96-well Solid-phase Extraction Research Articles

Ultra sensitive measurement of endogenous epinephrine and norepinephrine in human plasma by semi-automated SPE-LC–MS/MS

Measurement of endogenous epinephrine (E) and norepinephrine (NE) in human plasma is very challenging due to lower endogenous concentrations as compared with animal plasma. An LC–MS/MS in combination with alumina-based SPE and derivatization procedure was validated for the measurement of E and NE in human plasma with acceptable intra-day and inter-day accuracy and precision. Sample was extracted with semi-automated alumina 96-well solid phase extraction (SPE) cartridge. The resulting eluent was dried and derivatized using d4-acetaldehyde. The analytes were separated on a monolithic C18 column. Extraction efficiencies were >66% for E and NE. The lower limit of quantitation (LLOQ) was 5.00pg/mL for E and 20.0pg/mL for NE.

Rapid quantitative analysis of clarithromycin in rat plasma by UPLC–MS/MS after intravenous injection of the clarithromycin-loaded ultrafine PLGA nanoparticles

Nanoparticles were designed to encapsulate drugs to alter their pharmacological behaviors, therefore, it is very essential to monitor the pharmacokinetic profile of drug encapsulated in nanoparticles in order to clarify and predict their efficacy and side effects. In this paper, we reported a simple, rapid μ-elution 96-well solid phase extraction (μSPE) method combining with ultra high performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for determination of nanoformulated drug in rat plasma. This method presented satisfactory results in terms of sensitivity, precision, accuracy, and recovery, for the first time, of quantitatively analyzing clarithromycin (CLA) in rat plasma after intravenous administration CLA-loaded ultrafine PLGA nanoparticles for pharmacokinetic study. This method has been proved to be fast, reliable and reproducible to accurately analyze drug encapsulated in polymeric nanoparticles sample for a pharmacokinetic study.

A solid phase extraction-liquid chromatographic–tandem mass spectrometry method for determination of concentrations of GDC-0941, a small molecule class I phosphatidylinositide 3-kinase inhibitor, to support clinical development

A solid phase extraction (SPE) liquid chromatographic–tandem mass spectrometry (LC–MS/MS) method for the determination of GDC-0941 concentrations in human plasma has been developed and validated to support clinical development. An Oasis MCX 10 mg 96-well SPE plate was used to extract plasma samples (50 μL) and the resulting extracts were analyzed using reverse-phase chromatography and mass spectrometer coupled with a turbo-ionspray interface. The method was validated over the calibration curve range 0.500–500 ng/mL with linear regression and 1/ x 2 weighting. Within-run relative standard deviation (%RSD) ranged from 1.5 to 11.5%, while the between-run %RSD varied from 0.0 to 4.4%. The accuracy ranged from 96.0% to 110.0% of nominal for within-run and 98.0% to 108.0% of nominal for between-run at all concentrations including the LLOQ quality control at 0.500 ng/mL. Extraction recovery of GDC-0941 was between 79.0% and 86.2%. Stability of GDC-0941 was established in human plasma for 602 days at −70 °C and 598 days at −20 °C, respectively, and established in reconstituted sample extracts for 167 h when stored at room temperature. Internal standard normalized matrix factor was 1.1, demonstrating that the use of the stable-labeled internal standard GDC-0941-d 8 effectively compensated observed matrix effect and resulting in no adverse impact on the quality of the data produced. This assay was used for the determination of GDC-0941 human plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Determination of cinacalcet hydrochloride in human plasma by liquid chromatography–tandem mass spectrometry

A sensitive and selective high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed to determine the cinacalcet hydrochloride in human plasma. The analyte was extracted from plasma samples using a 96-well plate automatic solid-phase extraction (SPE) device and chromatographed on an Inertsil SIL-150 (2.1 mm × 50 mm, i.d. 5 μm) column using acetonitrile–water–formic acid (90:10:1) as the mobile phase with an isocratic flow rate of 0.35 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). The method was validated over the concentration range of 0.1–25 ng/mL. The indicators of inter- and intra-day precision (RSD%) were all within 15.1%, and the accuracy (RE%) was within ±15%. The lower limit of quantitation (LLOQ) was 0.1 ng/mL. The average extraction recovery was 51.7%, and the detection was not affected by the matrix. The method was successfully applied to a pharmacokinetic study of cinacalcet hydrochloride in healthy Chinese volunteers.

