the major metabolite was comparatively high, and the peak plasma concentration was 87.0 +/- 29.1 ng/ml. The Tmax was late (7.85 +/- 1.62 h), and part of individual pharmacokinetic profiles showed double peaks, which indicated scutellarin could be absorbed into the intestine after hydrolysis to its aglycone by bacterial enzymes. This was followed by reconjugation in the intestinal cell and/or liver with glucuronic acid catalyzed by the phase II enzyme, which showed regioselectivity and species difference. The regioselectivity of glucuronoconjugation for scutellarin may be of importance for pharmacological activity. Plasma concentration of isoscutellarin can be used as a biomarker of scutellarin intake.

Pharmacokinetics of Dihydroergocristine and Its Major Metabolite 8- Hydroxy-Dihydroergocristine in Human Plasma

Dihydroergocristine (DHEC) is a semi-synthetic drug mainly used for age-related cognitive impairment. In this study, its major metabolite 8'-hydroxy-dihydroergocristine (8'-OH-DHEC) was produced in incubates of a bovine liver preparation using dihydroergocristine mesylate (DHECM) as substrate. Purification was achieved by flash silica gel column and reverse phase liquid chromatographies, and identification was based on accurate molecular mass measurements, mass fragmentation spectra and NMR ((1)H/(13)C) chemical shifts. By using the substance produced in vitro, a fast, sensitive, specific and robust LC/MS/MS method for the simultaneous determination of DHEC and its major metabolite in human plasma was developed and validated. Bromocriptine was used as internal standard and limits of quantification for DHEC and 8'-OH-DHEC were 10 pg/ml and 20 pg/ml, respectively. Pharmacokinetic parameters were investigated on 12 male healthy volunteers to whom a single dose of 18 mg DHECM was administered in tablets (Iskevert). The peak of DHEC was 0.28 +/- 0.22 microg/l, the t(max) 0.46 +/- 0.26 h, the AUC(last) 0.39 +/- 0.41 microg/l.h and the terminal elimination half-life 3.50 +/- 2.27 h. The peak of 8'-OH-DHEC was 5.63 +/- 3.34 microg/l, the t(max) 1.04 +/- 0.66 h, the AUC(last) 13.36 +/- 5.82 microg/l.h and the terminal elimination half-life 3.90 +/- 1.07 h. Dosing of 18 mg DHECM was well tolerated, causing no adverse events.

HPLC assay for bupropion and its major metabolites in human plasma

Bupropion is used clinically as an antidepressant and in smoking cessation. As it is metabolised to hydroxybupropion specifically by CYP2B6, bupropion has also been used as a probe to assess CYP2B6 activity. A specific and reproducible HPLC assay has been developed to simultaneously quantify bupropion and its major metabolites hydroxybupropion, threohydrobupropion and erythrohydrobupropion in human plasma. The analysis was performed on an Aqua C18 HPLC column, with a mobile phase consisting of 45:55 of methanol:0.05 M phosphate buffer (pH 5.5) and simultaneous UV detection at 214 nm (bupropion metabolites) and 254 nm (bupropion, internal standard timolol maleate). The assay showed a linear response for bupropion (2.5–250 ng/mL), threohydrobupropion (5–250 ng/mL), erythrohydrobupropion (10–250 ng/mL) and hydroxybupropion (10–1000 ng/mL). Extraction recovery was reproducible and greater than 55% for each analyte. The inter- and intra-day assay variability (measured as percent coefficient of variation; %CV) was less than 15% for all analytes. Limit of quantification was 2.5 ng/mL for bupropion, 5 ng/mL for threohydrobupropion and 10 ng/mL for hydroxybupropion and erythrohydrobupropion. This assay is more sensitive than currently published methods using HPLC with UV detection for the simultaneous quantitation of bupropion and metabolites and can be used for assessing CYP2B6 activity in vivo following a single dose of bupropion.

Simultaneous determination of clobazam and its major metabolite in human plasma by a rapid HPLC method

A rapid and specific HPLC method has been developed and validated for simultaneous determination of clobazam, the anticonvulsant agent, and its major metabolite in human plasma. The sample preparation was a liquid–liquid extraction with tuloene yielding almost near 100% recoveries of two compounds. Chromatographic separation was achieved with a Chromolith™ Performance RP-18e 100 mm × 4.6 mm column, using a mixture of a phosphate buffer (pH 3.5; 10 mM)–acetonitrile (70:30, v/v), in isocratic mode at 2 ml/min at a detection wave-length of 228 nm. The calibration curves were linear ( r 2 > 0.998) in the concentration range of 5–450 ng ml −1. The lower limit of quantification was 5 ng ml −1 for two compounds studied. The within- and between-day precisions in the measurement of QC samples at four tested concentrations were in the range of 0.89–9.1% and 2.1–10.1% R.S.D., respectively. The developed procedure was applied to assess the pharmacokinetics of clobazam and its major metabolite following administration of a single 10 mg oral dose of clobazam to healthy volunteers.

