Li-Heng Pao

High-performance liquid chromatographic method for the simultaneous determination of nalbuphine and its prodrug, sebacoyl dinalbuphine ester, in dog plasma and application to pharmacokinetic studies in dogs

For the determination of nalbuphine and its long acting prodrug, sebacoyl dinalbuphine ester (SDN), in biological samples, a reversed-phase high-performance liquid chromatographic method using dual detectors was established. Ultraviolet and fluorescence detectors were connected in series for determining SDN and nalbuphine, respectively. The two analytes and internal standard were extracted from plasma by alkaline liquid-liquid extraction using n-hexane-isoamyl alcohol (9:1, v/v). The calibration curve for nalbuphine was linear over the range from 10 to 2,500 ng/ml, while the range was 25 to 2,500 ng/ml for SDN. The within- and between-day precision and accuracy were all within 10% for both nalbuphine and SDN over these concentrations. The method was applied successfully to a pharmacokinetic study of SDN administered at 20 mg/kg to two beagle dogs. Pharmacokinetic analysis revealed that SDN followed a linear one-compartment model with an elimination half-life of 74.7 min. Formation of nalbuphine after intravenous administration of SDN was observed in the first time point sample (5 min). These results indicate that SDN is rapidly metabolized to its active moiety, nalbuphine, in dogs and no other metabolites are detected in plasma.

Validated LC -MS-MS Method for the Determination of Quetiapine in Human Plasma: Application to a Pharmacokinetic Study

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the determination of quetiapine was developed and validated over the linearity range 1-1500 ng/mL with 0.1 mL of plasma using clozapine as the internal standard. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive electrospray ionization and quantification was performed by selected reaction monitoring mode. The MS-MS ion transitions monitored were m/z 384.1 → 253.1 and 327.0 → 270.0 for quetiapine and clozapine, respectively. The between- and within-run precision was less than 7.44% and accuracy was less than 10.2%. The lower limit of quantification was 1 ng/mL. The extraction recoveries of quetiapine were over 90%. The method is proved to be accurate and specific, and was applied to the pharmacokinetic study in healthy Chinese volunteers.

Quantitative rat liver function test by galactose single point method

Summary The purpose of this study was to investigate the galactose single point (GSP) method, a residual liver function test recently recommended by the US Food and Drug Administration, which can be a useful tool for rat liver function measurement. Rats were treated either with carbon tetrachloride (CCl4) alone (1 mL/kg, intraperitoneally [i.p.]) for one day or with isoniazid (INH) alone (150 mg/kg, i.p.) or (in order to ameliorate the effects of INH) with a combination of INH and bis-p-nitrophenyl phosphate (BNPP) (25 mg/kg, i.p.) for 21 days. Hepatotoxicity was assayed by plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and scores of histological activity index-necroinflammation (HAI-NI) of the respective liver specimens. The GSP method in rats was defined by the galactose blood level after 60 min. Significant differences in GSP values were observed between controls and the CCl4-treated rats. After 21 days of treatment, no significant changes in AST and ALT values were observed among the control, INH and INH-BNPP groups. There were significant differences in average GSP values for controls (P < 0.001) and INH-BNPP (P < 0.001) compared with INH alone. Highly significant correlations (P < 0.001) were obtained between GSP and scores of HAI-NI for all the groups. GSP was concluded to be a more sensitive biomarker of INH-induced hepatotoxicity than AST or ALT in the rats. The GSP method has been proved to be a simple and useful tool for the quantitative determination of liver function in rats, which can possibly be extended to other animals.

Simultaneous determination of triazolam and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of triazolam and its metabolites, alpha-hydroxytriazolam (alpha-OHTRZ) and 4-hydroxytriazolam (4-OHTRZ), was developed and validated. Triazolam-D4 was used as the internal standard (IS). This analysis was carried out on a Thermo C(18) column and the mobile phase was composed of acetonitrile:H(2)O:formic acid (35:65:0.2, v/v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) and quantification was performed by multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 343.1-->308.3, 359.0-->308.3, 359.0-->111.2 and 347.0-->312.0 for triazolam, alpha-OHTRZ, 4-OHTRZ and triazolam-D4, respectively. LLOQ of the analytical method was 0.05 ng/mL for triazolam and 0.1 ng/mL for alpha-OHTRZ and 4-OHTRZ. The within- and between-run precisions were less than 15.26% and accuracy was -8.08% to 13.33%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of triazolam in healthy Chinese volunteers.

Validated liquid chromatography–tandem mass spectrometry method for determination of totally nine probe metabolites of cytochrome P450 enzymes and UDP-glucuronosyltransferases