Direct sampling and analysis from solid‐phase extraction cards using an automated liquid extraction surface analysis nanoelectrospray mass spectrometry system

Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as a liquid extraction surface analysis (LESA) mode on a commercially available chip-based infusion nanoelectrospray ionization (nanoESI) system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom-made solid-phase extraction (SPE) cards, with each well consisting of either a 1 or a 2 mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the commercial nanoESI system coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer or a linear ion trap mass spectrometer. The extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10× increase in signal and cleanup of micromolar angiotensin II from a concentrated salt solution was demonstrated. In addition, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS(3) spectra for compound identification in the presence of isobaric interferences.

Analysis of urinary nucleosides as potential tumor markers in human breast cancer by high performance liquid chromatography/electrospray ionization tandem mass spectrometry

BackgroundBreast cancer is the most common female cancer and the fourth leading cause of cancer death among women in Taiwan. We measured urinary nucleoside levels in female breast cancer patients (n=36) to evaluate the diagnostic value of nucleosides as potential tumor markers. MethodsPurification of urinary nucleosides was performed using a 96-well solid phase extraction (SPE, cation-exchange column) procedure to decrease the variation between the single column preparations and to shorten the pretreatment time. Cation-exchange allows for the comprehensive purification of modified nucleosides, such as 2-deoxynucleosides, that are not purifiable by phenylboronic acid-based SPE. High-performance liquid chromatography (HPLC) coupled with mass spectrometry (MS) in selected reaction monitoring (SRM) mode was used to quantify multiple nucleosides. Tubercidin was used as an internal standard. The qualitative parameters, retention time, and the parent and daughter ions used revealed that the method was more specific and sensitive than traditional UV detection. ResultsUrinary levels of 3 nucleosides, cytidine, 3-methylcytidine, and inosine were significantly higher in breast cancer patients than in normal controls (p<0.01). The discriminative powers of cytidine, 3-methylcytidine, and inosine were 58%, 58%, and 62%, respectively. ConclusionsLC/MS/MS is a highly specific and sensitive method for rapidly screening a large number of urinary nucleosides that may be potential cancer markers. The 3-methylcytidine may be a candidate marker for breast cancer.

A rugged and accurate liquid chromatography–tandem mass spectrometry method for quantitative determination of BMS-790052 in plasma

To support toxicokinetic assessments, a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of BMS-790052 in rat, dog, monkey, rabbit and mouse K 2EDTA plasma. The drug was isolated from buffered samples using ISOLUTE C8 96-well solid phase extraction (SPE) plates. Chromatographic separation was achieved on a Waters Atlantis dC18 analytical column (2.1 mm × 50 mm, 5 μm) with detection accomplished using an API 4000 tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 5.00 to 2000 ng/mL for BMS-790052, were fitted to a 1/ x 2 weighted linear regression model. The intra-assay precision (%CV) and inter-assay precision (%CV) were within 8.5%, and the assay accuracy (%Dev) was within ±7.1 for rat, dog, monkey, rabbit and mouse K 2EDTA plasma. This accurate, precise, and selective SPE/LC–MS/MS method has been successfully applied to analyze several thousands of non-clinical study samples.

Simultaneous determination of nine tyrosine kinase inhibitors by 96-well solid-phase extraction and ultra performance LC/MS-MS

