Validation of a sensitive and automated 96-well solid-phase extraction liquid chromatography-tandem mass spectrometry method for the determination of desloratadine and 3-hydroxydesloratadine in human plasma

To support clinical development, a liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed and validated for the determination of desloratadine (descarboethoxyloratadine) and 3-OH desloratadine (3-hydroxydescarboethoxyloratadine) concentrations in human plasma. The method consisted of automated 96-well solid-phase extraction for sample preparation and liquid chromatography/turbo ionspray tandem mass spectrometry for analysis. [2H(4)]Desloratadine and [2H(4)]3-OH desloratadine were used as internal standards (I.S.). A quadratic regression (weighted 1/concentration(2)) gave the best fit for calibration curves over the concentration range of 25-10000 pg/ml for both desloratadine and 3-OH desloratadine. There was no interference from endogenous components in the blank plasma tested. The accuracy (%bias) at the lower limit of quantitation (LLOQ) was -12.8 and +3.4% for desloratadine and 3-OH desloratadine, respectively. The precision (%CV) for samples at the LLOQ was 15.1 and 10.9% for desloratadine and 3-OH desloratadine, respectively. For quality control samples at 75, 1000 and 7500 pg/ml, the between run %CV was 相似文献   

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(2)EDTA 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(2)EDTA plasma. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to analyze several thousands of non-clinical study samples.     

Sensitive and specific LC-MS/MS method for the simultaneous measurements of viramidine and ribavirin in human plasma

Viramidine is a prodrug of ribavirin. To facilitate pharmacokinetics studies of viramidine in man, a sensitive and specific LC-MS/MS method for the simultaneous analyses of viramidine and ribavirin in human plasma was developed and validated. The method involved the addition of [13C]viramidine and [13C]ribavirin as internal standards, protein precipitation with acetonitrile, HPLC separation, and quantification by MS/MS system using positive electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor-->product ion transitions were monitored at 245-->113, 250-->113, 244-->112, and 249-->112 for ribavirin, [13C]ribavirin, viramidine, and [13C]viramidine, respectively. The calibration curves for viramidine and ribavirin were linear over a concentration range of 1-1000 ng/mL. For both viramidine and ribavirin, the lower limit of quantification (LLOQ) was 1 ng/mL. For viramidine, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated good precision (%CV between 1.0 and 7.0%) and accuracy (%bias between -4.3 and 5.2%). For ribavirin, intra- and inter-day analyses of QC samples at 1, 5, 250, and 1000 ng/mL indicated similar precision (%CV between 0.8 and 8.3%) and accuracy (%bias between -5.8 and 9.4%). Both viramidine and ribavirin were stable in human plasma stored at room temperature for at least 3 h, 4 degrees C for at least 6 h, and for at least three freeze-thaw cycles. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of viramidine and ribavirin in man following administration of viramidine.     

Development of a liquid chromatography-tandem mass spectrometry assay of six antimicrobials in plasma for pharmacokinetic studies in premature infants

This method provides a simple extraction procedure, as well as a validated, sensitive, and specific liquid chromatography-tandem mass spectrometry assay for the simultaneous quantification of ampicillin, piperacillin, tazobactam, meropenem, acyclovir, and metronidazole in human plasma. The method was validated over concentration ranges specific for each compound, with a lower limit of quantification of 50-300 ng/mL and a sample volume of 50 μL. The method is accurate and precise, with within- and between-day accuracy ranging from 85 to 110% and 92 to 110%, respectively, and within- and between-day precision of 89-111% and 91-109%, respectively. Simplicity, low plasma volume, and high throughput make this method suitable for clinical pharmacokinetic studies in premature infants.     

