Pharmacokinetic Study of Conjugated Equine Estrogens in Healthy Chinese Postmenopausal Women Using a Parallel Two-Column LC-MS/MS Method.

BACKGROUND AND OBJECTIVE: Postmenopausal women often require estrogen supplementation to improve menopausal and postmenopausal vasomotor symptoms and maintain hormonal balance. Conjugated equine estrogens extracted from the urine of pregnant mares are commonly used to provide this estrogen replacement therapy. The complex composition of this mixture of animal sulfated metabolites makes its bioanalysis challenging such that its detailed pharmacokinetics has not been fully characterized. The purpose of this work is to reveal the pharmacokinetic behavior of conjugated equine estrogens in healthy Chinese postmenopausal women by a parallel two-column LC-MS/MS method. METHODS: An open-label study was carried out in 35 Chinese healthy postmenopausal women who received a single dose of Premarin® 0.625 mg. A high-throughput column-switching liquid chromatography-tandem mass spectrometry method was developed to determine four conjugated estrogens and two unconjugated estrogens formed by hydrolysis in vivo. The method multiplexes two high-performance liquid chromatography systems into one mass spectrometer and incorporates the positive/negative ion switching acquisition mode of mass spectrometry to significantly increase analysis efficiency. Pharmacokinetics was determined using non-compartmental methods. RESULTS: Both conjugated and unconjugated estrogens can be analyzed simultaneously in a single run with an analysis time of 13.0 minutes in the column-switching liquid chromatography-tandem mass spectrometry method as opposed to 23.0 minutes in a single-column liquid chromatography-tandem mass spectrometry system. The exposures (maximum concentration and area under the curve) of estrone and equilin in Chinese women were higher than those in the North American women. CONCLUSIONS: The fully validated assay was successfully applied to a pharmacokinetic study in healthy postmenopausal Chinese women after oral administration of a conjugated equine estrogen tablet. This study suggests that Chinese postmenopausal women achieve the same level of unconjugated estrogens in plasma at a lower dose of conjugated equine estrogens than North American women.

Biological fate and interaction with cytochromes P450 of the nanocarrier material, d-α-tocopheryl polyethylene glycol 1000 succinate.

d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS, also known as vitamin E-TPGS) is a biodegradable amphiphilic polymer prepared by esterification of vitamin E with polyethylene glycol (PEG) 1000. It is approved by the US Food and Drug Administration (FDA) and has found wide application in nanocarrier drug delivery systems (NDDS). Fully characterizing the in vivo fate and pharmacokinetic behavior of TPGS is important to promote the further development of TPGS-based NDDS. However, to date, a bioassay for the simultaneous quantitation of TPGS and its metabolite, PEG1000, has not been reported. In the present study, we developed such an innovative bioassay and used it to investigate the pharmacokinetics, tissue distribution and excretion of TPGS and PEG1000 in rat after oral and intravenous dosing. In addition, we evaluated the interaction of TPGS with cytochromes P450 (CYP450s) in human liver microsomes. The results show that TPGS is poorly absorbed after oral administration with very low bioavailability and that, after intravenous administration, TPGS and PEG1000 are mainly distributed to the spleen, liver, lung and kidney before both being slowly eliminated in urine and feces as PEG1000. In vitro studies show the inhibition of human CYP450 enzymes by TPGS is limited to a weak inhibition of CYP3A4. Overall, our results provide a clear picture of the in vivo fate of TPGS which will be useful in evaluating the safety of TPGS-based NDDS in clinical use and in promoting their further development.

Cell-based, animal and H1 receptor binding studies relative to the sedative effects of ketotifen and norketotifen atropisomers.

