Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

High-performance liquid chromatography method with ultraviolet detection for the quantification of the BCR-ABL inhibitor nilotinib (AMN107) in plasma, urine, culture medium and cell preparations

An isocratic and sensitive HPLC assay was developed allowing the determination of the new anticancer drug nilotinib (AMN107) in human plasma, urine, culture medium and cell samples. After protein precipitation with perchloric acid, AMN107 underwent an online enrichment using a Zirchrom-PBD precolumn, was separated on a Macherey-Nagel C18-HD column and finally quantified by UV-detection at 258 nm. The total run time is 25 min. The assay demonstrates linearity within a concentration range of 0.005-5.0 microg/ml in plasma (r(2)=0.9998) and 0.1-10.0 microg/ml in urine (r(2)=0.9913). The intra-day precision expressed as coefficients of variation ranged depending on the spiked concentration between 1.27-9.23% in plasma and 1.77-3.29% in urine, respectively. The coefficients of variation of inter-day precision was lower than 10%. Limit of detection was 0.002 microg/ml in plasma and 0.01 microg/ml in urine. The described method is stable, simple, economic and is routinely used for in vivo and in vitro pharmacokinetic studies of AMN107.     

A sensitive gas chromatographic/mass spectrometric method for the resolution and quantification of ethosuximide enantiomers in biological fluids

A modified specific, sensitive and reproducible chiral gas chromatographic (GC) method for the resolution and quantification of ethosuximide enantiomers in urine and plasma was developed. The samples were extracted by liquid-liquid extraction, using diethylether and the enantiomers were separated and quantified on a chiral gas chromatographic column (25QC2 / CYDEX- beta 0.25). The method involved the use of GC/MS instrumentation for the acquisition of data in the electron impact selective-ion monitoring mode, collecting ions characteristic of both ethosuximide and alpha, alpha - dimethyl - beta - methylsuccinimide, the internal standard and of mass-to-charge ratio (m/z) exactly equal to 55 and 70 units. The limit of quantitation of the method was 2.5 microg/ml for both urine and plasma with both enantiomers. The method proved to be linear, precise and reproducible in the 5-300 microg/ml concentration range for urine samples and in the 10-250 microg/ml concentration range for plasma samples. Future research work envisaged the application of this method in pharmacokinetic and pharmacodynamic studies.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

Liquid chromatography method for determination of bivalirudin in human plasma and urine using automated ortho-phthalaldehyde derivatization and fluorescence detection

A high-performance liquid chromatographic (HPLC) method was developed using solid-phase extraction, o-phthalaldehyde (OPA) derivatization and fluorescence detection for the determination of the direct thrombin inhibitor bivalirudin in human plasma and urine. The use of this assay will facilitate the study of the pharmacodynamics of bivalirudin in studies of special patient populations. A C(18) bioanalytical column at a flow rate of 1 ml/min with an aqueous trifluoroacetic acid (0.1% TFA in deionized water, pH 2.2, v/v) mobile phase and methanol gradient was used. The assay demonstrated linearity from 3 to 20 microg/ml bivalirudin in plasma, with a detection limit of 1 microg/ml. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of bivalirudin in patients undergoing percutaneous coronary interventions (PCIs).     

High-performance liquid chromatographic determination of 2-hydroxypropyl-gamma-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-gamma-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5-15 microg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.     

Liquid chromatographic assay for dicloxacillin in plasma

A simple high-performance liquid chromatographic method for the determination of dicloxacillin in plasma has been developed. The method only requires 0.5 ml of plasma, phosphate buffer solution (pH = 4.7), acidification with 0.5N hydrochloride acid and liquid extraction with dichloromethane. Posterior evaporation of organic under nitrogen steam and redissolution in mobile phase is carried out. The analysis was performed on a Spherisorb C18 (5 microm) column, using methanol -0.05 M phosphate buffer, pH = 4.7 (75:25; v/v) as mobile phase, with ultraviolet detection at 220 nm. Results showed that the assay is sensitive: 0.5 microg/ml. The response is linear in the range of 0.5 - 10 microg/ml. Maximum inter-day coefficient of variation was 12.4%. Mean extraction recovery obtained was 96.95%. Stability studies showed that the loss was not higher than 10%, samples are stable at room temperature for 6 h, at -20 Celsius for 2 months, processed samples were stable at least for 24 h and also after two freeze-thaw cycles. The method has been used to perform pharmacokinetic and bioequivalence studies in humans.     