High-throughput determination of fudosteine in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A 96-well protein precipitation, liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated for the determination of fudosteine in human plasma. After protein precipitation of the plasma samples (50 microL) by the methanol (150 microL) containing the internal standard (IS), erdosteine, the 96-well plate was vortexed for 5 min and centrifuged for 15 min. The 100 microL supernatant and 100 microL mobile phase were added to another plate and mixed and then the mixture was directly injected into the LC-MS/MS system in the negative ionization mode. The separation was performed on a XB-CN column for 3.0 min per sample using an eluent of methanol-water (60:40, v/v) containing 0.005% formic acid. Multiple reaction monitoring (MRM) using the precursor-product ion transitions m/z 178-->91 and m/z 284-->91 was performed to quantify fudosteine and erdosteine, respectively. The method was sensitive with a lower limit of quantification (LLOQ) of 0.02 microg mL(-1), with good linearity (r>0.999) over the linear range of 0.02-10 microg mL(-1). The within- and between-run precision was less than 5.5% and accuracy ranged from 94.2 to 106.7% for quality control (QC) samples at three concentrations of 0.05, 1 and 8 microg mL(-1). The method was employed in the clinical pharmacokinetic study of fudosteine formulation product after oral administration to healthy volunteers.     

Quantitative HPLC-UV method for the determination of firocoxib from horse and dog plasma

A sensitive reversed-phase HPLC-UV method was developed for the determination of firocoxib, a novel and highly selective COX-2 inhibitor, in plasma. A 1.0 mL dog or horse plasma sample is mixed with water and passed through a hydrophobic-lipophilic copolymer solid-phase extraction column to isolate firocoxib. Quantitation is based on an external standard curve. The method has a validated limit of quantitation of 25 ng/mL and a limit of detection of 10 ng/mL. The validated upper limit of quantitation was 2500 ng/mL for horses and 10,000 ng/mL for dogs. The average recoveries ranged from 88-93% for horse plasma and 96-103% for dog plasma. The coefficient of variation in all cases was less than 10%. This method is suitable for the analysis of clinical samples from pharmacokinetic and bioequivalence studies and drug monitoring.     

High performance liquid chromatography-electrospray ionization mass spectrometric determination of balofloxacin in human plasma and its pharmacokinetics

A selective and sensitive high performance liquid chromatography-electrospray ionisation-mass spectrometry method has been developed for the determination of balofloxacin (BLFX) in human plasma. The sample preparation was a liquid-liquid extraction, and chromatographic separation was achieved with an Agilent ZORBAX 300SB C18 2.1 mm x 150 mm column using a mobile phase comprised of methanol-water (10 mM CH(3)COONH(4), pH 3.0)=40:60 (v/v). Standard curves were linear (r=0.9992) over the concentration range of 0.03-3 microg/ml and had good accuracy and precision. The within- and between-batch precisions were within 10% relative standard deviation (R.S.D.). The limit of detection (LOD) was 0.02 microg/ml. The validated HPLC-electrospray ionization (ESI)-MS method has been used successfully to study balofloxacin pharmacokinetics in healthy volunteers.     

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the determination of salidroside in rat plasma: application to the pharmacokinetics study

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the identification and quantification of salidroside, a major active constituent from Rhodiola rosea L., in rat plasma using helicid as an internal standard. The method involves a simple single-step liquid-liquid extraction with n-butanol. The analytes were separated by isocratic gradient elution on a Shim-pack ODS (4.6 microm, 250 mmx2.0 mm i.d.) column and analyzed in selected ion monitoring (SIM) mode with a negative electrospray ionization (ESI) interface using the respective [M+Cl]- ions, m/z 335 for salidroside, m/z 319 for internal standard. The method was validated over the concentration range of 5-2000 ng/mL for salidroside. Within- and between-batch precision (R.S.D.%) were all within 6% and accuracy ranged from 96 to 112%. The lower limits of quantification was 5 ng/mL. The extraction recovery was on average 86.6% for salidroside. The validated method was used to study the pharmacokinetic profile of salidroside in rat plasma after intravenous and oral administration of salidroside. The bioavailability of salidroside in rats is 32.1%.     

