Simple high-performance liquid chromatographic determination of buspirone in human plasma

A simple, rapid and sensitive high-performance liquid chromatographic method for the determination of buspirone in plasma was developed. Buspirone was isolated from plasma using protein precipitation by acetonitrile and the recovery was complete. Citalopram was used as internal standard. The chromatographic conditions were as follows: analytical 125 x 4 mm, i.d. Nucleosil C18 column (5 microm particle size), mobile phase consisting of sodium dihydrogen phosphate buffer/acetonitrile (60:40, v/v) adjusted to pH 5.5 at a flow rate of 1.0 ml min(-1), UV detection at 235 nm. The quantification limit for buspirone in plasma was 0.5 ng ml(-1).The calibration curve was linear over the concentration range 0.5-10 ng ml(-1). The inter- and intra-day assay coefficients of variation were found to be less than 8%. The present validated method was successfully used for bioequivalence studies of buspirone in human subjects.     

Liquid chromatographic-electrospray ionization mass spectrometric quantitative analysis of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in rat plasma

A liquid chromatographic-mass spectrometric method with electrospray ionization is presented for the simultaneous determination of buprenorphine, nordiazepam and their pharmacologically active metabolites, norbuprenorphine and oxazepam, in rat plasma. The drugs were extracted from plasma by liquid-liquid extraction and chromatographically separated using a gradient elution of aqueous ammonium formate and acetonitrile. Following electrospray ionization, the analytes were quantified in the single ion storage mode. The assay was validated according to current acceptance criteria for bioanalytical method validation. It was proved to be linear from 0.7 to 200 ng/ml plasma for buprenorphine, 1.0 to 200 ng/ml for norbuprenorphine, 2.0 to 200 ng/ml for nordiazepam, and from 5.0 to 200 ng/ml for oxazepam. The average recoveries of buprenorphine, norbuprenorphine, nordiazepam and oxazepam were 89, 39, 88 and 82%, respectively, with average coefficients of variation ranging from 1.8 to 14.3%. The limits of quantitation for these drugs were 0.7, 1.0, 2.0 and 5.0 ng/ml, respectively, with associated precisions within 17% and accuracies within +/-18% of the nominal values. Both the intra- and inter-assay precision values did not exceed 11.3% for the four analytes. Intra- and inter-assay accuracies lay within +/-15% of the nominal values. The validated method was applied to the determination of buprenorphine, norbuprenorphine, nordiazepam and oxazepam in plasma samples collected from rats at various times after intravenous administration of buprenorphine and nordiazepam.     

Determination of rabeprazole enantiomers and their metabolites by high-performance liquid chromatography with solid-phase extraction

Here, we describe the development of a rapid, simple and sensitive high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of rabeprazole enantiomers (1a,b) and their metabolites, rabeprazole-thioether (2) and rabeprazole sulfone (3), in human plasma. Analytes and the internal standard (omeprazole-thioether) were separated using a mobile phase of 0.5 M NaClO4-acetonitrile (6:4, v/v) over a Chiral CD-Ph column. Analysis required only 100 microl of plasma and involved solid-phase extraction with an Oasis HLB cartridge, which gave high recovery (>91.8%) with good selectivity for all analytes. The lower limit of quantification was 5 ng/ml for analytes 1a, 1b and 3 and 10 ng/ml for 2. Linearity of this assay was determined to lie between 5 and 1000 ng/ml for 1a, 1b and 3 and 10 and 1000 ng/ml for 2 (r2>0.982 of the regression line). Inter- and intra-day coefficients of variation were less than 7.8% and accuracies were within 8.4% over the linear range for all analytes. Our results indicate that this method is applicable to the simultaneous monitoring of plasma levels of rabeprazole enantiomers and associated metabolites in human plasma.     

