Determination of beta-artemether and its main metabolite dihydroartemisinin in plasma employing liquid-phase microextraction prior to liquid chromatographic-tandem mass spectrometric analysis

A method for the determination of artemether (ART) and its main metabolite dihydroartemisinin (DHA) in plasma employing liquid-phase microextraction (LPME) for sample preparation prior to liquid chromatography-tandem mass spectrometry (LC-MS-MS) was developed. The analytes were extracted from 1 nil, of plasma utilizing a two-phase LPME procedure with artemisinin as internal standard. Using the optimized LPME conditions, mean absolute recovery rates of 25 and 32% for DHA and ART, respectively, were achieved using toluene-n-octanol (1:1, viv) as organic phase with an extraction time of 30 min. After extraction, the analytes were resolved within 5 min using a mobile phase consisting of methanol-ammonium acetate (10 mmol L(-1) pH 5.0, 80:20. v/v) on a laboratory-made column based on poly(methyltetradecylsiloxane) attached to a zirconized-silica support. MS-MS detection was employed using an electrospray interface in the positive ion mode. The method developed was linear over the range of 5-1000 ng mL(-1) for both analytes. Precision and accuracy were within acceptable levels of confidence (<15%). The assay was applied to the determination of these analytes in plasma from rats treated with ART. The two-phase LPME procedure is affordable and the solvent consumption was very low compared to the traditional methods of sample preparation. (C) 2010 Elsevier B.V. All rights reserved.

Two-step liquid-phase microextraction and high-performance liquid chromatography for the simultaneous analysis of the enantiomers of mefloquine and its main metabolite carboxymefloquine in plasma

A method for the simultaneous analysis of the enantiomers of mefloquine (MQ) and its main metabolite carboxymefloquine (CMQ) in plasma is described for the first time. The assay involves two-step liquid-phase micro-extraction (LPME) and enantioselective high-performance liquid chromatography. In the first LPME step, the enantiomers of MQ were extracted from an alkalinized sample through a thin layer of di-n-hexyl ether immobilized in the pores of the hollow fiber and into 0.01 M perchloric acid as acceptor solution. In the second LPME step, the same sample was acidified to enable the extraction of CMQ using the same organic solvent and 0.05 M sodium hydroxide as acceptor phase. The analytes were resolved on a Chirobiotic T column in the polar-organic mode of elution and detected at 285 nm. The recovery rates from 1 mL of plasma were in the range 35-38%. The method presented limits of quantification of 50 ng/mL for all analytes and was linear up to 1,500 and 3,000 ng/mL for the enantiomers of MQ and CMQ, respectively. The plasmatic concentrations of (+)-(RS)-MQ were higher than those of (-)-(SR)-MQ after oral administration of the racemic drug to rats.

Fungal tyrosine betaine, a novel secondary metabolite from conidia of entomopathogenic Metarhizium spp. fungi

Fungi, including the entomopathogenic deuteromycete Metarhizium anisopliae, produce a wide diversity of secondary metabolites that either can be secreted or stored in specific developmental structures, e.g., conidia. Some secondary metabolites, such as pigments, polyols and mycosporines, are associated with pathogenicity and/or fungal tolerance to several stress-inducing environmental factors, including temperature and solar radiation extremes. Extracts of M. anisopliae var. anisopliae (strain ESALQ-1037) conidia were purified by chromatographic procedures and the isolated compounds analyzed by (1)H and (13)C nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. LC-MS analyses were carried out to search for mycosporines (the initial targets), but no compounds of this class were detected. A molecule whose natural occurrence was previously undescribed was identified. It consists of betaine conjugated with tyrosine, and the structure was identified as 2-([1-carboxy-2-(4-hydroxyphenyl)ethyl]amino)-N,N,N-trimethyl-2-oxoethanammonium. mannitol was the predominant compound in the alcoholic conidial extract, but no amino acids other than tyrosine were found to be conjugated with betaine in conidia. The fungal tyrosine betaine was detected also in conidial extracts of three other M. anisopliae var. anisopliae (ARSEF 1095, 5626 and 5749) and three M. anisopliae var. acridum isolates (ARSEF 324, 3391 and 7486), but it was not detected in Aspergillus nidulans conidial extract (ATCC 10074). (C) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Metabolite mean transit times in the liver as predicted by various models of hepatic elimination

