Clinical Bioequivalence Studies Research Articles

Method Development, Validation and Pharmacokinetics for Ramipril and Hydrochlorothiazide in Human Plasma and Application in a Bioequivalence Study Based on Healthy Jordanian Volunteers

A simple liquid chromatography mass spectrometric method was developed for quantitative determination of Ramipril (RAM) and Hydrochlorothiazide (HCTZ) simultaneously in human plasma. RAM, HCTZ and the internal standards (RAM-D5, HCTZ-13C, D2) were extracted from human plasma by direct protein precipitation. The chromatographic separation was achieved using ACE 5 C8, (50 mm x 4.6 mm, 5µm) column with gradient elution mobile phase of 0.25 mM Ammonium chloride and methanol. The method validation intends to investigate sensitivity, linearity, precision and accuracy, recovery, matrix effect, specificity and stability according to the European Medicines Agency (EMA) guideline. Standard calibration levels were prepared in pooled human plasma to attain final dynamic range of 0.2 - 20.0 ng/mL for RAM and 1.0 - 150 ng/mL for HCTZ. Clinical bioequivalence study was successfully investigated by application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products for 5mg RAM/ 25mg Hydrochlorothiazide single dose.

Mathematical Model-Based Accelerated Development of Extended-release Metformin Hydrochloride Tablet Formulation.

A computational fluid dynamic (CFD) model was developed to predict metformin release from a hydroxypropylmethylcellulose (HPMC) matrix-based extended-release formulation that took into consideration the physical and chemical properties of the drug substance, composition, as well as size and shape of the tablet. New high dose strength (1000mg) tablet geometry was selected based on the surface area/volume (SA/V) approach advocated by Lapidus/Lordi/Reynold to obtain the desired equivalent metformin release kinetics. Maintaining a similar SA/V ratio across all extended-release metformin hydrochloride (Met XR) tablet strengths that had different geometries provided similar simulations of dissolution behavior. Experimental dissolution profiles of three lots of high-strength tablets agreed with the simulated release kinetics. Additionally, a pharmacokinetic absorption model was developed using GastroPlus™ software and known physicochemical, pharmacokinetic, and in vitro dissolution properties of metformin to predict the clinical exposure of the new high strength (1000mg) tablet prior to conducting a human clinical bioequivalence study. In vitro metformin release kinetics were utilized in the absorption model to predict exposures in humans for new 1000-mg Met XR tablets, and the absorption model correctly projected equivalent in vivo exposure across all dose strengths. A clinical bioequivalence study was pursued based on the combined modeling results and demonstrated equivalent exposure as predicted by the simulations.

Simultaneous determination of capecitabine and its three nucleoside metabolites in human plasma by high performance liquid chromatography-tandem mass spectrometry.

Capecitabine (Cape) is a prodrug that is metabolized into 5'-deoxy-5-fluorocytidine (DFCR), 5'-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5-FU) after oral administration. A liquid chromatography-tandem mass spectrometry method for the simultaneous determination of capecitabine and its three metabolites in human plasma was developed and validated. The ex vivo conversion of DFCR to DFUR in human blood was investigated and an appropriate blood sample handling condition was recommended. Capecitabine and its metabolites were extracted from 100 μL of plasma by protein precipitation. Adequate chromatographic retention and efficient separation were achieved on an Atlantis dC18 column under gradient elution. Interferences from endogenous matrix and the naturally occurring heavy isotopic species were avoided. Detection was performed in electrospray ionization mode using a polarity-switching strategy. The method was linear in the range of 10.0-5000 ng/mL for Cape, DFCR, and DFUR, and 2.00-200 ng/mL for 5-FU. The LLOQ was established at 10.0 ng/mL for Cape, DFCR, and DFUR, and 2.00 ng/mL for 5-FU. The inter- and intra-day precisions were less than 13.5%, 11.1%, 9.7%, and 11.4%, and the accuracy was in the range of -13.2% to 1.6%, -2.4% to 2.5%, -7.1% to 8.2%, and -2.0% to 3.8% for Cape, DFCR, DFUR, and 5-FU, respectively. The matrix effect was negligible under the current conditions. The mean extraction recoveries were within 105-115%, 92.6-101%, 94.0-100%, and 85.1-99.9% for Cape, DFCR, DFUR, and 5-FU, respectively. Stability testing showed that the four analytes remained stable under all relevant analytical conditions. This method has been applied to a clinical bioequivalence study.