Determination of depleted uranium, pyridostigmine bromide and its metabolite in plasma and urine following combined administration in rats

A simple and reliable method was developed for the quantification of depleted uranium, the anti nerve agent drug pyridostigmine bromide (PB;3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) and its metabolite N-methyl-3-hydroxypyridinium bromide in rat plasma and urine. The method involved using solid phase extraction and spectrophotometric determination of uranium, and high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 280 nm for PB and its metabolite. Uranium was derivatized using dibenzoylmethane (DBM) then the absorbance was measured at 405 nm. PB and its metabolite were separated using a gradient of 1--40% acetonitrile in 0.1% triflouroacetic acid water solution (pH 3.2) at a flow rate of 0.8 ml/min in a period of 14 min. Limits of detection were 2 ng/ml for uranium and 50 ng/ml for PB and its metabolite. Limits of quantitation were between 10 and 100 ng/ml for uranium and the other two analytes, respectively. Average percentage recovery of five spiked plasma samples were 83.7+/-8.6, 76.8+/-6.7, 79.1+/-7.1, and from urine 82.7+/-8.6, 79.3+/-9.5 and 78.0+/-6.2, for depleted uranium, PB and N-methyl-3-hydroxypyridinium bromide, respectively. The relationship between peak areas and concentration was linear for standards between 100 and 1000 ng/ml for all three analytes. This method was applied to analyze the above chemicals and metabolites following combined administration in rats.     

Determination of fexofenadine enantiomers in human plasma with high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatography (HPLC) method was developed as an assay for fexofenadine enantiomers in human plasma. Fexofenadine enantiomers were separated using a mobile phase of 0.5% KH(2)PO(4)-acetonitrile (65:35, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 ml/min and measurement at 220 nm. Analysis required 400 microl of plasma and involved solid-phase extraction with an Oasis HLB cartridge, which gave recoveries for both enantiomers from 67.4 to 71.8%. The lower limit of quantification was 25 ng/ml for (R)- and (S)-fexofenadine. The linear range of this assay was between 25 and 625 ng/ml (regression line r(2)>0.993). Inter- and intra-day coefficients of variation were less than 13.6% and accuracies were within 8.8% over the linear range for both analytes. This method can be applied effectively to measure fexofenadine enantiomer concentrations in clinical samples.     

Evaluation of Mutual Drug–Drug Interaction within Geneva Cocktail for Cytochrome P450 Phenotyping using Innovative Dried Blood Sampling Method

Cytochrome P450 (CYP) activity can be assessed using a ‘cocktail’ phenotyping approach. Recently, we have developed a cocktail (Geneva cocktail) which combines the use of low‐dose probes with a low‐invasiveness dried blood spots (DBS) sampling technique and a single analytical method for the phenotyping of six major CYP isoforms. We have previously demonstrated that modulation of CYP activity after pre‐treatment with CYP inhibitors/inducer could be reliably predicted using Geneva cocktail. To further validate this cocktail, in this study, we have verified whether probe drugs contained in the latter cause mutual drug–drug interactions. In a randomized, four‐way, Latin‐square crossover study, 30 healthy volunteers received low‐dose caffeine, flurbiprofen, omeprazole, dextromethorphan and midazolam (a previously validated combination with no mutual drug–drug interactions); fexofenadine alone; bupropion alone; or all seven drugs simultaneously (Geneva cocktail). Pharmacokinetic profiles of the probe drugs and their metabolites were determined in DBS samples using both conventional micropipette sampling and new microfluidic device allowing for self‐sampling. The 90% confidence intervals for the geometric mean ratios of AUC metabolite/AUC probe for CYP probes administered alone or within Geneva cocktail fell within the 0.8–1.25 bioequivalence range indicating the absence of pharmacokinetic interaction. The same result was observed for the chosen phenotyping indices, that is metabolic ratios at 2 hr (CYP1A2, CYP3A) or 3 hr (CYP2B6, CYP2C9, CYP2C19, CYP2D6) post‐cocktail administration. DBS sampling could successfully be performed using a new microfluidic device. In conclusion, Geneva cocktail combined with an innovative DBS sampling device can be used routinely as a test for simultaneous CYP phenotyping.     