OBJECTIVES: Ketotifen (K) and its active metabolite norketotifen (N) exist as optically active atropisomers. They both have antihistaminic and anti-inflammatory properties but the S-atropisomer of N (SN) causes less sedation than K and RN in rodents. This study investigated whether this could be related to a lower concentration of SN in brain or a lower affinity of SN for rat brain H1 receptors. METHODS: Ketotifen and norketotifen atropisomers were quantified using a validated chiral HPLC assay. RBE4 and Caco-2 cell monolayers were used in uptake and permeability studies, respectively. Free and total brain-to-plasma (B/P) ratios were determined after injecting racemic K and N into rat tail veins. Affinity for rat brain H1 receptors (KI ) was determined using the [3 H]mepyramine binding assay. KEY FINDINGS: Uptake and permeation studies indicate no stereoselective transport for K or N. B/P ratios reveal the brain concentration of N is lower than K with no stereoselective transport into brain. Finally, the [3 H]mepyramine binding assay shows SN has the lowest affinity for rat brain H1 receptors. CONCLUSION: The lower sedative effect of SN in rodents is probably due to a combination of a lower uptake of N than K into the brain and less affinity of SN for CNS H1 receptors.

Uptake and release profiles of PEGylated liposomal doxorubicin nanoparticles: A comprehensive picture based on separate determination of encapsulated and total drug concentrations in tissues of tumor-bearing mice.

The PEGylated liposomal nanoparticle has been widely used as a carrier in drug delivery system. To become biologically active, the encapsulated drug must be released from the nanoparticle vehicle. However, due to limitations of current bioanalytical methods, the characterization of this release process has been restricted to determination of total drug in tissues and tumor. As a result, the fate of liposomal nanoparticles including their uptake into target tissue has not been fully characterized. In this study, we developed a novel two-step solid phase extraction on two separated columns procedure to separate liposomes from tissues and tumors without liposomal leakage. This allowed us to determine encapsulated drug, total drug and, by difference, released drug and compare the release and uptake profiles of PEGylated liposomal doxorubicin in tissues and tumor of tumor-bearing mice with corresponding profiles for free doxorubicin. The liposomal nanoparticles released doxorubicin into tumor efficiently and, compared with administration of free drug, increased doxorubicin uptake into tumor by 1.8-fold. It also decreased doxorubicin uptake into heart (0.78-fold lower) with the potential to reduce doxorubicin cardiotoxicity. Drug release reached constant levels in tissues and tumor after 12 h with released doxorubicin concentration remaining at 70-80% of total doxorubicin concentration and in tumor at 86% of total drug concentration. The assay also included determination of the main doxorubicin metabolites. Determination of the metabolites showed that liposomal entrapment delays and decreases the metabolism of doxorubicin but does not alter the metabolic pathway. These results provide a clear and comprehensive picture of the biodistribution of doxorubicin administered in liposomal nanoparticles which may assist in the rational design of other liposomal nanoparticles.

Ultrahigh-throughput absolute quantitative analysis of linezolid in human plasma by direct analysis in real time mass spectrometry without chromatographic separation and its application to a pharmacokinetic study.

Therapeutic drug monitoring (TDM) is necessary in the clinical management of linezolid to improve its efficacy and reduce the risk of time- and dose-dependent toxicity. A novel and ultrahigh-throughput analytical method for the determination of linezolid in human plasma was developed based on direct analysis in real-time tandem mass spectrometry (DART-MS/MS) without chromatographic separation. After solid-phase extraction with Waters Oasis HLB, the linezolid and internal standard linezolid-d3 were detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 338.1 → 296.2 and 341.2 → 297.3, respectively. The use of DART-MS obviates the need for chromatographic separation and allowed determination of linezolid in a total run time of only 24 s per sample. The method was linear in the concentration range 0.20-25 µg mL-1 with intraday and interday precision <14.5% and accuracy ranging from -3.85% to 12.7%. The method was successfully applied to a pharmacokinetic study of linezolid in healthy male volunteers after oral administration of a 600 mg tablet. DART-MS/MS provides a rapid and sensitive method for the determination of linezolid that does not require chromatographic separation. It is eminently suitable to meet the high-throughput challenge of clinical TDM. Graphical abstract.