Predicted area under curve (AUC), mean transit time (MTT) and normalized variance (CV2) data have been compared for parent compound and generated metabolite following an impulse input into the liver, Models studied were the well-stirred (tank) model, tube model, a distributed tube model, dispersion model (Danckwerts and mixed boundary conditions) and tanks-in-series model. It is well known that discrimination between models for a parent solute is greatest when the parent solute is highly extracted by the liver. With the metabolite, greatest model differences for MTT and CV2 occur when parent solute is poorly extracted. In all cases the predictions of the distributed tube, dispersion, and tasks-in-series models are between the predictions of the rank and tube models. The dispersion model with mixed boundary conditions yields identical predictions to those for the distributed tube model (assuming an inverse gaussian distribution of tube transit times). The dispersion model with Danckwerts boundary conditions and the tanks-in series models give similar predictions to the dispersion (mixed boundary conditions) and the distributed tube. The normalized variance for parent compound is dependent upon hepatocyte permeability only within a distinct range of permeability values. This range is similar for each model but the order of magnitude predicted for normalized variance is model dependent. Only for a one-compartment system is the MIT for generated metabolite equal to the sum of MTTs for the parent compound and preformed metabolite administered as parent.

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

Investigation of the antinociceptive efficacy and relative potency of extended duration injectable 3-acylmorphine-6-sulfate prodrugs in rats

This investigation was designed to examine the antinociceptive activity in rats of 3-O-acyl prodrugs of M6S relative to the parent drug, after intravenous and intramuscular injection, using the tail flick latency test of antinociception. M6S, 3-acetylmorphine-6-sulfate (3AcM6S), 3-propionylmorphine-6-sulfate (3PrM6S), 3-butanoylmorphine-6-sulfate (3BuM6S) and 3-heptanoylmorphine-6-sulfate (3HpM6S) were administered by the IV route in a dose of 4.10 mu mol/kg. Relatively high levels of antinociception (>40% Maximum Possible Effect) were achieved following administration of M6S, 3AcM6S and 3PrM6S, whereas insignificant antinociception (<20%MPE) was achieved following administration of 3BuM6S or 3HpM6S. Although the mean duration of action for 3AcM6S (6 h) was longer than for M6S or 3PrM6S (4 h), the mean area (+/- S.E.M.) under the degree of antinociception versus time curve (AUG) for 3AcM6S (151.6 +/- 6.9%MPE h) was not significantly different (p <0.05) from that for M6S (120.8 +/- 32.7%MPE h) or for 3PrM6S (106.0 +/- 21.3%MPE h). The mean ED50 (range) doses for M6S, 3AcM6S and 3PrM6S were calculated to be 4.16 (3.61-4.48), 4.32 (3.55-5.09) and 4.54 (4.21-4.79) mu mol/kg, respectively. Preliminary studies were conducted on potential long-acting formulations containing 8 x ED50 doses of M6S and the 3-acetyl and 3-propionyl esters suspended in soybean oil. These showed that 3PrM6S gave a greater AUC (mean + S.E.M.) (1087.4 +/- 97.4%MPE h) and longer duration of action (20 h) than did M6S (613.1 +/- 155.9%MPE h; 10 h duration) or 3AcM6S (379.3 + 114.2%MPE h: 8 h duration). Further studies are needed to more fully investigate these findings. (C) 1998 Elsevier Science B.V. All rights reserved.

Hydromorphone-3-glucuronide: Biochemical synthesis and preliminary pharmacological evaluation