Simultaneous determination of albendazole metabolites, praziquantel and its metabolite in plasma by high-performance liquid chromatography-electrospray mass spectrometry

The analysis of albendazole sulfoxide, albendazole sulfone, praziquantel and trans-4-hydroxypraziquantel in plasma was carried out by high-performance liquid chromatography-mass spectrometry ((LC-MS-MS). The plasma samples were prepared by liquid-liquid extraction using dichloromethane as extracting solvent. The partial HPLC resolution of drug and metabolites was obtained using a cyanopropyl column and a mobile phase consisting of methanol:water (3:7, v/v) plus 0.5% of acetic acid, at a flow rate of 1.0 mL/min. Multi reaction monitoring detection was performed by electrospray ionization in the positive ion mode, conferring additional selectivity to the method. Method validation showed relative standard deviation (precision) and relative errors (accuracy) lower than 15% for all analytes evaluated. The quantification limit was 5 ng/mL and the linear range was 5-2500 ng/mL for all analytes. The method was used for the determination of drug and metabolites in swine plasma samples and proved to be suitable for pharmacokinetic studies.     

Determination of omeprazole, hydroxyomeprazole and omeprazole sulfone using automated solid phase extraction and micellar electrokinetic capillary chromatography

A sensitive method for the determination of omeprazole and its metabolites has been developed. It involves an automated solid phase extraction (SPE) procedure and capillary electrophoresis with UV detection. Omeprazole, hydroxyomeprazole and omeprazole sulfone could be separated by micellar electrokinetic capillary chromatography using a background electrolyte composed of 20 mM borate buffer and 30 mM sodium dodecyl sulfate, pH 9.5. The isolation of omeprazole and its metabolites from plasma was automatically accomplished with an original SPE procedure using surface-modified styrene-divinylbenzene polymer cartridges. Good recovery data and satisfactory precision values were obtained. Responses were linear for the three analytes, from 0.08 to 2.0 microg/mL of plasma. Intra- and inter-day precision values of about 1.6% R.S.D. (n=10) and 2.5% R.S.D. (n=36), respectively, were obtained. The method is highly robust and no breakdown of the current or capillary blockages were observed during several weeks of operation. The validated method was applied to the determination of omeprazole in pharmaceutical preparations and for the analysis of plasma samples obtained from three volunteers who received oral doses of omeprazole.     

Advanced method for determination of omeprazole in plasma by HPLC

An advanced and sensitive high-performance liquid chromatographic (HPLC) method for determination of omeprazole in human plasma has been developed. After omeprazole was extracted from plasma with diethylether, the organic phase was transferred to another tube and trapped back with 0.1 N NaOH solution. The alkaline aqueous layer was injected into a reversed-phase C8 column. Lansoprazole was used as an internal standard. The mobile phase consisted of 30% of acetonitrile and 70% of 0.2 M KH2P04, pH 7.0. Recoveries of the analytes and internal standard were >75.48%. The coefficients of variation of intra- and inter-day assay were <5.78 and 4.59% for plasma samples. The detection limit in plasma was 2 ng/ml. The clinical applicability of this assay method was evaluated by determining plasma concentration-time courses of the respective analytes in 24 healthy volunteers after oral administration 40 mg of omeprazole. The present assay is considered to be simple, accurate, economical and suitable for the study of the kinetic disposition of omeprazole in the body.     

The quantitative determination of cilostazol and its four metabolites in human liver microsomal incubation mixtures by high-performance liquid chromatography

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150 x 4.6 mm, 5 microm) column and a Cosmil C-18 column (150 x 4.6 mm, 5 microm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile in acetate buffer (pH = 6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43-17.59. The components of liver microsomal incubation mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml(-1) for cilostazol and 100 ng ml(-1) for each of the metabolites. This method has been validated for linear ranges of 100-4000 ng ml(-1) for OPC-13213, OPC-13217 and OPC-13326; 100-2000 ng ml(-1) for OPC-13015; and 1000-20000 ng ml(-1) for cilostazol. The percent relative recovery of this method was established to be 81.2-101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8-7.7%. The autosampler stability of the analytes was evaluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, accurate and reproducible.     

Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

OBJECTIVE: This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. METHODS: Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. RESULTS: The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n = 8), or CYP2C19*17*2 (n = 1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n = 9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. CONCLUSIONS: Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R = -0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.     

Determination of fexofenadine in human plasma using 96-well solid phase extraction and HPLC with tandem mass spectrometric detection

A fast and sensitive HPLC-MS/MS method, utilizing atmospheric pressure chemical ionization, for the determination of fexofenadine in human plasma is described. A deuterated analog, d6-fexofenadine is used as the internal standard (IS). Plasma samples are prepared using 96-well solid phase extraction with plates containing Waters Oasis HLB sorbent. The analytes are chromatographed on a Restek Ultra IBD column (3.2 mm x 50 mm, 3 microm) using a mobile phase consisting of a mixture of 90% acetonitrile and 10% 10 mM ammonium acetate buffer and 0.1% formic acid. Quantitation of the analyte is based on the response from the multiple reaction monitoring of the precursor to product ion pairs for fexofenadine (m/z 502 --> 466) and d6-fexofenadine (m/z 508 --> 472). The assay has been validated over the concentration range of 1-200 ng/ml based on the analysis of 0.5 ml aliquots of plasma. Within-day assay accuracy was between 97 and 102% of nominal, while within-day precision was better than 3.5% CV at all points on the standard curve. Analyte extraction recovery was better than 70% over the range of the standard curve. The method was found to be suitable for the analysis of human plasma samples obtained 24 h following the administration of a single 60 mg dose of fexofenadine.     

Liquid chromatographic tandem mass spectrometric assay for simultaneous quantification of compound 97/78 and its in vivo metabolite 97/63, a novel trioxane antimalarial, in human plasma and its application to a protein binding study

A sensitive, selective and specific LC-MS/ MS assay for simultaneous quantification of compound 97/78 and its active in vivo metabolite 97/63, a novel 1,2,4-trioxane antimalarial, in human plasma has been developed and validated using alpha-arteether as internal standard (IS). Extraction from plasma involves a simple protein precipitation method. The analytes were chromatographed on a Columbus C18 column with guard by isocratic elution with acetonitrile:ammonium acetate buffer (10 mM, pH 4.0) (80:20 v/v) as mobile phase at a flow rate of 0.45 mL min(-1) and analyzed in multiple reaction-monitoring (MRM) positive ion mode. The chromatographic run time was 4.0 min. The weighted (1/x2) calibration curves were linear over a range of 1.56-200 ng mL(-1) with correlation coefficients > 0.998. For both analytes, the limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.5 ng mL(-1) and 1.56 ng mL(-1), respectively. The recovery of 97/78, 97/63 and IS from spiked control samples were > 90% and their matrix suppression obtained were < 8 %. The accuracy (% bias) and precision (%RSD) for both analytes were < 6.78%. Both analytes were stable after three freeze-thaw cycles (% deviation < 12.80), long-term for 30 days in plasma at -60 degrees C (% deviation < 14.38), for 8 h on bench top in plasma at ambient temperature (% deviation < 1.52) and also in the auto-sampler for 12 h (% deviation < 3.9%). The validated method was successfully applied to a protein binding study of compound 97/78 and metabolite 97/63 in human plasma. Furthermore, the validated method will be applicable to pharmacokinetics, bioavailability and metabolism in various clinical phases and in drug interaction studies.     