Impact of Discordance Among Regulations for Biopharmaceutics Classification System-Based Waivers of Clinical Bioequivalence Studies

Impact of Discordance Among Regulations for Biopharmaceutics Classification System-Based Waivers of Clinical Bioequivalence Studies

7th GCC Insights: incurred samples use; fit-for-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing.

The Global CRO Council for Bioanalysis (GCC), a global independent consortium bringing together many senior level CRO representatives, was created in 2010 in order to openly discuss and share opinions on scientific and regulatory issues specific to the bioanalytical field [1]. Since its formation, the GCC members have met on a regular basis to discuss various topics and challenges faced by bioanalytical CRO companies. Several conference reports of past GCC meetings were published to share the discussions held during these events [2–4]. In addition, the GCC also published several influential White Papers on topics of widespread interest in bioanalysis [5–9]. These White Papers provide unified GCC recommendations that are helpful to the global bioanalytical community. The 7th GCC Closed Forum for Bioanalysis took place in Long Beach, CA, USA on 8 April 2013, one day before the start of the 7th Workshop on Recent Issues in Bioanalysis (WRIB). In attendance were 46 senior-level participants from six countries, representing 37 bioanalytical CRO companies/sites. This event represented a good opportunity for bioanalytical experts from CROs to share and discuss these issues of concern for the outsourcing industry. The 7th GCC Closed Forum was chaired by Mario Rocci, who started the meeting by communicating the official admonition statement, as has been done in all previous editions [1]. Prior to initiating discussions, participants introduced themselves. As in previous GCC meetings, multiple topics of 7th GCC Insights: incurred samples use; fitfor-purpose validation, solution stability, electronic laboratory notebook and hyperlipidemic matrix testing

<b>健康成人被験者で高含量の向精神薬の生物学的同等性試験を行うことの妥当性</b>

<b>健康成人被験者で高含量の向精神薬の生物学的同等性試験を行うことの妥当性</b>

Simultaneous determination of amlodipine and atorvastatin with its metabolites; ortho and para hydroxy atorvastatin; in human plasma by LC–MS/MS

A simple liquid chromatography/ion trap mass spectrometry method for the quantification of amlodipine and atorvastatin with its metabolites, ortho and para hydroxy atorvastatin, simultaneously in human plasma was developed. Analytes with internal standard were extracted by protein direct precipitation with acetonitrile. Adequate chromatographic separation was achieved using Phenomenex Synergi 4u polar-RP 80A (150mm×4.6mm, 4μm) column in the isocratic elution mode and the eluent was water/methanol (14:86%, v/v) adjusted by trichloroacetic acid to pH 3.2 which was delivered isocratically at constant flow rate of 0.50mL/min. Standard solutions for the analytes were prepared using amlodipine besylate, atorvastatin calcium, ortho-hydroxy atorvastatin dihydrate monosodium salt, para-hydroxy atorvastatin disodium salt, and pravastatin sodium as an internal standard. The method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect and stability according to USFDA guideline. Standard calibration levels were prepared by pooled human plasma to attain final dynamic range of 0.2-20.0ng/mL for amlodipine, 1.5-150ng/mL for atorvastatin, 1.0-100.0ng/mL for ortho-hydroxy atorvastatin and 0.2-20.0ng/mL for para-hydroxy atorvastatin. Clinical bioequivalence study was successfully investigated by the application of this validated bioanalytical method in order to evaluate bioequivalence of two commercial products 10mg amlodipine/80mg atorvastatin in a single dose. In this study, 29 healthy volunteers were participated in randomized, two periods, double blend, open label cross over design. Pharmacokinetic parameters of C(max), AUC(0-t) and AUC(0-∞) were calculated to compare a test product with CADUET(®) reference product.