Hydromorphone-3-glucuronide (H3G) was synthesized biochemically using rat liver microsomes, uridine-5'-diphosphoglucuronic acid (UDPGA) and the substrate, hydromorphone. Initially, the crude putative H3G product was purified by ethyl acetate precipitation and washing with acetonitrile, Final purification was achieved using semi-preparative high-performance-liquid-chromatography (HPLC) with ultraviolet (UV) detection. The purity of the final H3G product was shown by HPLC with electrochemical and ultraviolet detection to be > 99.9% and it was produced in a yield of approximate to 60% (on a molar basis). The chemical structure of the putative H3G was confirmed by enzymatic hydrolysis of the glucuronide moiety using P-glucuronidase, producing a hydrolysis product with the same HPLC retention time as the hydromorphone reference standard. Using HPLC with tandem mass spectrometry (HPLC-MS-MS) in the positive ionization mode, the molecular mass (M+1) was found to be 462 g/mol, in agreement with H3G's expected molecular weight of 461 g/mol. Importantly, proton-NMR indicated that the glucuronide moiety was attached at the 3-phenolic position of hydromorphone. A preliminary evaluation of H3G's intrinsic pharmacological effects revealed that following icy administration to adult male Sprague-Dawley rats in a dose of 5 mu g, H3G evoked a range of excitatory behavioural effects.including chewing, rearing, myoclonus, ataxia and tonic-clonic convulsions, in a manner similar to that reported previously for the glucuronide metabolites of morphine, morphine-3-glucuronide and normorphine-3-glucuronide.

Hepatic structure-pharmacokinetic relationships: The hepatic disposition and metabolite kinetics of a homologous series of O-acyl derivatives of salicylic acid

1 The hepatic disposition and metabolite kinetics of a homologous series of O-acyl (acetyl, propionyl, butanoyl, pentanoyl, hexanoyl and octanoyl) esters of salicylic acid (C2SA, C3SA, C4SA, C5SA, C6SA and C8SA, respectively) was determined using a single-pass, in-sills rat liver preparation. 2 The hepatic venous outflow profiles for the parent esters and the generated metabolite, salicylic acid (SA) were analysed by HPLC. Non-parametric moments analysis was used to determine the area under the curve (AUC'), mean transit time (MTT) and normalized variance (CV2) for the parent esters and generated SA. 3 Pregenerated SA ([C-14]-salicylic acid) was injected into each liver with the parent ester to determine its distribution characteristics. 4 The overall recovery of ester plus metabolite was 89% of the ester dose injected and independent of the ester carbon number, suggesting that ester extraction was due to hepatic metabolism to salicylic acid. 5 The metabolite AUC' value increased directly with the lipophilicity of the parent ester (from 0.12 for C2SA to 0.95 for C8SA). By contrast, the parent AUC' decreased with the lipophilicity (from 0.85 for C2SA to zero for C8SA). The metabolite MTT value also showed a trend to increase with the lipophilicity of the parent ester (from 15.72 s for C3SA to 61.97 s for C8SA). However, the parent MTT value shows no significant change across the series. 6 The two-compartment dispersion model was used to derive the kinetic parameters for parent ester, pregenerated SA and generated SA. Consequently, these parameters were used to estimate the values of AUG', MITT and CV2 for the parent ester and metabolite. The moments values obtained using the two-compartment dispersion model show similar trends to the corresponding moments values obtained from the outflow profiles using a non-parametric approach. 7 The more lipophilic aspirin analogues are more confined to the portal circulation after oral administration than aspirin due to their more extensive hepatic elimination avoiding systemic prostacyclin inhibition. Given that aspirin's selectivity as an anti-thrombotic agent has been postulated to be due to selective anti-platelet effects in the portal circulation, the more lipophilic and highly extracted analogues are potentially more selective anti-thrombotic agents than aspirin.

Hepatic disposition and metabolite kinetics of a homologous series of diflunisal esters

The hepatic disposition and metabolite kinetics of a homologous series of diflunisal O-acyl esters (acetyl, butanoyl, pentanoyl, anti hexanoyl) were determined using a single-pass perfused in situ rat liver preparation. The experiments were conducted using 2% BSA Krebs-Henseleit buffer (pH 7.4), and perfusions were performed at 30 mL/min in each liver. O-Acyl esters of diflunisal and pregenerated diflunisal were injected separately into the portal vein. The venous outflow samples containing the esters and metabolite diflunisal were analyzed by high performance liquid chromatography (HPLC). The normalized outflow concentration-time profiles for each parent ester and the formed metabolite, diflunisal, were analyzed using statistical moments analysis and the two-compartment dispersion model. Data (presented as mean +/- standard error for triplicate experiments) was compared using ANOVA repeated measures, significance level P < 0.05. The hepatic availability (AUC'), the fraction of the injected dose recovered in the outflowing perfusate, for O-acetyldiflunisal (C2D = 0.21 +/- 0.03) was significantly lower than the other esters (0.34-0.38). However, R-N/f(u), the removal efficiency number R-N divided by the unbound fraction in perfusate f(u), which represents the removal efficiency of unbound ester by the liver, was significantly higher for the most lipophilic ester (O-hexanoyldiflunisal, C6D = 16.50 +/- 0.22) compared to the other members of the series (9.57 to 11.17). The most lipophilic ester, C6D, had the largest permeability surface area (PS) product (94.52 +/- 38.20 mt min-l g-l liver) and tissue distribution value VT (35.62 +/- 11.33 mL g(-1) liver) in this series. The MTT of these O-acyl esters of diflunisal were not significantly different from one another. However, the metabolite diflunisal MTTs tended to increase with the increase in the parent ester lipophilicity (11.41 +/- 2.19 s for C2D to 38.63 +/- 9.81 s for C6D). The two-compartment dispersion model equations adequately described the outflow profiles for the parent esters and the metabolite diflunisal formed from the O-acyl esters of diflunisal in the liver.