BackgroundTyrosine Kinase Inhibitors (TKIs) are a class of targeted drugs for the treatment of malignant pathologies. The metabolic profile of these drugs can result in great interindividual variability, thus therapeutic drug monitoring (TDM) is of importance. Here, a rapid and specific method for quantification of nine TKIs in plasma samples is described. MethodsChromatography was performed on a Waters Acquity-UPLC® system with BEH C18-50*2.1mm column, under a gradient of ammonium formate–acetonitrile. An Acquity-TQD® with electrospray ionization was used for detection. Samples were prepared by solid phase extraction (Oasis® MCX μElution) and eluate was injected in the system. ResultsCalibration curves ranged from 10 to 5000ng/mL for imatinib, its metabolite, nilotinib, lapatinib, erlotinib and sorafenib and from 0.1 to 200ng/mL for dasatinib, axitinib, gefitinib and sunitinib. Peaks of each compound (retention time from 0.76 to 2.51min) were adequately separated. The mean relative extraction recovery was in the range of 90.3–106.5% thanks to the use of stable isotopes as internal standard. There was no significant ion suppression observed at the respective TKI retention times. ConclusionThis rapid, sensitive and specific UPLC/MS-MS method is able to perform simultaneous quantification of nine TKIs in human plasma and usable for routine TDM.

Quantification of midazolam, morphine and metabolites in plasma using 96‐well solid‐phase extraction and ultra‐performance liquid chromatography–tandem mass spectrometry

Currently, pharmacokinetic-pharmacodynamic studies of sedatives and analgesics are performed in neonates and children to find suitable dose regimens. As a result, sensitive assays using only small volumes of blood are necessary to determine drug and metabolite concentrations. We developed an ultra-performance liquid chromatographic method with tandem mass spectrometry detection for quantification of midazolam, 1-hydroxymidazolam, hydroxymidazolamglucuronide, morphine, morphine-3-glucuronide and morphine-6-glucuronide in 100 microL of plasma. Cleanup consisted of 96 wells micro-solid phase extraction, before reversed-phase chromatographic separation (ultra-performance liquid chromatography) and selective detection using electrospray ionization tandem mass spectrometry. Separate solid-phase extraction methods were necessary to quantify morphine, midazolam and their metabolites because of each group's physicochemical properties. Standard curves were linear over a large dynamic range with adequate limits of quantitation. Intra- and interrun accuracy and precision were within 85-115% (of nominal concentration using a fresh calibration curve) and 15% (coefficient of variation, CV) respectively. Recoveries were >80% for all analytes, with interbatch CVs (as a measure of matrix effects) of less than 15% over six batches of plasma. Stability in plasma and extracts was sufficient, allowing large autosampler loads. Runtime was 3.00 min per sample for each method. The combination of 96-well micro-SPE and UPLC-MS/MS allows reliable quantification of morphine, midazolam and their major metabolites in 100 microL of plasma.

Quantitative Determination of ABT-925 in Human Plasma by On-Line SPE and LC-MS/MS: Validation and Sample Analysis in Phase II Studies

A fully automated 96-well On-Line Solid Phase Extraction (SPE) followed by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometric (MS/MS) method for the determination of ABT-925 (2-{3-[4-(2-tert-Butyl-6-trifluoromethyl-pyrimidin-4-yl)-piperazin-1-yl)-propyl-sulfanyl}-3H-pyrimidin-4-one fumarate) in human plasma was developed, validated and utilized in Phase II clinical studies. 50 µL of plasma sample was fortified with internal standard (IS, d8-ABT-925) and extracted on-line with Cohesive Turbo Flow Cyclone P HTLC column. The chromatographic separation was performed on Aquasil C18 (3 μm 50 × 3 mm) HPLC column with a mobile phase consisting of 50/50/0.1 (v/v/v) ACN/H2O/formic acid. The mass spectrometric measurement was conducted under positive ion mode using multiple reaction monitoring (MRM) of m/z 457.4 → 329.4 for analyte and m/z 465.5 → 337.5 for IS. The peak area ratio (analyte/IS) was used to quantitate ABT-925. A dynamic range of 0.0102 μg/mL to 5.24 μg/mL was established after the validation. The validated method was then used for two Phase II studies. To demonstrate the method reproducibility, approximately 10% of the incurred samples from one study were repeated in singlet. The repeated values were compared to the initial values. All repeated values agreed within ±15% of the mean values.