Determination of Amlodipine in Human Plasma by LC-MS/MS and Its Bioequivalence Study in Healthy Chinese Subjects

A sensitive and reproducible liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of amlodipine in human plasma, with gliclazide as an internal standard (IS). The analyte was extracted with ethyl acetate and analyzed on a Diamond C18 (150 mm× 4.6 mm, 5 μm) column. The mobile phase was composed of methanol ?10 mM ammonium acetate with gradient flow rates and gradient conditions. Amlodipine and IS were ionized by positive ion pneumatically assisted electrospray and detected in the multi-reaction monitoring (MRM) mode using precursor→productions of m/z 409.2→238.1, 294.1 and m/z 324.2→127.3, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision, and stabilities were all validated over the concentration range of 0.05 - 12 ng/mL. The 90% confidence interval (CI) for AUC0-t, AUC0-∞ and Cmax ratios (test: reference) were all within 80% - 125% interval proposed by FDA. It is concluded that the validated method can successfully fulfill the requirement of clinical bioequivalence study of amlodipine in healthy Chinese subjects after administration of amlodipine/losartan combination tablets and amlodipine tablets, and the test and reference tablets were bioequivalent.

Quantification of human serum insulin concentrations in clinical pharmacokinetic or bioequivalence studies: what defines the “best method”?

Historically, quantitative clinical diagnostic assays (QCDAs) have not been accepted for use in pharmacokinetic or bioequivalence studies because they do not fully comply with the US Food and Drug Administration (FDA) Guidance for Industry: Bioanalytical Method Validation (e.g., full calibration curve not generated with each analytical run). Samples from a bioequivalence study were analysed for insulin and C-peptide concentrations with QCDAs and guidance-conforming radioimmunoassays (RIAs) and the results compared across and within assays. Serum samples (n=1913) from study MKC-TI-142 were analysed first using the Roche E170 electrochemiluminescence immunoassay (ECLIA) for insulin concentration and the Immulite 2000 chemiluminescence immunoassay (CLIA) for C-peptide, and then using the corresponding Millipore RIAs. The insulin assays were highly correlated: r2=0.92 excluding samples requiring dilution and R2=0.88 including all samples. There was increasing negative bias of the ECLIA compared with the RIA with increasing insulin, especially with samples that required dilution for the RIA. The ECLIA had significantly fewer below-quantifiable-limit samples, a larger dynamic analysis range without dilution, and tighter agreement within incurred sample reanalysis (ISR) as compared with the RIA. The C-peptide assays showed good agreement but the CLIA method produced ISR results that were in closer agreement with the original values. The science indicates that the QCDAs are appropriate for the quantification of serum insulin (ECLIA) and C-peptide (CLIA) concentrations in human pharmacokinetic and bioequivalence studies even though the calibration curve is not generated in each analytical run.

High-Performance Liquid Chromatography-Tandem Mass Spectrometry for the Determination of Trospium Chloride in Human Plasma and Its Application in a Bioequivalence Study

A selective, rapid, and sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed and validated for the quantification of trospium chloride in human plasma. With tramadol chloride as the internal standard, sample pretreatment involved a simple liquid-liquid extraction with chloroform-isopropyl alcohol (40/2, v/v) of 0.5 ml plasma. The analysis was carried out on a Hypsil C18 column (150 mm × 2.1 mm, 5.0 μm) with a flow rate of 0.2 ml/min. The mobile phase was methanol-5% acetic acid-20 mM ammonium methylate (55/30/15, v/v/v). The detection was performed by a selected reaction monitoring (SRM) mode via electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 0.2–50.0 ng/ml, with a lower limit of quantification of 0.2 ng/ml. The intra- and inter-day precision (RSD) values were below 15%. The method was successfully applied to a clinical bioequivalence study of trospium chloride in Chinese healthy volunteers following oral administration.