Separation and simultaneous quantitation of PGF2α and its epimer 8-iso-PGF2α using modifier-assisted differential mobility spectrometry tandem mass spectrometry.

Because many therapeutic agents are contaminated by epimeric impurities or form epimers as a result of metabolism, analytical tools capable of determining epimers are increasingly in demand. This article is a proof-of-principle report of a novel DMS-MS/MS method to separate and simultaneously quantify epimers, taking PGF2α and its 8-epimer, 8-iso-PGF2α, as an example. Good accuracy and precision were achieved in the range of 10-500 ng/mL with a run time of only 1.5 min. Isopropanol as organic modifier facilitated a good combination of sensitivity and separation. The method is the first example of the quantitation of epimers without chromatographic separation.

Correction to: Beta2-Agonist Doping Control and Optical Isomer Challenges.

Page 1789, table 1, 'Carmoterol' row: The cell entry in the 'Stereochemistry' column, which previously read.

Differential mobility spectrometry followed by tandem mass spectrometry with multiple ion monitoring for bioanalysis of eptifibatide in rat plasma.

Eptifibatide is a therapeutic cyclic peptide with poor collision-induced dissociation (CID) efficiency for multiple reaction monitoring (MRM), which limits the development of a traditional liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioassay with MRM. In this study, a method combining differential mobility spectrometry (DMS) with liquid chromatography-multiple ion monitoring (LC-DMS-MIM) was developed for the quantitation of eptifibatide in rat plasma. After solid phase extraction (SPE) of 100 µL plasma on an Oasis® HLB cartridge, the analyte and I.S. (octreotide) were analyzed using a SCIEX QTRAP 6500 operated in the positive ion mode and preceded by a DMS device. The lower limit of quantitation (LLOQ) for eptifibatide was 0.5 ng/mL using only 100 µL plasma. The method was linear in the concentration range 0.5-300 ng/mL with good precision and accuracy. Compared to regulated quantitative LC-MS/MS bioanalysis of eptifibatide, the LC-DMS-MIM method effectively overcomes the sensitivity challenge in the LC-MRM method and reduces the high background noise and matrix interference in LC-MIM method without DMS. The method was successfully applied to a pharmacokinetic study involving intravenous injection of eptifibatide to Wistar rats.

Determination of rabeprazole enantiomers in dog plasma by supercritical fluid chromatography tandem mass spectrometry and its application to a pharmacokinetic study.

Rabeprazole is a novel benzimidazole proton pump inhibitor used for the treatment of gastrointestinal disorders. It is a chiral molecule that gives rise to the possibility of stereoselective pharmacokinetics. To investigate this phenomenon, a rapid and sensitive chiral assay based on supercritical fluid chromatography tandem mass spectrometry was developed and applied to the determination of (R)-rabeprazole and (S)-rabeprazole in dog plasma. Sample preparation involved protein precipitation with acetonitrile after the addition of (R)-lansoprazole as internal standard. Baseline separation of enantiomers in 4.5 min was achieved on an Acquity UPC2 system using an ACQUITY UPC2 Trefoil CEL2 column maintained at 60°C and a mobile phase consisting of methanol/CO2 (30:70, v/v) delivered at 2.5 mL/min. Detection was achieved by multiple reaction monitoring of the transitions at m/z 360.0→242.2 (rabeprazole) and 370.3→252.0 (internal standard) in the positive ion mode. The assay was linear in the range of 1-1000 ng/mL and free of matrix effects. Intra- and interday precisions were less than 10.0% with accuracy in the range of -2.6 to 3.1%. The method was successfully applied to a pharmacokinetic study of rabeprazole enantiomers after administration of a single oral dose of 10 mg racemate to beagle dogs.

Beta2-Agonist Doping Control and Optical Isomer Challenges.