Evaluation of an immunoassay (EMIT) for mycophenolic acid in plasma from renal transplant recipients compared with a high-performance liquid chromatography assay

Mycophenolic acid is an immunosuppressant administered as a bioavailable ester, mycophenolate mofetil. The pharmacokinetics of mycophenolic acid have been reported to be variable. Accurate measurement of concentrations of this drug could be important to adjust doses. The aim of this study was to compare the enzyme-multiplied immunoassay technique (EMIT [Dade Behring; San Jose, CA, U.S.A.]) for mycophenolic acid with a high-performance liquid chromatographic (HPLC) assay using samples collected from renal transplant recipients. The HPLC assay used solid phase extraction and a C18 stationary phase with ultraviolet (UV) detection (254 nm). The immunoassay required no manual sample preparation. Plasma samples (n = 102) from seven patients, collected at various times after a dose, were analyzed using both methods. Both assays fulfilled quality-control criteria. Higher concentrations were consistently measured in patient samples when using EMIT. The mean (+/- standard deviation [SD]) bias (EMIT-HPLC) was 1.88 +/- 0.86 mg/L. The differences in concentrations were higher in the middle of a dosage interval, suggesting that a metabolite might have been responsible for overestimation. Measurement of glucuronide concentrations by HPLC demonstrated only a weak correlation between assay differences and glucuronide concentrations. If the crossreacting substance is active, EMIT could provide a superior measure of immunosuppression; if inactive, further work is needed to improve antibody specificity. In conclusion, it was found that EMIT overestimates the concentration of mycophenolic acid in plasma samples from renal transplant recipients compared with HPLC analysis.

Cerebrospinal fluid and plasma concentrations of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in patients before and after initiation of intracerebroventricular morphine for cancer pain management

Twenty-three patients treated with intracerebroventricular (ICV) morphine in this study not only obtained excellent pain relief without rapid increases in dose, but also experienced a reduction in morphine-related side effects. By 24 h after initiation of ICV morphine, the mean trough cerebrospinal fluid (CSF) morphine concentration (approximately 20 mu M) was 50-fold higher than the baseline concentration (approximately 0.4 mu M), and the CSF concentration of morphine-6-glucuronide (M6G) was undetectable (

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1(R)) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was

Brain region-specific studies of the excitatory behavioral effects of morphine-3-glucuronide

This study was designed to determine in rats whether morphine-3-glucuronide (M3G) produces its neuro-excitatory effects most potently in the ventral hippocampus (as has been reported previously for subanalgesic doses of opioid peptides). Guide cannulae were implanted into one of seven regions of the rat brain: lateral ventricle; ventral, CA1 and CA2-CA3 regions of the hippocampus; amygdala; striatum or cortex. After a 7 day recovery period, rats received intracerebral injections of (i) M3G (1.1 or 11 nmol) (ii) DADLE ([D-Ala(2),D-Leu(5)]enkephalin), (45 nmol, positive controls) or (iii) vehicle (deionised water), and behavioral excitation was quantified over 80 min. High-dose M3G (11 nmol) evoked behavioral excitation in all brain regions but the onset, severity and duration of these effects varied considerably among brain regions. By contrast, low-dose M3G (1.1 nmol) evoked excitatory behaviors only when administered into the ventral hippocampus and the amygdala, with the most potent effects being observed in the ventral hippocampus. Prior administration of the nonselective opioid antagonists, naloxone and beta-funaltrexamine into the ventral hippocampus, markedly attenuated low-dose M3G's excitatory effects but did not significantly alter levels of excitation evoked by high-dose M3G. Naloxone given 10 min after M3G (1.1 or 11 nmol) did not significantly attenuate behavioral excitation. Thus, M3G's excitatory behavioral effects occur most potently in the ventral hippocampus as reported previously for subanalgesic doses of opioid peptides, and appear to be mediated through at least two mechanisms, one possibly involving excitatory opioid receptors and the other, non-opioid receptors.