Implications of Differences in Bioanalytical Regulations Between Canada, USA and South America

To complete globally, pharmaceutical companies desire to use bioanalytical data and reports as a single version for all filings; not revising for specific countries or regions. Historically, this meant following the US FDA and International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidance/guidelines; finding them sufficient to achieve global acceptance. However, a growing challenge of the past decade has been additional country-specific and regional regulations that have been released. The differences between the bioanalytical regulations among countries have been recognized as a challenge to the pharmaceutical industry and its CRO partners. Harmonization of the regulations at a global level has been the subject of a number of recent articles and editorials, and the topic has been vigorously discussed at several conferences over the past year. Since all have been in agreement about the need to harmonize regulations, this article will not focus on harmonization but rather it will provide a comparison of the USA/Canadian regulations versus those of South America, in particular Brazil, noting the additional work needed to achieve compliance with country-specific regulations. All countries discussed have specific guidance or regulations on clinical bioequivalence studies, and due to the higher standards for these studies, the regulations for bioequivalence studies will be used as the basis for comparison in the article.

SPE and LC–ESI–MS for Quantitative Analysis of Mitiglinide in Human Plasma in a Bioequivalence Study

Liquid chromatography–electrospray ionization mass spectrometry has been used for rapid, selective, and sensitive quantitative analysis of mitiglinide in human plasma. Sample pretreatment involved solid-phase extraction from plasma with gliclazide as internal standard. Separation was performed on a C18 column (150 × 2.0 mm) with 71:29 (v/v) acetonitrile–water (containing 0.1% formic acid and 0.2 mmol L−1 ammonium acetate) as mobile phase at a flow rate of 0.2 mL min−1. The method was validated then successfully applied to a clinical bioequivalence study of mitiglinide in 20 healthy volunteers after oral administration.

Comparing Generic and Innovator Drugs: A Review of 12 Years of Bioequivalence Data from the United States Food and Drug Administration

In the US, manufacturers seeking approval to market a generic drug product must submit data demonstrating that the generic formulation provides the same rate and extent of absorption as (ie, is bioequivalent to) the innovator drug product. Thus, most orally administered generic drug products in the US are approved based on results of one or more clinical bioequivalence studies. To evaluate how well the bioequivalence measures of generic drugs approved in the US over a 12-year period compare with those of their corresponding innovator counterparts. This retrospective analysis compared the generic and innovator bioequivalence measures from 2070 single-dose clinical bioequivalence studies of orally administered generic drug products approved by the Food and Drug Administration (FDA) from 1996 to 2007 (12 y). Bioequivalence measures evaluated were drug peak plasma concentration (C(max)) and area under the plasma drug concentration versus time curve (AUC), representing drug rate and extent of absorption, respectively. The generic/innovator C(max) and AUC geometric mean ratios (GMRs) were determined from each of the bioequivalence studies, which used from 12 to 170 subjects. The GMRs from the 2070 studies were averaged. In addition, the distribution of differences between generic means and innovator means was determined for both C(max) and AUC. The mean +/- SD of the GMRs from the 2070 studies was 1.00 +/- 0.06 for C(max) and 1.00 +/- 0.04 for AUC. The average difference in C(max) and AUC between generic and innovator products was 4.35% and 3.56%, respectively. In addition, in nearly 98% of the bioequivalence studies conducted during this period, the generic product AUC differed from that of the innovator product by less than 10%. The criteria used to evaluate generic drug bioequivalence studies support the FDA's objective of approving generic drug formulations that are therapeutically equivalent to their innovator counterparts.