The World Anti-Doping Agency (WADA) currently allows therapeutic use of the beta2-agonists salbutamol, formoterol and salmeterol when delivered via inhalation despite some evidence suggesting these anti-asthma drugs may be performance enhancing. Beta2-agonists are usually administered as 50:50 racemic mixtures of two enantiomers (non-superimposable mirror images), one of which demonstrates significant beta2-adrenoceptor-mediated bronchodilation while the other appears to have little or no pharmacological activity. For salbutamol and formoterol, urine thresholds have been adopted to limit supratherapeutic dosing and to discriminate between inhaled (permitted) and oral (prohibited) use. However, chiral switches have led to the availability of enantiopure (active enantiomer only) preparations of salbutamol and formoterol, which effectively doubles their urine thresholds and provides a means for athletes to take supratherapeutic doses for doping purposes. Given the availability of these enantiopure beta2-agonists, the analysis of these drugs using enantioselective assays should now become routine. For salmeterol, there is currently only a therapeutic dose threshold and adoption of a urinary threshold should be a high priority for doping control.

Application of a high-throughput, parallel HPLC system for quantitative chiral analysis of pantoprazole.

BACKGROUND: Chromatographic separation of enantiomers is considered a task in analytical chemistry particularly for high sample throughput. This paper describes a high-throughput parallel HPLC-MS/MS method for the determination of pantoprazole enantiomers. RESULTS: Baseline separation of pantoprazole enantiomers was achieved on a Chiralcel OZ-RH column in a run time of 4.5 min. Assays for enantiomers were linear with satisfactory intra- and inter-day precision and accuracy. The assay was suitable for high-throughput analysis as shown by its successful application to a chiral PK study in beagle dog. CONCLUSION: A high-throughput parallel HPLC-MS/MS assay for pantoprazole has been developed and validated. This method provides nearly twofold increased sample throughput, and was shown to be suitable for application in PK studies.

Simultaneous determination of carboprost methylate and its active metabolite carboprost in dog plasma by liquid chromatography-tandem mass spectrometry with positive/negative ion-switching electrospray ionization and its application to a pharmacokinetic study.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method using positive/negative electrospray ionization (ESI) switching for the simultaneous quantitation of carboprost methylate and carboprost in dog plasma has been developed and validated. After screening, the esterase inhibitor, dichlorvos was added to the whole blood at a ratio of 1:99 (v/v) to stabilize carboprost methylate during blood collection, sample storage and LLE. Indomethacin was added to plasma to inhibit prostaglandins synthesis after sampling. After liquid-liquid extraction of 500µL plasma with ethyl ether-dichloromethane (75:25, v/v), analytes and internal standard (IS), alprostadil-d4, were chromatographed on a CAPCELL PAK Phenyl column (150×2.0mm, 5µm) using acetonitrile-5mM ammonium acetate as mobile phase. Carboprost methylate was detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 400.5→329.3; the carboprost and IS were detected by negative ion electrospray ionization followed by MRM of the transitions at m/z 367.2→323.2, and 357.1→321.2, respectively. The method was linear for both analytes in the concentration range 0.05-30ng/mL with intra- and inter-day precisions (as relative standard deviation) of ≤6.75% and accuracy (as relative error) of ≤7.21% and limit of detection (LOD) values were 10 and 20pg/mL, respectively. The method was successfully applied to a pharmacokinetic study of the analytes in beagle dogs after intravaginal administration of a suppository containing 0.5mg carboprost methylate.

Determination of thymopentin in beagle dog blood by liquid chromatography with tandem mass spectrometry and its application to a preclinical pharmacokinetic study.

The pentapeptide thymopentin (Arg-Lys-Asp-Val-Tyr, RKDVY) corresponds to amino acids 32-36 of the 49 amino acid immunomodulatory polypeptide, thymopoietin, whose biological activity is partially reproduced. Thymopentin is widely used in the clinic and represents a promising target for drug design but bioanalytical and pharmacokinetic data are limited due to its enzymatic instability. This paper reports a rapid and sensitive method based on liquid chromatography with tandem mass spectrometry for the determination of thymopentin in beagle dog blood. To inactivate peptidases and stabilize thymopentin, acetonitrile was added to blood samples immediately after collection followed by addition of stable isotope-labeled thymopentin as internal standard and washing with dichloromethane. Chromatography was carried out on an Ascentis Express Peptide ES-C18 column using gradient elution with methanol and aqueous 0.1% formic acid at a flow rate of 0.6 mL/min. Positive electrospray ionization mass spectrometry with selected reaction monitoring achieved linearity in the range of 1.5-800 ng/mL with good accuracy/precision and minimal matrix effects. The method was successfully applied to a pharmacokinetic study in beagle dogs after intravenous administration of 0.2 mg/kg thymopentin.