A highly sensitive LC–MS–MS assay for analysis of midazolam and its major metabolite in human plasma: Applications to drug metabolism

The present report describes a rapid, selective and a highly sensitive assay for midazolam (MDZ) and its major metabolite 1-hydroxymidazolam (1-OH-MDZ) in human plasma employing liquid chromatography–tandem mass spectrometry (LC–MS–MS) detection. The method involves liquid–liquid extraction sample clean-up, separation on a Purospher RP 18-e column and detection with an electrospray interface in the positive ion mode. The overall recoveries were about 100% and 80% for midazolam and 1-hydroxymidazolam, respectively. Accuracy, precision and linearity were acceptable for biological samples with quantitation limits of 0.1–100 ng mL −1 plasma for both analytes. The validated method was successfully applied to quantify plasma concentration of midazolam and 1-hydroxymidazolam in authentic samples from a healthy volunteer following a single 15 mg oral dose of midazolam (apparent total clearance: 3.47 L h −1 kg −1 and AUC 0 − α IOH-MDZ/MDZ: 0.338).

Simultaneous selected reaction monitoring, MS/MS and MS3 quantitation for the analysis of pharmaceutical compounds in human plasma using chip‐based infusion

An assay method with mass spectrometric detection was developed for the quantitative analysis of a pharmaceutical compound and its major metabolite in human plasma using chip-based infusion. Liquid-liquid extraction sample preparation was found to be essential to minimize matrix suppression and to achieve a limit of quantitation (LOQ) of 2.5 ng/mL using a 100 microL plasma aliquot. The potential for simultaneous quantitation in selected reaction monitoring (SRM), tandem mass spectrometry (MS/MS) (enhanced product ion), and MS(3) was investigated and found to be very beneficial in improving assay selectivity. A novel concept for monitoring quantitative assay performance using a SRM/MS(3) ratio is proposed.

Fast high-performance liquid chromatographic analysis of mianserin and its metabolites in human plasma using monolithic silica column and solid phase extraction

A fast high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of mianserin (MIAN) and its metabolites desmethylmianserin (DMM), 8-hydroxymianserin (HM) and mianserin- N-oxide (MNO) in human plasma. Each compound, together with internal standard (propranolol) was extracted from the plasma matrix using solid phase extraction. Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column ( 100 mm×4.6 mm i.d.) under isocratic conditions using a mobile phase of 74:26 (v/v) 25 mM phosphate buffer (pH 5.3 adjusted with phosphoric acid): acetonitrile. The elution of the analytes were monitored at 292 mm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 3.5 ml/min. The total run time of the assay was 5 min. The method was validated over the range of 10–200 ng/ml for MIAN, 10–150 ng/ml for DMM, 20–300 ng/ml for HM and 25–500 ng/ml for MNO. The method proved to be precise (within-run precision ranging from 1.6 to 6.9% R.S.D. and between-run precision ranging from 1.3 to 7.2% R.S.D.) and accurate (within-run accuracies ranged from 1.4 to 6.4% and between-run accuracies ranging from 1.5 to 4.5%). The mean absolute recoveries were 95.7, 94.8, 99.6, and 102.6% for MIAN, DMM, HM and MNO, respectively. The limit of quantitation (LOQ) for MIAN and DMM was 10 ng/ml and for HM and MNO were 20 and 25 ng/ml in human plasma, respectively. The limit of detection (LOD) for MIAN, DMM, HM and MNO was 5, 2.5, 10 and 15 ng/ml, respectively. The described method demonstrates the feasibility for employing monolithic columns to effect rapid bioanalytical HPLC analysis for the quantitative determination of MIAN and its major metabolites in human plasma.