Evaluation of a multi-class, multi-residue liquid chromatography-tandem mass spectrometry method for analysis of 120 veterinary drugs in bovine kidney

Traditionally, regulatory monitoring of veterinary drug residues in food animal tissues involves the use of several single-class methods to cover a wide analytical scope. Multi-class, multi-residue methods (MMMs) of analysis tend to provide greater overall laboratory efficiency than the use of multiple methods, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of targeted drug analytes usually provides exceptional performance even for complicated sample extracts. In this work, an LC-MS/MS method was optimized and validated in a test of 120 drug analytes from 11 different classes in bovine kidney. The method used 10 ml of 4/1 acetonitrile/water for extraction of 2 g samples and cleanup with hexane partitioning. Quantitative and qualitative performance was assessed for the analytes at fortification levels of 10, 50, 100, and 200 ng/g. With the method, 66 drugs gave 70-120% recovery with ≤ 20% RSD at all levels over the course of 3 days. At the 200 ng/g level, 89 drugs met these same standards. Limits of detection were ≤ 10 ng/g for 109 of the analytes in the kidney matrix in validation experiments. Qualitatively, MS/MS identification criteria were set that ion ratios occur within ± 10% (absolute value) from those of the analyte reference standards. At the 10 ng/g level, 57% of the drugs met the identification criteria, which improved to 84% at the 200 ng/g level. The method serves as an efficient and useful additional option among the current monitoring methods available.     

Determination of tamsulosin in human aqueous humor and serum by liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.     

Cocktail approach for in vivo phenotyping of 5 major CYP450 isoenzymes: development of an effective sampling, extraction, and analytical procedure and pilot study with comparative genotyping

In this study, the authors developed a phenotyping method for CYP1A2, 2C9, 2C19, 2D6, and 3A4 using a cocktail of 100 mg caffeine, 125 mg tolbutamide, 20 mg omeprazole, 30 mg dextromethorphan, and 2 mg midazolam. A simple sampling scheme was established collecting 3 blood samples at 0, 4, and 24 hours followed by solid-phase extraction and liquid chromatography/tandem mass spectrometry analysis. After addition of 8 deuterated internal standards and extraction, the analytes were separated using gradient elution with ammonium acetate and methanol. Data acquisition was performed on a triple quadrupole linear ion trap mass spectrometer in multiple-reaction monitoring mode with positive electrospray ionization. The assay was validated according to international guidelines: limits of quantification (LOQs) were between 0.25 and 1.0 ng/mL for all analytes, except for paraxanthine and caffeine (20 ng/mL). Extraction efficiencies ranged between 77% and 103% and matrix effects between 23% and 95%; precision and accuracy data fulfilled accepted criteria. Calibration curves from LOQ to 1000 ng/mL were established for undiluted and 1:10 diluted plasma (r > 0.998). The method was tested in a pilot study with 14 volunteers. Additional genotyping of the probands generally demonstrated good accordance with the measured phenotyping indices but also disclosed certain contradictory results.     

Determination of oxycodone, noroxycodone, oxymorphone, and noroxymorphone in human plasma by liquid chromatography-electrospray-tandem mass spectrometry

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of oxycodone, noroxycodone, oxymorphone, and noroxymorphone. Following solid-phase extraction, the analytes were separated on a reverse-phase column by gradient elution and analyzed by MS/MS. The analytes were found to be stable in plasma for at least three freeze-thaw cycles and 5 hours at room temperature, and also in the reconstitution solution at 8 degrees C for at least 48 hours. The lower limits of quantification were 0.1 ng/mL for oxycodone and oxymorphone and 0.25 ng/mL for noroxycodone and noroxymorphone. All calibration curves were linear up to 100 ng/mL. The extraction recoveries were more than 85%, the intraday and interday coefficients of variation were <15%, and the accuracy was >90% for all analytes at relevant plasma concentrations. The method has been used in the therapeutic drug monitoring of more than 1000 clinical plasma samples. About 50 concomitantly used drugs were tested for possible ion suppression and found not to interfere with the method. In conclusion, this method is suitable for pharmacokinetic studies in patients and healthy volunteers, and it can be applied to therapeutic monitoring of oxycodone.     