A New, High-Throughput High-Performance Liquid Chromatographic/Mass Spectrometric Assay for Therapeutic Level Monitoring of Digoxin in Human Plasma

A new, high-throughput HPLCIMS assay for the quantification of digoxin in human plasma was developed and validated. The separation was performed on a Zorbax SB-C18 column under isocratic conditions using a 55 + 45 (v/v) mixture of methanol and 0.1% (v/v) formic acid in 10 microM sodium acetate as the mobile phase at 45 degrees C with a flow rate of 1 mL/min. The detection of digoxin was performed in the selected ion monitoring mode (m/z 803.5). The human plasma samples (0.2 mL) were deproteinized with 7% perchloric acid in water, and aliquots of 20 microL from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method showed good linearity (r > 0.9926), precision (CV > 18.9%), and accuracy (bias > 5.6%) over the range of 0.5-50 ng/mL plasma. The lower LOQ was 0.5 ng/mL, and the recovery was between 92 and 106%. The method is not expensive, it needs a minimum time for plasma sample preparation, and it has a run time of 2.3 min for instrument analysis (the retention time of digoxin was 1.9 min). The developed and validated high-throughput method is very simple, rapid, and efficient, with wide applications in clinical level monitoring, pharmacokinetics, and bioequivalence studies.

Spreadability Measurements to Assess Structural Equivalence (Q3) of Topical Formulations—A Technical Note

Due to the disadvantages associated with comparative clinical trials (e.g., extensive time and cost involved), in vitro methods are of high interest for evaluating the bioequivalence of topical formulations (1,2). The analysis of spreadability is one such method that can help assess bioequivalence of formulations that are qualitatively (Q1) and quantitatively (Q2) equivalent. The FDA will usually not request additional bioequivalence testing for solution formulations that are Q1 and Q2 equivalent. However, semi-solid formulations that are Q1 and Q2 equivalent may have differences in the physical attributes and state of the product (arrangement of matter, aggregation) that reflect changes in the manufacturing method or the physical state of starting materials. Thus, structural similarity (Q3) can be defined as a third aspect of equivalence (3). The rheology of semi-solids is sensitive to changes in the microstructure and is potentially able to detect Q3 differences. In addition, the viscosity of a topical formulation will affect its application and delivery of the active agent to and across the skin, resulting in variance in its therapeutic performance. Subjective spreadability has been shown to be related to rheology through a material’s yield stress, the minimum shear stress required to initiate flow (σο). Specifically, the spreadability is inversely proportional to σο. (4,5) Extensive studies have been completed for the yield stress analysis of semi-solid foods that can be extended to semi-solid pharmaceutical formulations (4,6–8). In this study, the vane method has been employed to determine the yield stress of Q1/Q2 topical formulations in an attempt to determine if they were Q3 equivalent. The vane method (Fig. 1) involves the immersion of the blades of a vane into a sample followed by slow rotation at a constant RPM until the torque exerted on the vane reaches a maximum value and the sample begins to flow. Torque vs. time curves are used to determine yield stress where Mo is the maximum torque exerted on the vane by the fluid. Fig. 1 A vane, used in the vane method, with blade height of H and blade diameter of d with the height (h) of vane immersed in a fluid. This geometry is used along with the maximum torque value obtained with the vane method to obtain the yield stress of the ... MATERIALS AND METHODS The materials used in these studies were four topical formulations containing the active ingredient Econazole Nitrate 1%, and these were obtained from the Food and Drug Administration. All three of the generic formulations were previously determined to be equivalent to the innovator product in clinical bioequivalence studies. In these studies, the creams were referred to as formulations A, B, C and D, where formulation C is the innovator and formulations A, B, and D are generic brands. These rheological flow tests were carried out on a Brookfield RV DV III + Digital Rheometer using a Brookfield cone (CPE 52) and plate apparatus. The rheological vane method experiments were carried out on a Brookfield RV DV III + Digital Rheometer using a vane spindle attachment. Flow Curves Procedure Flow curves for these formulations were constructed by discharging 0.5 mL of each formulation onto the center of the plate. The gap was then set for the cone and plate apparatus so that the cone was at an appropriate and consistent distance from the plate for each trial. The rheometer was then programmed with increasing RPM (shear rate) over a range of 1 to 24 s−1 at constant temperature (25°C).