The novel sensitive and high throughput determination of cefepime in mouse plasma by SCX-LC/MS/MS method following off-line μElution 96-well solid-phase extraction to support systemic antibiotic programs

A sensitive and high throughput off-line μElution 96-well solid-phase extraction (SPE) followed by strong cation exchange (SCX) liquid chromatography with tandem mass spectrometry (LC/MS/MS) quantification for determination of cefepime has been developed and validated in mouse plasma. Using the chemical analog, ceftazidime as an internal standard (IS), the linear range of the method for the determination of cefepime in mouse plasma was 4–2048 ng/mL with the lower limit of quantitation level (LLOQ) of 4 ng/mL. The inter- and intra-assay precision and accuracy of the method were below 9.05% and ranged from 95.6 to 113%, respectively, determined by quality control (QC) samples at five concentration levels including LLOQ. After μElution SPE, 71.1% of cefepime was recovered. The application of the validated assay for the determination of cefepime in mouse pharmacokinetics (PK) samples after intravenous (IV) and subcutaneous (SC) doses was demonstrated.

Determination of GDC-0449, a small-molecule inhibitor of the Hedgehog signaling pathway, in human plasma by solid phase extraction-liquid chromatographic-tandem mass spectrometry

To support clinical development, a solid phase extraction (SPE) liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the determination of GDC-0449 concentrations in human plasma has been developed and validated. Samples (200 μl) were extracted using an Oasis MCX 10 mg 96-well SPE plate and the resulting extracts were analyzed using reverse-phase chromatography coupled with a turbo-ionspray interface. The method was validated over calibration curve range 5–5000 ng/mL. Quadratic regression and 1/ x 2 weighing were used. Within-run relative standard deviation (%RSD) was within 10.1% and accuracy ranged from 88.6% to 109.0% of nominal. Between-run %RSD was within 8.6% and accuracy ranged from 92.4% to 105.3% of nominal. Extraction recovery of GDC-0449 was between 88.3% and 91.2% as assessed using quality control sample concentrations. GDC-0449 was stable in plasma for 315 days when stored at −70 °C and stable in reconstituted sample extracts for 117 h when stored at room temperature. Quantitative matrix effect/ion suppression experiment was performed and no significant matrix ion suppression was observed. This assay allows for the determination of GDC-0449 plasma concentrations over a sufficient time period to determine pharmacokinetic parameters at relevant clinical doses.

Automated solid-phase microextraction and thin-film microextraction for high-throughput analysis of biological fluids and ligand–receptor binding studies

This protocol describes how to perform automated solid-phase microextraction (SPME) and thin-film microextraction (TFME) in a 96-well plate format for high-throughput analysis of drugs, metabolites and any other analytes of interest in biological fluids using liquid chromatography-electrospray tandem mass spectrometry. Sample preparation time required is typically 1 min per sample; hence, the throughput achievable with automated SPME/TFME is comparable with automated 96-well liquid-liquid extraction and solid-phase extraction methods, but greater than most online solid-phase extraction methods. The technique is applicable to complex samples such as whole blood without additional pretreatment. The amount of analyte extracted by SPME/TFME is proportional to the free (unbound) concentration of the analyte; hence, SPME/TFME can be used to determine both total and free concentrations of analytes from a single biofluid sample and to perform automated ligand-receptor binding studies in order to determine binding affinity and/or overall extent of ligand binding to a complex biofluid.

Epicatechin, procyanidins, and phenolic microbial metabolites after cocoa intake in humans and rats

Proanthocyanidins, flavonoids exhibiting cardiovascular protection, constitute a major fraction of the flavonoid ingested in the human diet. Although they are poorly absorbed, they are metabolized by the intestinal microbiota into various phenolic acids. An analytical method, based on an optimized 96-well plate solid-phase extraction (SPE) procedure and liquid chromatography tandem mass spectrometry (SPE-LC-MS/MS) for the analysis of 19 phenolic microbial metabolites and monomeric and dimeric flavanols in urine samples, was developed and validated. Human urine samples were obtained before and after ingestion of an acute consumption of 40 g of soluble cocoa powder and rat urines before and after the prolonged administration (2 weeks) of different diets composed of natural cocoa powder. The mean recovery of analytes using the new SPE-LC-MS/MS method ranged from 87% to 109%. Accuracy ranged from 87.5% to 113.8%, and precision met acceptance criteria (<15% relative standard deviation). Procyanidin B2 has been detected and quantified for the first time in human and rat urine after cocoa consumption. Changes in human and rat urinary levels of microbial phenolic acids and flavanols were in the range of 0.001-59.43 nmol/mg creatinine and of 0.004-181.56 nmol/mg creatinine, respectively. Major advantages of the method developed include reduction of laboratory work in the sample preparation step by the use of 96-well SPE plates and the sensitive measurement of a large number of metabolites in a very short run time, which makes it ideal for use in epidemiological studies.