Kinetic and equilibrium studies of bile salt-liposome interactions.

Research has suggested that exposure to sub-micellar concentrations of bile salts (BS) increases the permeability of lipid bilayers in a time-dependent manner. In this study, incubation of soy phosphatidylcholine small unilamellar vesicles (liposomes) with sub-micellar concentrations of cholate (C), deoxycholate (DC), 12-monoketocholate (MKC) or taurocholate (TC) in pH 7.2 buffer increased membrane fluidity and negative zeta potential in the order of increasing BS liposome-pH 7.2 buffer distribution coefficients (MKC < C ≈ TC < DC). In liposomes labeled with the dithionite-sensitive fluorescent lipid N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE) in both leaflets and equilibrated with sub-micellar concentrations of BS, fluorescence decline during continuous exposure to dithionite was biphasic involving a rapid initial phase followed by a slower second phase. Membrane permeability to dithionite as measured by the rate of the second phase increased in the order control < MKC < TC ∼ C < DC. In liposomes labeled with NBD-PE in the inner leaflet only and incubated with the same concentrations of C, DC and MKC, membrane permeability to dithionite initially increased very rapidly in the order MKC < C < DC before impermeability to dithionite was restored after which fluorescence decline was consistent with NBD-PE flip-flop. For liposomes incubated with TC, membrane permeability to dithionite was only slightly increased and the decline in fluorescence was mainly the result of NBD-PE flip-flop. These results provide evidence that BS interact with lipid bilayers in a time-dependent manner that is different for conjugated and unconjugated BS. MKC appears to cause least disturbance to liposomal membranes but, when the actual MKC concentration in liposomes is taken into account, MKC is actually the most disruptive.

Determination of a deuterohemin-peptide conjugate in rat plasma by liquid chromatography-tandem mass spectrometry and application to a preclinical pharmacokinetic study.

The deuterohemin-peptide conjugate (DhHP-6) is a microperoxidase mimetic, which has demonstrated substantial benefits in vivo as a scavenger of reactive oxygen species. This paper reports the development of a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of DhHP-6 in rat plasma using triptorelin as an internal standard (IS). 50µL plasma was used in sample preparation, and a simple protein precipitation procedure with acetonitrile was involved. Satisfactory peak shapes of analyte and IS were obtained on an Agilent HC-C18 column by using a gradient elution with 10mM ammonium acetate-0.5% formic acid (v:v) and acetonitrile, there was no significant interference impacting the determination. A calibration curve obtained from this method was linear within the concentration range 10-3000ng/mL with intra- and inter-day precisions of 4.2-6.8% and 3.2-8.9%, respectively and accuracy of -1.3% to 2.1%. The recovery was above 80% with low matrix effects. The method was successfully applied to support a preclinical pharmacokinetic study in rat.

A rapid and sensitive UFLC-MS/MS method for the simultaneous determination of gentiopicroside and swertiamarin in rat plasma and its application in pharmacokinetics.