Systemic coadministration of chloramphenicol with intravenous but not intracerebroventricular morphine markedly increases morphine antinociception and delays development of antinociceptive tolerance in rats

Chloramphenicol, an in vitro inhibitor of the glucuronidation of morphine to its putative antianalgesic metabolite, morphine-3-glucuronide (M3G), was coadministered with morphine in adult male Sprague-Dawley rats to determine whether it inhibited the in vivo metabolism of morphine to M3G, thereby enhancing morphine antinociception and/or delaying the development of antinociceptive tolerance. Parenteral chloramphenicol was given acutely (3-h studies) or chronically (48-h studies). Morphine was administered by the i.v. or i.c.v. route. Control rats received chloramphenicol and/or vehicle. Antinociception was quantified using the hotplate latency test. Coadministration of chloramphenicol with i.v. but not i.cv. morphine increased the extent and duration of morphine antinociception by approximate to 5.5-fold relative to rats that received i.v. morphine alone. Thus, the mechanism through which chloramphenicol enhances i.v. morphine antinociception in the rat does not directly involve supraspinal opioid receptors. Acutely, parenteral coadministration of chloramphenicol and morphine resulted in an approximate to 75% increase in the mean area under the serum morphine concentration-time curve but for chronic dosing there was no significant change in this curve, indicating that factors other than morphine concentrations contribute significantly to antinociception. Antinociceptive tolerance to morphine developed more slowly in rats coadministered chloramphenicol, consistent with our proposal that in vivo inhibition of M3G formation would result in increased antinociception and delayed development of tolerance. However, our data also indicate that chloramphenicol inhibited the biliary secretion of M3G. Whether chloramphenicol altered the passage of M3G and morphine across the blood-brain barrier remains to be investigated.

Incomplete, asymmetric, and route-dependent cross-tolerance between oxycodone and morphine in the Dark Agouti rat

Our previous studies indicate that oxycodone is a putative kappa-opioid agonist, whereas morphine is a well documented mu-opioid agonist. Because there is limited information regarding the development of tolerance to oxycodone, this study was designed to 1) document the development of tolerance to the antinociceptive effects of chronically infused i.v. oxycodone relative to that for i.v. morphine and 2) quantify the degree of antinociceptive cross-tolerance between morphine and oxycodone in adult male Dark Agouti (DA) rats. Antinociceptive testing was performed using the tail-flick latency test. Complete antinociceptive tolerance was achieved in 48 to 84 h after chronic infusion of equi-antinociceptive doses of i.v. oxycodone (2.5 mg/24 h and 5 mg/24 h) and i.v. morphine (10 mg/24 h and 20 mg/24 h, respectively). Dose-response curves for bolus doses of i.v. and i.c.v. morphine and oxycodone were produced in naive, morphine-tolerant, and oxycodone-tolerant rats. Consistent with our previous findings that oxycodone and morphine produce their intrinsic antinociceptive effects through distinctly different opioid receptor populations, there was no discernible cross-tolerance when i.c.v. oxycodone was given to morphine-tolerant rats. Similarly, only a low degree of cross-tolerance (approximate to 24%) was observed after i.v. oxycodone administration to morphine-tolerant rats. By contrast, both i.v. and i.c.v. morphine showed a high degree of cross-tolerance (approximate to 71% and approximate to 54%, respectively) in rats rendered tolerant to oxycodone. Taken together, these findings suggest that, after parenteral but not supraspinal administration, oxycodone is metabolized to a mu-opioid agonist metabolite, thereby explaining asymmetric and incomplete cross-tolerance between oxycodone and morphine.