Enantioselective quantitation of the ecstasy compound ( R)- and ( S)- N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma and urine

An enantioselective HPLC method has been developed and validated for the stereospecific analysis of N-ethyl-3,4-methylenedioxyamphetamine (MDE) and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA). These compounds have been analyzed both from human plasma and urine after administration of 70 mg pure MDE-hydrochloride enantiomers to four subjects. The samples were prepared by hydrolysis of the o-glucuronate and sulfate conjugates using β-glucuronidase/arylsulfatase and solid-phase extraction with a cation-exchange phase. A chiral stationary protein phase (chiral-CBH) was used for the stereoselective determination of MDE, HME and MDA in a single HPLC run using sodium dihydrogenphosphate, ethylendiaminetetraacetic acid disodium salt and isopropanol as the mobile phase (pH 6.44) and fluorimetric detection ( λ ex 286 nm, λ em 322 nm). Moreover, a suitable internal standard ( N-ethyl-3,4-methylenedioxybenzylamine) was synthesized and qualified for quantitation purposes. The method showed high recovery rates (>95%) and limits of quantitation for MDE and MDA of 5 ng/ml and for HME of 10 ng/ml. The RSDs for all working ranges of MDE, MDA and HME in plasma and urine, respectively, were less than 1.5%. After validation of the analytical methods in plasma and urine samples pharmacokinetic parameters were calculated. The plasma concentrations of ( R)-MDE exceeded those of the S-enantiomer (ratio R: S of the area under the curve, 3.1) and the plasma half time of ( R)-MDE was longer than that of ( S)-MDE (7.9 vs. 4.0 h). In contrast, the stereochemical disposition of the MDE metabolites HME and MDA was reversed. Concentrations of the ( S)-metabolites in plasma of volunteers were much higher than those of the ( R)-enantiomers.

Simultaneous determination of levomepromazine, midazolam and their major metabolites in human plasma by reversed-phase liquid chromatography

A sensitive and reliable high-performance liquid chromatographic (HPLC) assay is a prerequisite for pharmacokinetic analysis of continuous infusion of levomepromazine adjuvant to midazolam. We developed such a method to determine the levels of levomepromazine, midazolam and their major metabolites (levomepromazinesulfoxide, desmethyl-, didesmethyllevomepromazine, O-desmethyllevomepromazine and α-hydroxy-midazolam) simultaneously. Desmethylclomipramine was used as an internal standard (I.S.). The lower limit of quantification of this assay was set for levomepromazine 4.1 μg/l, levomepromazinesulfoxide 4.9 μg/l, O-desmethyllevomepromazine 18.4 μg/l, α-hydroxymidazolam 26.6 μg/l, midazolam 23.4 μg/l, didesmethyllevomepromazine 15.8 μg/l, and desmethyllevomepromazine 6.6 μg/l. The between- and within day assay variations were commonly below 5%. The recovery in human plasma for the different analytes varied between 85 and 11%. The accuracy of this assay varied between 95 and 105% for the different concentrations. The linearity of this assay was set between 25 and 800 μg/l ( r 2>0.999 of the regression line). The first results of pharmacokinetic analysis of midazolam indicated that half-life varied between 1.1 and 1.9 h. Pharmacokinetic analysis using a one-compartment model of levomepromazine revealed that the apparent volume of distribution was 4.1±2.4 l per kg lean body mass and the metabolic clearance was 309±225 l per hour per 70 kg. This assay proved to be robust and reproducible. It can reliably be used for further study of the pharmacokinetics of continuous infusion of levomepromazine.

Quantitative determination of rivastigmine and its major metabolite in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive, specific, accurate and reproducible LC–MS–MS method was developed and validated for the simultaneous determination of rivastigmine and its major metabolite NAP 226-90 in human plasma according to International Regulatory Requirements. After addition of their respective labelled internal standards, the compounds were extracted from plasma using methyl- tert.-butyl ether at basic pH with a simultaneous derivatization of NAP 226-90 with propionic anhydride, and backextracted into an acidic solution. After re-extraction the compounds were analyzed on a 3-μm Purospher Star RP-18 column interfaced with a MDS Sciex API 3000 triple quadrupole mass spectrometer. Positive atmospheric chemical ionization was employed as the ionization source. The analytes and their internal standards were detected by use of multiple reaction monitoring mode. Intra- and inter-day accuracy and precision were found suitable over the range of concentrations between 0.200 and 30.0 ng/ml. The LC–MS–MS method was crossvalidated with a previously developed in-house GC–MS method by the analysis of plasma samples obtained from patients after administration of Exelon ® capsules and showed excellent correlation between the methods.

Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet–visible high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C 16 column (250×3 mm I.D., 5 μm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV–visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 μg/mL, with intra-day and inter-day coefficients of variation and percent error ≤10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.