A New High-Throughput LC-MS/MS Assay for Therapeutic Level Monitoring of Valproic Acid in Human Plasma

A new high-throughput liquid chromatographic tandem mass spectrometric (LC-MS/MS) assay for the quantification of valproic acid in human plasma was developed and validated. The separation was performed on a Zorbax SB-C18 column under isocratic conditions using a 48:52 (v/v) mixture of acetonitrile and 0.1% (v/v) acetic acid in water at 45 °C with a flow rate of 0.8 mL/min. The detection of valproic acid was performed in SIM mode (m/z 143.1). The human plasma samples (0.2 mL) were deproteinized with methanol and aliquots of 2 μL from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r > 0.9972), precision (CV > 7.8 %) and accuracy (bias > 5.7 %) over the range of 5-200 μg/mL plasma. Lower limit of quantification (LLOQ) was 5 μg/mL and the recovery was between 98-106 %. The method is not expensive, it needs a minimum time for plasma sample preparation and has a run-time of 2.4 min for instrument analysis (retention time of valproic acid was 1.8 min). The developed and validated high-throughput method is very simple, rapid and efficient, with wide applications in clinical level monitoring, pharmacokinetics and bioequivalence studies.

A new esomeprazole packet (sachet) formulation for suspension: in vitro characteristics and comparative pharmacokinetics versus intact capsules/tablets in healthy volunteers

Background: A packet (sachet) formulation of esomeprazole for suspension has been developed for use in patients who have difficulty swallowing. Objectives: This article reports the in vitro characteristics of the new esomeprazole formulation, including stability in suspension and suitability for administration orally or via enteral tubes. It also describes the pharmacokinetic profile of the esomeprazole 40-mg packet compared with that of existing solid dosage forms (capsules and tablets) in a clinical bioequivalence study. Methods: The stability in suspension of the packet formulation was assessed after reconstitution at various strengths (2.5, 10, and 40 mg) and a different pH (3.4–5.0) in strength-appropriate volumes of water held at temperatures ranging from 5°C to 37°C for up to 60 minutes. Suitability for oral administration was examined in terms of reconstitution time and the actual dose delivered after simulated oral administration, as well as in terms of the actual dose delivered by enteral tubes ranging in diameter from 6 to 20 Fr. Chemical stability and suspension characteristics were also analyzed using alternative reconstitution vehicles (applesauce, apple juice, and orange juice). The comparative pharmacokinetics of the packet, capsule, and tablet formulations of esomeprazole were evaluated in a randomized, open-label, 3-way crossover study in healthy volunteers, who received single 40-mg doses of each formulation. Bioequivalence was assumed if the 90% CIs for the ratios of the geometric mean AUC and CmaX were between 0.80 and 1.25. Reversephase liquid chromatography with ultraviolet detection was used to assess the esomeprazole content and/or degradation products of esomeprazole in the tests for in-suspension stability, dose delivery, and acid resistance. Normal-phase liquid chromatography was used to assess the esomeprazole content of the plasma samples in the bioequivalence study. Results: At the pH and temperature ranges investigated, the packet formulation was stable for up to 60 minutes after reconstitution. Chemical degradation was low (<0.1%) for all reconstitution vehicles investigated. Reconstitution time was 2 minutes with water and 9 to 10 minutes with apple or orange juice. Dose delivery was ≥98% after simulated oral administration and was generally ≥96% after administration via enteral tubes. Ninety-six healthy volunteers (56 women, 40 men; mean age, 24.9 years; mean weight, 68.9 kg) participated in the randomized, crossover, comparative pharmacokinetic study of the packet and capsule/tablet formulations. The estimated ratios of the geometric mean AUC and C max for the packet:capsule and packet: tablet formulations were 0.98 (90% CI, 0.93–1.03) and 0.99 (90% CI, 0.94–1.04), respectively. Conclusions: In these analyses, the packet (sachet) formulation of esomeprazole was chemically stable in suspension and when administered orally and via enteral tubes. The formulation had a short reconstitution time, remaining fully dispersed in water for at least 30 minutes, and was dispersed in applesauce, apple juice, or orange juice without compromising its stability or dispersion characteristics. The packet formulation met the regulatory definition for bioequivalence to the tablet and capsule formulations.