A sensitive method for the determination of entecavir at picogram per milliliter level in human plasma by solid phase extraction and high-pH LC–MS/MS

Entecavir is a guanine nucleoside analogue used in the treatment of hepatitis B virus (HBV) infection. In this paper, we describe an LC–MS/MS method that was developed and validated for the quantitation of entecavir in human EDTA plasma with both high sensitivity (lower limit of quantitation (LLOQ) of 5 pg/mL) and a wide concentration range (5000-fold) intended for low dose ascending clinical studies. High enrichment was achieved by taking advantage of the excellent loading capacity and reproducibility of Oasis HLB 96-well solid phase extraction plate, which allowed 1 mL of plasma samples to be processed in two equal sequential loading steps. Lobucavir, a structural analogue, was used as the internal standard. A filtration step following the reconstitution proved to be vital for the method robustness. The analyte and internal standard were separated on an Xterra MS C18 column with a gradient elution and high-pH mobile phases. Analytes were detected by positive ion electrospray tandem mass spectrometry. The high-pH mobile phase provided both excellent analyte on-column retention and peak shape, leading to the desired sensitivity. Validation results show good intra-assay (12.3%CV) and inter-assay (3.1%CV) precisions, and good assay accuracy (±7.6%Dev). Recovery was high (∼80%), however, the large volume of plasma used did result in a considerable matrix effect (∼0.45) which was well compensated by the analog internal standard. The method was applied to sample analysis of a Phase I clinical study.

Quantification of Organophosphorus Nerve Agent Metabolites Using a Reduced-Volume, High-Throughput Sample Processing Format and Liquid Chromatography-Tandem Mass Spectrometry

A reduced-volume, high-throughput analytical method has been developed for the quantification of organophosphorus (OP) nerve agent metabolites in human urine. Metabolites of soman, sarin, cyclohexyl-sarin, VX, and Russian-VX were quantified down to a lowest reportable limit of 1 ng/mL in human urine. One hundred microliter urine samples were preconcentrated using normal-phase 96-well solid-phase extraction silica sorbent beds. Dual-column hydrophilic interaction liquid chromatography was applied in a 2.5-min isocratic separation followed by negative electrospray isotope-dilution multiple-reaction-monitoring mass spectrometry. Method validation included the characterization of two synthetic urine pools, relative recovery experiments, and calculation of the method limit of detection. All liquid handling steps were processed in a high-density 96-well format, including sample aliquoting, extraction, dry-down, and reconstitution. This allows up to 3840 unknown samples, plus calibrators and quality control materials, to be prepared on a single liquid handler in a 24-h period. In a public health emergency involving OP-nerve agents, this method provides the sample preparation and analytical capacity to respond rapidly to a large number of patient samples.

Determination of ulifloxacin, the active metabolite of prulifloxacin, in human plasma by a 96-well format solid-phase extraction and capillary zone electrophoresis

This paper described a method for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma by capillary zone electrophoresis using lomefloxacin as the internal standard. The separation was carried out at 25 °C in a 60.2 cm × 75 μm fused-silica capillary with an applied voltage of 20 kV using 200 mM borate buffer (pH 10.5). The detection wavelength was 275 nm. Clean-up and preconcentration of the samples were developed by 96-well format solid-phase extraction. 0.25 ml of plasma sample and 0.25 ml of IS were loaded onto the preconditioned wells, and the wells were washed using 1 ml of 20% methanol in acid water (1% phosphoric acid), and the analytes were eluted using 1 ml of 95/5 methanol/ammonia water. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and robustness. The calibration graph was linear for ulifloxacin from 0.02 to 2 μg/ml. The lower limit of quantification was 0.02 μg/ml. The intra- and inter-day precisions were within 4.0 and 8.2%, respectively. The method developed was successfully applied to the evaluation of clinical pharmacokinetic study of prulifloxacin formulation product after oral administration to healthy volunteers.