A simplified, rapid, and selective liquid chromatography-tandem mass spectrometry method for the determination of the activities of cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGTs) in two separate settings was developed and successfully applied to 8 CYP isoenzymes and UGT2B7 enzyme activities in rat liver microsomes. The triple-quadrupole mass spectrometric detection was operated in positive mode for the probe metabolites: CYP1A2 (resorufin), CYP2B6 (hydroxybupropion), CYP2C19 (5-hydroxyomeprazole), CYP2D6 (dextrophan), CYP3A4 (6β-hydroxytestosterone), and UGT2B7 (morphine-3-glucuronide); also in negative mode for CYP2C9 (4-hydroxytolbutamide), CYP2E1 (6-hydroxychloroxazone), and CYP4A (hydroxylauric acid). The metabolic reactions were terminated with acetonitrile, containing metoprolol and acetaminophen as the internal standard for positive and negative ion electrospray ionization, respectively. The method was validated over the concentration range of 25-2500 ng/mL for 5-hydroxyomeprazole, dextrophan, hydroxylauric acid, and morphine-3-glucuronide; 5-500 ng/mL for resorufin; 3-300 ng/mL for hydroxybupropion; 10-1000 ng/mL for 4-hydroxytolbutamide; 40-4000 ng/mL for 6-hydroxychloroxazone; and 63-6300 ng/mL for 6β-hydroxytestosterone. All of the extraction recoveries of these analytes were greater than 85%, except for hydroxylauric acid at mid-concentration with a recovery of 83.2% ± 3.2%. The matrix effects were between 85.8% and 119.9%; the respective within- and between-run precisions were 0.9-12.0% and 2.0-13.9%; and the within- and between-run accuracy levels were 0.6-17.2% and 0.1-15.1%, respectively, for all these analytes. All of the analytes were stable during the assay and storage in the liver microsomes of Sprague-Dawley rats. The measurement activity of multiple enzymes was feasible using a cocktail approach. This method proved to be a robust, fast, accurate, specific and sensitive assay, and was successfully used to investigate in vivo enzyme activities of 8 major CYP isoenzymes and UGT2B7 in Sprague-Dawley rats with fatty livers. By the end of the eighth week, the CD-fed induced fatty liver rats showed a significant decrease in the activities of CYP1A2 and UGT2B7 as compared to the standard diet group.

HPLC ASSAY FOR BASIC AMINE DRUG IN PLASMA USING A SILICA GEL COLUMN AND AN AQUEOUS MOBILE PHASE -- APPLICATION IN A PILOT BIOAVAILABILITY STUDY OF CHLORPHENIRAMINE CONTROLLED-RELEASE DOSAGE FORM

A high performance liquid chromatographic (HPLC) method that involves the use of a silica gel column and an aqueous mobile phase for simultaneous separation of chlorpheniramine, pseudoephedrine and terfenadine in plasma is presented. Alkalized samples are cleaned by extraction with n-hexane, and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by a HPLC system with a silica gel column, an aqueous mobile phase containing 55% CH3CN and 45% (NH4)H2PO4(pH 4.0), and UV detection at 210 nm. The low detection limits of the method in plasma are 1 ng, 4 ng and 0.5 ng for chlorpheniramine, pseudoephedrine and terfenadine, respectively. In this study, terfenadine acts as an internal standard. The coefficient of variance on the results of intraday and interday precision and the accuracy on control samples of chlorpheniramine and pseudoephedrine were all within 10%. We have used this method successfully in a ...

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry.

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C18 column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple-quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2-->120.2 and 152.1-->110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1-500 microg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.

Stability of 11-Nor-delta-9-Tetrahydrocan nabinol-9-carboxylic Acid from Urine Samples Stored in Polypropylene, Polyethylene and Polystyrene Containers at Different Temperatures

The stability of 11-Nor-9-delta-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) from urine specimens stored in polypropylene (PP), polyethylene (PE) and polystyrene (PS) containers was examined. Urinesamples containing 100μg/m L TI-ICCOOH were stored at -20°C, 4°C and 25°C up to 180 days. The samples were analyzed at 0,5, 10, 20, 30, 60, 90, 105, 135, 150 and 180 days, and alteration of the analyte pattern during storage were determined by a validated liquid chromatography-tandem mass (LC-MS/MS). The concentration of THCCOOH significantly decreased to approximately 20% of its initial concentration within 5 days in all three containers at all temperature conditions. All loses of THCCOOH were relatively stabilized after 5 days. The results indicated that THCCOOH was preferred to store in PP and PE containers at 4 °C.

Simultaneous Determination of Ketamine, Norketamine and 6-Monoacetlymorphine in Urine Specimens by Liquid Chromatography-Tandem Mass Spectrometry: Application to a Stability Test of Various Materials

Aliquid chromatography-tandem spectrometry (LC-MS/MS) method to determine ketamine, norketamine and 6-monoacetylmorphine (6-MAM) in urine was developed. Ketamine-d4 was used as the internal standard. The study was performed by using a C18 column. The mobile phase consisted of 20mMammonium formate in H2O (pH 4.3, adjusted with formic acid) and ACN containing 0.1% formic acid. The gradient started at 10% organic phase and changed linearly to 85% organic phase in 2 min, after being maintained at 85% organic phase for 2 min, it then returned to the initial condition in 5 min. Ionization was conducted in the positive mode and quantification was performed by multiple reaction monitoring (MRM) mode. The within- and between-run precisions of the three analytes were less than 14.3% and that of accuracies were -7% to 12%. The retention time was 3.34, 3.28, 2.86 and 3.36 min for ketmaine, norketmaine, 6-MAM and ketamined4, respectively. The analyticalmethod was applied to a stability study of the effect of different materials of containers, polypropylene (PP), polyethylene (PE) and polystyrene (PS), on ketmaine, norketmaine, and 6-MAM at ambient temperature. The result indicated that only 6-MAM would decrease to 50% of its original concentration within 30 days in the three containers.