Validated liquid chromatographic-ultraviolet method for the quantitation of tadalafil in human plasma using liquid-liquid extraction

A highly selective, sensitive and rapid HPLC method has been developed and validated to quantify tadalafil in human plasma. The tadalafil and internal standard (loratadine, I.S.) were extracted by liquid-liquid extraction technique followed by an aqueous back-extraction allowing injection of an aqueous solvent in the HPLC system. The chromatographic separation was performed on a reverse phase BDS Hypersil C-18 column (250 mm x 4.6 mm, 5 microm, Thermo Separation Co., USA) with a mobile phase of acetonitrile and aqueous solution containing 0.012 M triethylamine+0.020 M orthophosphoric acid (50/50, v/v). The analytes were detected at 225 nm. The assay exhibited a linear range of 5-600 ng/mL for tadalafil in human plasma. The lower limit of quantitation (LLOQ) was 5 ng/mL. The within- and between batch precision (expressed as coefficient of variation, C.V.) did not exceed 10.3% and the accuracy was within -7.6% deviation of the nominal concentration. The recovery of tadalafil from plasma was greater than 66.1%. Stability of tadalafil in plasma was excellent with no evidence of degradation during sample processing (auto-sampler) and 30 days storage in a freezer. This validated method is applied for the clinical study of the tadalafil in human volunteers.     

Simultaneous quantification of emtricitabine and tenofovir in human plasma using high-performance liquid chromatography after solid phase extraction

An accurate, sensitive and simple reverse-phase (RP) high-performance liquid chromatography (HPLC) assay for the simultaneous quantitative determination of emtricitabine and tenofovir in human blood plasma is described. Using 200 microL of plasma and BOND ELUT-C18 Varian columns, the solid phase extraction (SPE) method results in a clean baseline and high extraction efficiencies (100% for emtricitabine and 98.6% for tenofovir). An Atlantistrade mark dC-18 analytical column is used along with an 18 min linear gradient elution of phosphate buffer (pH 5.7) and methanol to provide sharp peaks for emtricitabine at 280 nm, tenofovir at 259 nm, and the internal standard 2',3'didoxyuridine (DDU) at 262 nm. The method was validated over the range of 10-10,000 ng/mL for both analytes, and is accurate (average accuracies of three different concentrations ranged from 98 to 105% for emtricitabine and 97 to 103% for tenofovir) and precise (within- and between-day precision ranged from 1.7 to 3.7% and 3.7 to 5.2%, respectively). This method is suitable for use in clinical pharmacokinetic studies and is nimble enough for therapeutic drug monitoring.     

Determination of remifentanil in human plasma by liquid chromatography-tandem mass spectrometry utilizing micro extraction in packed syringe (MEPS) as sample preparation

Remifentanil is a synthetic short-acting opioid with a short half-life that is being used during anaesthesia of small children. In this work an LC-MS/MS method for remifentanil quantification in 20 μL volume of human plasma was developed and validated in connection with a clinical study on neonatal children. Sample preparation was performed with micro extraction in packed syringe (MEPS), which is a miniaturization of solid phase extraction. For this method a mixed phase sorbent M1 (C8, cation exchange), and a protocol for basic compound extraction was followed. Remifentanil-(13)C(6) was used as internal standard. For chromatographic separation, a C18 analytical column with gradient elution was used with mobile phase consisting of aqueous 0.1% formic acid and methanol. The total analysis time was 5.0 min and the measuring range was between 0.05 and 50 ng/mL. Precision and accuracy were with acceptance criteria of ±15%. Plasma samples were stable for 5 weeks at -20°C and for 4h at room temperature while 50% was lost after 24h. This method was successfully applied for remifentanil determination in clinical samples and results agreed with a reference method. With this method using MEPS, a low limit of quantification and much reduced sample volume was obtained as compared with previous methods.     