Simultaneous determination of metformin and gliclazide in human plasma by liquid chromatography–tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy volunteers

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine gliclazide and metformin in human plasma using huperzine A as the internal standard (IS). After acetonitrile-induced protein precipitation of the plasma samples, gliclazide, metformin and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of 2.0 min and linear calibration curves over the concentration ranges of 10-10,000 ng ml(-1) for gliclazide and 7.8-4678.9 ng ml(-1) for metformin. The recoveries of the method were found to be 71-104%. The lower limits of quantification (LOQ) of the method were 10.0 and 7.8 ng ml(-1) for gliclazide and metformin, respectively. The intra- and interday precision was less than 15% for all quality control samples at concentrations of 100, 500, and 2000 ng ml(-1). The validated LC/MS/MS method has been used to study bioequivalence in healthy volunteers. These results indicate that the method was efficient with a very short running time (2.0 min) for metformin and gliclazide compared to the methods reported in the literature. The presented method had acceptable accuracy, precision and sensitivity and was used in clinical bioequivalence study.

Simultaneous determination of paracetamol and dextropropoxyphene in human plasma by liquid chromatography/tandem mass spectrometry: application to clinical bioequivalence studies

A liquid chromatography/mass spectrometry method for simultaneous determination of paracetamol and dextropropoxyphene in human plasma is described. Paracetamol and dextropropoxyphene, together with their internal standards (tolbutamide and pyrroliphene), were extracted from 0.5 mL of plasma using solid-phase extraction. The chromatography was performed using a Thermo Hypersil APS-2 Amino column (250 mm x 4.6 mm, 5 microm) with a mobile phase consisting of acetonitrile and 0.4% glacial acetic acid in water (20:80). The total run time was 6 min for each sample. The triple-quadrupole mass spectrometer was operated in both positive (for detection of dextropropoxyphene and its IS pyrroliphene) and negative (for detection of paracetamol and its IS tolbutamide) modes using a polarity-switching technique. Multiple reaction monitoring was used for quantification. The method was linear over the concentration range of 0.1-20 microg/mL for paracetamol and 0.5-80 ng/mL for dextropropoxyphene. The intra- and inter-day precision were less than 10%, and the accuracy ranged from 92.2-110.9%. The lower limits of quantification were 0.1 microg/mL for paracetamol and 0.5 ng/mL for dextropropoxyphene. The present method provides a robust, fast and sensitive analytical tool for both paracetamol and dextropropoxyphene, and has been successfully applied to a clinical bioequivalence study in 14 subjects.

Development of a New Controlled-Release Formulation of Chlorpheniramine Maleate Using In Vitro/In Vivo Correlations

□ Development of a controlled-released formulation of chlorpheniramine maleate is described, using in vitro/in vivo correlates, according to a process that has been termed "biorelevant dissolution". The process begins with simulations using several possible input rates combined with known disposition parameters of chlorpheniramine maleate. Based on desired plasma concentrations, an input rate is selected for further development which consists of a combination of clinical bioequivalence studies and further in vitro testing and simulations. The method is designed to reduce the requirements for trial and error clinical bioequivalence testing of a new controlled-release formulation.