Determination of L-threonate in human plasma and urine by high performance liquid chromatography-tandem mass spectrometry

A fast and selective HPLC-MS-MS method was established to determine L-threonate in human plasma and urine. Plasma and urine samples were extracted by protein precipitation and diluted with water, then chromatographed on an YMC J'Sphere C(18) column with methanol-acetonitrile-10mM ammonium acetate (20:5:75, v/v) as mobile phase, and at a flow rate of 0.2 ml/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using negative electrospray ionization (ESI). Multiple reactions monitoring (MRM) was used and L-threonate was quantified by monitoring the ion transition of m/z 134.5-->74.7. The linear calibration curves of L-threonate in plasma and urine were obtained over the concentration range of 0.25-50 microg/ml and 2.5-500 microg/ml, respectively. Lower limit of quantitation was 0.25 and 2.5 microg/ml, respectively. Accuracy was within 85-115%, and intra- and inter-batch precision (R.S.D.%) were within +/-15%. The method proved to be accurate and specific, and was applied to the pharmacokinetic study of L-threonate in Chinese healthy subjects.     

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase

The simultaneous determination of the enantiomers of the β₁-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline preextraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3, 5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (?)-(S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses. © 1993 Wiley-Liss, Inc.     

Chlorpheniramine : I. Rapid quantitative analysis of chlorpheniramine in plasma,saliva and urine by high-performance liquid chromatography

A method was developed for the rapid quantitative analysis of chlorpheniramine in plasma, saliva and urine using high-performance liquid chromatography. A diethyl ether or hexane extract of the alkalinized biological samples was extracted with dilute acid which was chromatographed on a reversed-phase column using mixtures of acetonitrile and ammonium phosphate buffer as the mobile phase. Ultraviolet absorption at 254 nm was monitored for the detection and brompheniramine was employed as the internal standard for the quantitation. The effects of buffer, pH, and acetonitrile concentration in the mobile phase on the chromatographic separation were investigated. A mobile phase 20% acetonitrile in 0.0075 M phosphate buffer at a flow-rate of 2 ml/min was used for the assays of plasma and saliva samples. A similar mobile phase was used for urine samples. The drug and internal standard were eluted at retention volumes of less than 17 ml. The method can also be used to quantify two metabolites, didesmethyl- and desmethylchlorpheniramine, in the urine. The method can accurately measure chlorpheniramine levels down to 2 ng/ml in plasma or saliva using 1 ml of sample, and should be adequate for biopharmaceutical and pharmacokinetic studies. Various precautions for using the assay are discussed.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Determination of Taxotere in human plasma by a semi-automated high-performance liquid chromatographic method

A rapid, selective and reproducible high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed for the determination of the anti-cancer agent Taxotere in biological fluids. The method involves a solid-phase extraction step (C₂ ethyl microcolumns) using a Varian Advanced Automated Sample Processor (AASP) followed by reversed-phase HPLC. The validated quantitation range of the method is 10–2500 ng/ml in plasma with coefficients of variation ≤ 11%. The method is also suitable for the determination of Taxotere in urine samples under the same conditions. The method was applied in a phase I tolerance study of Taxotere in cancer patients, allowing the pharmacokinetic profile of Taxotere to be established.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

Determination of lefucoxib in rat plasma, urine, and feces by high-performance liquid chromatography with fluorescence detection: application in pharmacokinetic studies