Quantitative determination of cholesterol, sitosterol, and sitostanol in cultured Caco-2 cells by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

In this study, we describe a simple liquid extraction (methanol/choloroform, 1:1, v/v) method for endogenous free cholesterol and administered sterols extracted from cultured Caco-2 cells. To quantify sterol contents in Caco-2 cells, a new HPLC-APCI-MS method was developed. All the sterols were baseline separated using reversed-phase column (C8, 2.1 mm x 150 mm, 3.5 microm) and isocratic conditions (90%, v/v, methanol-water mixture containing 0.2 mM ammonium acetate). The full scan mass spectra of sterols were measured by an ion trap mass spectrometer equipped with an APCI ion source. The intense fragment ions resulting from the loss of water [M+H-H2O]+ (m/z 369, 395, 397 and 399 for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively) were used for determinations. The absolute extraction recovery of sterols from the spiked cell samples were 109.7+/-26.2, 105.7+/-5.1, 109.8+/-5.0 and 99.0+/-7.0% for cholesterol, stigmasterol, sitosterol, and sitostanol, respectively. Furthermore, no significant matrix effect was observed for the sterols in the cell samples. The sample assay was based on the internal standard method using stigmasterol as an internal standard. The method was linear over the concentration ranges of 0.45-9.0 microM (cholesterol) and 0.225-7.2 microM (sitosterol and sitostanol). The within- and between-day precision was less than 7% and accuracy ranged from 93.51 to 101.77%. The lowest limit of quantitation (LLOQ) was 0.225 microM for sitosterol and sitostanol, and 0.45 microM for cholesterol. The accuracy range was 95-106% and precision was lower than 9% for all LLOQ values.     

Validation of high-performance liquid chromatography assay for quantification of formoterol in urine samples after inhalation using UV detection technique

A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb (250 mm x 4.6 mm x 5 microm) analytical column maintained at 30 degrees C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n=10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.     

High-throughput solid-phase extraction for the determination of cimetidine in human plasma

For the implementation and validation of an automated `high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C₁₈ sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.     

Determination of plasma testosterone using a simple liquid chromatographic method

A simple and sensitive high-performance liquid chromatographic (HPLC) method using ultraviolet detection was developed for the determination of testosterone in human plasma. Testosterone and the internal standard, griseofulvin, were extracted from 0.50 ml plasma sample using a mixture of dichloromethane-2,2,4-trimethylpentane (3:2, v/v). The mobile phase, consisted of 0.02 M sodium dihydrogenphosphate-acetonitrile-methanol (51:47:2, v/v) adjusted to pH 3.1 and delivered to a C(18) analytical column (150 x 4.6 mm I.D., 4 microm particles) at a flow-rate of 1 ml/min while the detection wavelength was set at 240 nm with a sensitivity range of 0.005 a.u.f.s. The method has a quantification limit of 1.6 ng/ml. Recoveries of testosterone were all greater than 92% over the linear concentration range of 1.6-400 ng/ml while that of griseofulvin was approximately 95%. The within- and between-day RSD values were all less than 8% while the accuracy values ranged from 96.0 to 106.0% over the concentration range studied. The method was applied to the analysis of early morning plasma testosterone levels of 12 healthy human male volunteers. The levels were found to range from 3.1 to 8.4 ng/ml, within the normal range reported in the literature.     

Determination of the HIV integrase inhibitor, MK-0518 (raltegravir), in human plasma using 96-well liquid-liquid extraction and HPLC-MS/MS