OBJECTIVES: Radix Gentianae is a traditional Chinese medicine derived from medicinal plants of the family Gentianaceae. Its pharmacological effects have been primarily attributed to the presence of a number of secoiridoid glycosides, in particular gentiopicroside and swertiamarin. In this study, a rapid and sensitive method based on ultrafast liquid chromatography-tandem mass spectrometry has been developed for the simultaneous determination of gentiopicroside and swertiamarin in rat plasma using paeoniflorin as internal standard (IS). METHODS: After liquid-liquid extraction with ethyl acetate-isopropanol (95 : 5, v/v), separation was achieved on a Shim-pack XR-ODS C18 column (75 mm × 3.0 mm, 2.2 µm) with a mobile phase consisting of methanol : 0.1% formic acid (30 : 70, v/v) at a flow rate of 0.4 ml/min. Detection on an API 3200 QTRAP mass spectrometer equipped with an electrospray ionization source operated in the negative ionization mode was performed by multiple reaction monitoring of the precursor-to-product ion transitions of gentiopicroside, swertiamarin and IS at m/z 401.0 → 179.0, 419.0 → 179.1 and 525.1 → 121.0 respectively. The calibration curves were linear over the concentration range of 20-10 000 and 2-1000 ng/ml for gentiopicroside and swertiamarin with corresponding lower limits of quantification of 20 and 2 ng/ml. The limits of detection were 4 and 0.5 ng/ml for gentiopicroside and swertiamarin, respectively. The intraday and interday precisions were below 11.9% for gentiopicroside and below 9.5% for swertiamarin in terms of relative standard deviation, and the accuracy was within ±8.3% for gentiopicroside and within ±10.2% for swertiamarin in terms of relative error. Extraction recovery, matrix effect and stability were satisfactory in rat plasma. The method was fully validated and applied to a pharmacokinetic study involving oral administration of a Radix Gentianae extract to groups of male and female rats. KEY FINDINGS: Results showed that in female rats, both compounds were absorbed to a greater extent and eliminated more slowly than in male rats, although the rate of absorption was similar in the two groups. CONCLUSIONS: There were remarkable differences in pharmacokinetic properties of gentiopicroside and swertiamarin between male and female rats. The results will provide helpful information for the development of suitable dosage forms and clinical references on rational administration.

Pharmacokinetic study of calenduloside E and its active metabolite oleanolic acid in beagle dog using liquid chromatography-tandem mass spectrometry.

Aralia mandshrica is a well-known traditional Chinese medicine from Northeast China commonly used to treat digestive, circulatory and immune system disorders. Calenduloside E is one of its bioactive components currently under evaluation as a pure drug. In this study, a highly sensitive and rapid method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous quantitation of calenduloside E and its active metabolite oleanolic acid in beagle dog plasma has been developed and validated. Samples containing the ammonium salt of simvastatin acid as internal standard (IS) were purified by solid phase extraction and separated on a SUPELCO Ascentis-C18 column (50mm×4.6mm i.d., 5µm) using gradient elution with 0.35% formic acid and acetonitrile. Analytes and IS were detected in a cycle time of 5min after ionization in the negative ion mode by multiple reaction monitoring of the precursor-to-product ion transitions at m/z 631.4→455.4 and m/z 435.4→319.0 for calenduloside E and IS respectively and by single ion monitoring of the ion at m/z 455.4 for oleanolic acid. The method was linear over the concentration range 0.4-100ng/mL for both analytes using 0.5mL plasma. Inter- and intra-day precisions were both <6.96% with accuracies <6.40%. In the pharmacokinetic (PK) study, beagle dogs were given oral doses of calenduloside E (1.05, 2.10 and 4.20mg/kg) and an intravenous injection of 2.10mg/kg. The absolute bioavailability of calenduloside E was only 0.58%. Area under the plasma concentration time curve (AUC(0-t)) for the oral doses of calenduloside E was approximately dose proportional while other PK parameters (t1/2, Tmax and MRT) showed no significant differences among the three doses (P>0.05). The PK data provide a useful platform on which to base future clinical studies of calenduloside E.

Phenolic esters of O-desmethylvenlafaxine with improved oral bioavailability and brain uptake.