Development and validation of a selective and robust LC-MS/MS method for quantifying ilaprazole and its metabolite ilaprazole sulfone in human plasma

LC-MS/MS方法测定人血浆中艾普拉唑及其代谢产物艾普拉唑砜的浓度@谭志荣$Pharmacogenetics Research Institute, Institute of Clinical Pharmacology, Central South University!Changsha 410078, Hunan, China @张伟$Pharmacogenetics Research Institute, Institute of C     

Sensitive LC-MS/MS methods for the quantification of RGH-188 and its active metabolites, desmethyl- and didesmethyl-RGH-188 in human plasma and urine

Selective and sensitive LC-MS/MS methods have been developed and validated for simultaneous determination of RGH-188, a novel atypical antipsychotic, and its two active metabolites, desmethyl- and didesmethyl-RGH-188 in human plasma and urine. Deuterated analytes, [2H6]-RGH-188, [2H3]-desmethyl-RGH-188 and [2H8]-didesmethyl-RGH-188 were used as internal standards (IS). The compounds were isolated from the alkalized biological matrix using liquid-liquid extraction (LLE) and the extracts were analysed by reversed-phase HPLC with MS/MS detection. The chromatographic run time was 5.0min per injection. The PE Sciex API 365 mass spectrometer was equipped with a TurboIonSpray interface and operated in positive-ion, multiple reaction monitoring (MRM) mode. The mass transitions monitored were m/z 427.3-->382.2, 413.2-->382.2, 399.2-->382.2, 433.3-->382.2, 416.2-->382.2 and 407.3-->390.2 for RGH-188, desmethyl-RGH-188, didesmethyl-RGH-188, [2H6]-RGH-188, [2H3]-desmethyl-RGH-188 and [2H8]-didesmethyl-RGH-188, respectively. The lower limit of quantification (LLOQ) was 0.05 and 0.1ng/ml for RGH-188 and its metabolites, respectively, using 1ml of plasma. LLOQ in 1ml of urine was 0.1ng/ml for all three analytes. The methods were validated for selectivity, linearity, accuracy and precision. The lower limit of quantification, dilution integrity, matrix effect, stability of the analytes in the biological matrix during short- and long-term storage and after three freeze-thaw cycles were also tested. The assays were simple, specific and robust enough to support clinical development of RGH-188.     

Effects of itraconazole and diltiazem on the pharmacokinetics of fexofenadine, a substrate of P-glycoprotein

AIMS: Fexofenadine is a substrate of several drug transporters including P-glycoprotein. Our objective was to evaluate the possible effects of two P-glycoprotein inhibitors, itraconazole and diltiazem, on the pharmacokinetics of fexofenadine, a putative probe of P-glycoprotein activity in vivo, and compare the inhibitory effect between the two in healthy volunteers. METHODS: In a randomized three-phase crossover study, eight healthy volunteers were given oral doses of 100 mg itraconazole twice daily, 100 mg diltiazem twice daily or a placebo capsule twice daily (control) for 5 days. On the morning of day 5 each subject was given 120 mg fexofenadine, and plasma concentrations and urinary excretion of fexofenadine were measured up to 48 h after dosing. RESULTS: Itraconazole pretreatment significantly increased mean (+/-SD) peak plasma concentration (Cmax) of fexofenadine from 699 (+/-366) ng ml-1 to 1346 (+/-561) ng ml-1 (95% CI of differences 253, 1040; P<0.005) and the area under the plasma concentration-time curve [AUC0,infinity] from 4133 (+/-1776) ng ml-1 h to 11287 (+/-4552) ng ml-1 h (95% CI 3731, 10575; P<0.0001). Elimination half-life and renal clearance in the itraconazole phase were not altered significantly compared with those in the control phase. In contrast, diltiazem pretreatment did not affect Cmax (704+/-316 ng ml-1, 95% CI -145, 155), AUC0, infinity (4433+/-1565 ng ml-1 h, 95% CI -1353, 754), or other pharmacokinetic parameters of fexofenadine. CONCLUSIONS: Although some drug transporters other than P-glycoprotein are thought to play an important role in fexofenadine pharmacokinetics, itraconazole pretreatment increased fexofenadine exposure, probably due to the reduced first-pass effect by inhibiting the P-glycoprotein activity. As diltiazem pretreatment did not alter fexofenadine pharmacokinetics, therapeutic doses of diltiazem are unlikely to affect the P-glycoprotein activity in vivo.