An HPLC-MS/MS method was developed for the determination of MK-0518 (raltegravir), an HIV integrase inhibitor, in human plasma over the concentration range of 2-1000 ng/mL. Stable isotope labeled (13)C(6)-MK-0518 was used as an internal standard. The sample preparation procedure utilized liquid-liquid extraction with hexane:methylene chloride in the 96-well format with a 200 microL plasma sample size. The compounds were chromatographed on an Ace C(18) (50 x 3.0 mm, 3 microm, titanium frits) column with 42.5/57.5 (v/v %) 0.1mM EDTA in 0.1% formic acid/methanol mobile phase at a flow rate of 0.5 mL/min. Multiple reaction monitoring of the precursor-to-product ion pairs for MK-0518 (m/z 445-->109) and (13)C(6)-MK-0518 (m/z 451-->367) on an Applied Biosystem API 4000 HPLC-MS/MS was used for quantitation. Intraday precision of standard curve concentrations in five different lots of control plasma was within 3.2%, while accuracy ranged from 94.8 to 106.8%. The mean extraction recovery of spiked plasma samples was 87%. Quality control (QC) samples were stored at -20 degrees C. Initial within day analysis showed QC accuracy within 7.5% of nominal with precision of 3.1% or less. The plasma QC samples were demonstrated to be stable for up to 23 months at -20 degrees C. The method described has been used to support over 18 clinical studies during Phase I through III of clinical development.     

Development of a high throughput 96-well plate sample preparation method for the determination of trileptal (oxcarbazepine) and its metabolites in human plasma

A high throughput preparation method for the determination of trileptal (oxcarbazepine, OXC) and its mono (MHD) and dihydroxy (DHD) metabolites in human plasma, using 96-well plate technology, has been developed and validated according to international regulatory requirements. Preparation of plasma samples (50 μl) containing the compounds to be analysed involved solid-phase extraction (SPE) on Empore C₁₈ 96-well SPE plates. Eluates from the plate were injected onto a reversed-phase column (Hypersil C₁₈, 3 μm) with UV detection at 210 nm. Detector response was linear over the ranges 0.2–10, 0.1–200 and 0.1–20 μmol/l, for OXC, MHD and DHD, respectively, with relative standard deviations from 1 to 10% and mean accuracies within 4% of the nominal values (number of standard curves=3 in duplicate). The limits of quantitation were 0.2, 0.1 and 0.1 μmol/l, respectively. The overall mean accuracies ranged from 96 to 106% and precision was in the range 4 to 11%. Cross validation indicated no significant difference between plasma concentrations obtained using the 96-well method and the previous method using a traditional SPE method with a 50 mg C₁₈ cartridge. About a threefold increase in sample throughput and a twofold decrease of plasma volume required for the assays, were the main advantages obtained from the previous method. The method was applied for the determination of 3000 plasma samples from clinical studies.     

Determination of grepafloxacin in plasma and urine by a simple and rapid high-performance liquid chromatographic method

A rapid, specific, sensitive and economical method has been developed and validated for the determination of grepafloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with UV detection. Plasma proteins were removed by a fast and efficient procedure that has eliminated the need for costly extraction and evaporation. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a reversed-phase TSK gel column with an isocratic mobile system. The method had a quantification limit of 0.05 μg/ml in plasma and 0.5 μg/ml in urine. The coefficients of variation (C.V.) were less than 4% for within- and between-day analyses. The method was successfully applied to a pharmacokinetic study, and was proved to be simple, fast and reproducible.     

Fast, fully automated analysis of voriconazole from serum by LC-LC-ESI-MS-MS with parallel column-switching technique

Voriconazole is a novel broad-spectrum antifungal agent. We developed an on-line LC-LC-MS-MS method for fully automated and direct analysis of voriconazole in raw human serum. After injection of human serum size-selective sample fractionation and analyte extraction was achieved using an extraction column (25 mm x 4 mm) packed with a restricted access material (RAM, LiChrospher ADS C(8), 25 microm). On-line transfer of voriconazole from the extraction column was followed by chromatography separation on a C(18) column. Detection was done by ESI-MS-MS. The total analysis time was 13 min, managed by parallel extraction and chromatographic separation. This LC-MS assay was fully validated. The lower limit of quantification was 0.05 microg/ml. The automated inline extraction of voriconazole described here eliminates the need for difficult and time-consuming sample pre-treatment. Other advantages of the new method are that only a small quantity (5 microl) of serum is needed and that the method is very specific.