A sensitive, specific, and reproducible high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of lefucoxib in rat plasma, urine, and feces. The method involved liquid-liquid extraction using methyl tert-butyl ether, and celecoxib was used as the internal standard. The chromatographic separation was performed on a Kromasil C18 column (250.0 mm x 4.6 mm, 5.0 microm) with a mobile phase gradient consisting of water and methanol at a flow rate of 1 ml min(-1). The assay was linear in the range of 5.0-1000.0 ng ml(-1) with a correlation coefficient (r) of 0.9994. The limit of quantification was 5.0 ng ml(-1). Inter- and intra-assay precisions were 相似文献   

Determination of disodium clodronate in human plasma and urine using gas-chromatography-nitrogen-phosphorous detections: validation and application in pharmacokinetic study

We present a specific method for the determination of disodium clodronate in human plasma and urine using a gas-chromatographic system with nitrogen phosphorus detector (NPD). The compound was extracted from plasma and urine samples by an anion-exchange resin and derivatizated with bistrimethylsilyltrifluoroacetamide (BSTFA). Sodium bromobisphosphonate was used as internal standard. The calibration curves were linear in both plasma and urine, with a regression coefficient r > 0.9975 in plasma and r > 0.9977 in urine.The limit of quantitation was 0.3 microg/ml in plasma and 0.5 microg/ml in urine. The method was validated by intra-day assays at three concentration levels. During the study we carried out inter-day assays to confirm the feasibility of the method. The precision in plasma at 0.5, 15, and 45 microg/ml was 12.4, 0.2, and 6.5% (n = 40), respectively; in urine at 0.8, 8, and 40 microg/ml it was 8.6, 6.4, and 9.3% (n = 40), respectively.The method was accurate and reproducible, and was successfully applied to determine the pharmacokinetic parameters of clodronate in healthy volunteers after intravenous infusion and intramuscular injection of 200 mg of the compound. The Cmax after intravenous infusion and intramuscular injection was 16.1 and 12.8 microg/ml, respectively. AUC(0-48 h) after infusion administration and intramuscular injection was 44.2 +/- 18.0 and 47.5 +/- 12.4 h microg/ml, respectively. The elimination half-life in both administrations was 6.31 +/- 2.7 h.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Stereospecific determination of mefloquine in biological fluids by high-performance liquid chromatography

A sensitive stereoselective HPLC method was developed for determination of mefloquine (MFQ) enantiomers in plasma, urine and whole blood. The assay involved liquid-liquid extraction of MFQ from biological fluids with a mixture of hexane and isopropanol in the presence of sodium hydroxide and derivatization of the residue by (+)-(S)-naphthylethylisocyanate (NEIC) as chiral derivatizing reagent. Separation of the resulting diastereomers was performed on a silica normal-phase column using chloroform-hexane-methanol (25:74:1) as the mobile phase with a flow-rate of 1 ml/min. Using 200 μl of plasma or whole blood, the limit of determination was 0.2 μg/ml with UV detection for both enantiomers. The limit of determination in 500 μl of urine was 0.08 μg/ml with UV detection.     

Method for monitoring alginate released in biological fluids by high-performance anion-exchange chromatography with pulsed amperometric detection

The use of alginate-entrapped cells in cell therapy requires a method for monitoring possible released compound within biological fluids following either their implantation or inoculation in artificial organs. Oligomannuronic and oligoguluronic acids were prepared by enzymatic depolymerization with alginate lyase from Pseudomonas alginovora, characterized by high-performance anion-exchange chromatography with pulsed amperometric detection and quantitated in human, pig and rabbit blood, urine and tissue samples. The method was tested for linearity and detection limit, accuracy, intra- and inter-day precision. The limit of detection was 3 microgram/ml in both urine and plasma and 5 mg/g of tissues. The relative standard deviations (RSDs) of intra-day precision were 6.0-16.6% and 4.8-8.7% in plasma and urine, respectively; the RSDs of inter-day precision were 5.1-14.4% and 5.0-11.6% in plasma and urine, respectively. Thus, this method appears suitable for the measurement of released alginate from entrapped cells used in cell therapy.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.