A new approach to the application of solid phase extraction disks with LC–MS/MS for the analysis of drugs on a 96-well plate format

A new 96-well disk solid phase extraction sample preparation technique which does not involve vacuum pumps integrated with liquid chromatographic tandem mass spectrometric (LC–MS/MS) was developed for high throughput determination of benzodiazepines (nordiazepam, diazepam, lorazepam and oxazepam). In addition, the method completely allows the re-use of the SPE disk membranes for subsequent analyses after re-conditioning. The method utilizes a robotic autosampler for parallel extractions in a 96-well plate format. Results have been presented for independent extractions from three matrices; phosphate buffer solution, urine, and plasma. Factors affecting data reproducibility, extraction kinetics, sample throughput, and reliability of the system were investigated and optimized. A total time required per sample was 0.94 min using 96-well format. Method reproducibility was ≤9% relative standard deviation for all three matrices. Limits of detection and quantitation recorded were respectively in the range 0.02–0.15 and 0.2–2.0 ng/mL with linearity ranging from 0.2 to 500 ng/mL for all matrices.

Development and validation of a method for fipronil residue determination in ovine plasma using 96-well plate solid-phase extraction and gas chromatography–tandem mass spectrometry

Fipronil, a phenylpyrazole insecticide introduced for pest control on a broad range of crops, undergoes a reinforcement of the regulation within the European Union (2007/52/EC directive) due to its potential effects on environment and human health. In order to assess the plasmatic concentrations of fipronil residues (sulfone, sulfide, fipronil, desulfinyl and amide) in ovine, a methodology based on gas chromatography coupled with tandem mass spectrometry (GC–MS/MS) was developed and validated according to the European standard (2002/657/EC). The proposed method allows a large number of samples to be treated concurrently ( n = 80) using a reduced sample amounts (0.2 mL), and consents to reach a level of quantification of 0.1 pg μL −1. The sample preparation consisted of a single solid-phase extraction (SPE) purification on a 96-well plate filled with a styrene-divinyl-benzene phase. Linearity was demonstrated all along the investigated range of concentrations, i.e. from 0.25 to 2000 pg μL −1, with coefficient of determination ( R 2) from 0.977 to 0.994, depending on target analytes. Calculated decision limit (CCα) and detection capability (CCβ) for fipronil, sulfone and sulphide were in the range 0.05–0.16 and 0.28–0.73 pg μL −1 respectively.

Determination of indapamide in human serum using 96-well solid-phase extraction and high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS)

A sensitive and specific method using high-performance liquid chromatography (LC)–electrospray tandem mass spectrometry (ESI–MS/MS) for the determination of indapamide in human serum was developed and validated. Indapamide and an internal standard (4-diethylaminobenzoic acid) were isolated from serum samples by solid-phase extraction (SPE) with Oasis ®HLB 96-well plates and determined by LC–MS/MS in multiple reaction monitoring (MRM) mode. The calibration curve of serum indapamide was linear in the range of 0.2–20 ng/ml with a correlation coefficient of 0.9999. The repeatability, intermediate precisions, and accuracies at 0.2, 5, and 20 ng/ml in serum were less than 15%. The absolute recoveries of indapamide and the internal standard were 79.4–81.5% and 87.5%, respectively, and the low limit of quantitation of serum indapamide was 0.2 ng/mL. The analytical method was applied to a bioequivalence study of KYD-041 (1 mg as film-coated tablets, test formulations) and Natrix ®Tab.1 (1 mg as sugar-coated tablets, reference formulation). The 90% confidence interval of the ratios (test formulation/reference formulation) for log( C max) and log(AUC t) were in the range log(0.80)–log(1.25), which supports the conclusion that KYD-041 is bioequivalent to Natrix ®Tab.1 with respect to the rate and extent of indapamide absorption.