O-Desmethylvenlafaxine (desvenlafaxine, ODV) is a recently approved antidepressant which in some clinical studies failed to meet a satisfactory end-point. The aim of this study was to prepare a series of phenolic esters of ODV and evaluate their potential as ODV prodrugs with improved brain uptake. Fifteen phenolic esters (compounds 1a-o) were synthesized and their pharmacokinetic profiles evaluated in rat. The four compounds producing the highest relative bioavailability of ODV in rat (compounds 1c, 1e, 1n, 1o) were then studied to evaluate their brain uptake. Of these four compounds, compound 1n (the piperonylic acid ester of ODV) demonstrated the highest C(max) of ODV both in the rat hypothalamus and total brain. Finally the pharmacokinetics of 1n were evaluated in beagle dog where the increase in relative bioavailability of ODV was found to be as great as in rat. This high relative bioavailability of ODV coupled with its good brain penetration make 1n the most promising candidate for development as an ODV prodrug.

A liquid chromatography-tandem mass spectrometric method for the simultaneous quantitation of five components of Ixeris sonchifoliain (Bge.) Hance in rat plasma and its application to a pharmacokinetic study.

A rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantitation of five major active ingredients of Ixeris sonchifolia (Bge.) Hance in rat plasma has been developed and validated. After liquid-liquid extraction of 50µL plasma with ethyl acetate, analytes and internal standard (I.S.), astilbin, were chromatographed on a Zorbax SB-C18 column (150mm×4.6mm, 5µm) using acetonitrile - 10mM ammonium acetate (60:40, v/v, pH 5.6) as mobile phase. The five analytes: chicoric acid, luteolin 7-O-ß-d-glucuronide, luteolin 7-O-ß-d-glucopyranoside, luteolin 7-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside, apigenin 7-O-ß-d-glucuronide and I.S., were detected by negative ion electrospray ionization followed by multiple reaction monitoring of the ions with m/z 473.0→311.0, 461.0→285.0, 447.0→285.0, 609.1→285.0, 445.1→269.0 and 449.1→150.9, respectively. The method was linear for all analytes in the concentration range 10-3000ng/mL with intra- and inter-day precision (as relative standard deviation) ≤8.99% and accuracy (as relative error) ≤4.00%. The limits of detection (LOD) were 5, 1, 5, 5, 2ng/mL for chicoric acid, luteolin 7-O-ß-d-glucuronide, luteolin 7-O-ß-d-glucopyranoside, luteolin 7-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside, apigenin 7-O-ß-d-glucuronide, respectively. The method was successfully applied to a pharmacokinetic study of the five analytes in rat after a single intravenous dose of Kudiezi Injection.

Effect of 12-oxochenodeoxycholate on the pharmacokinetics and pharmacodynamics of morphine 6-glucuronide in Wistar rats.

OBJECTIVES: The semi-synthetic bile salt, 12-oxochenodeoxycholate (OCDC also known as 12-monoketocholate), has been shown to enhance drug permeation across biological membranes with low cytotoxicity. Its effect on the analgesic potency and brain concentration of morphine 6-glucuronide (M6G) was studied in male Wistar rats. METHODS: Four groups of animals (n = 8) were given 5, 10 or 20 mg/kg OCDC or normal saline (control) by subcutaneous injection 30 min before a subcutaneous injection of 5 mg/kg M6G after which the hotplate test was performed on each rat at various times. After a 2 week wash-out period, the same rats (n = 30) were randomized to two equal groups and given OCDC (20 mg/kg) or normal saline 30 min before 5 mg/kg M6G. At five time points up to 3 h after M6G administration, three rats from each group were euthanized and blood and brain analyzed for M6G. KEY FINDINGS: The area under the analgesic effect versus time curve (AUAE) was found to be significantly (P < 0.05) greater in rats given 20 mg/kg OCDC than in control rats. Area under the curve (AUC) for M6G in both plasma and brain was greater in OCDC-treated rats than in control rats, but the brain : plasma AUC ratio was lower. CONCLUSIONS: OCDC enhances the analgesic effect of M6G but gives a lower brain : plasma ratio due to increasing M6G plasma levels probably by reducing its renal clearance.