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.     

Quantification of sunitinib in human plasma by high-performance liquid chromatography-tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using LC/MS/MS for determination of sunitinib in human plasma. Sample preparation involved a liquid-liquid extraction by the addition of 0.2mL of plasma with 4.0mL tert-butyl-methyl-ether extraction solution containing 25ng/mL of the internal standard clozapine. Separation of compounds was achieved on a C18 (50mmx2.1mm i.d., 3.5microm) analytical column using a mobile phase consisting of acetonitrile/H20 (65:35, v/v) containing 0.1% formic acid and isocratic flow at 0.150mL/min for 3min. The analytes were monitored by tandem-mass spectrometry with electrospray positive ionization. Linear calibration curves in human plasma were generated over the range of 0.2-500ng/mL with values for the coefficient of determination of >0.9950. Within- and between day precision and accuracy were < or =10%. The method was applied to the quantitation of sunitinib in plasma samples from a patient receiving daily oral therapy with sunitinib.     

Liquid chromatography-tandem mass spectroscopy assay for quercetin and conjugated quercetin metabolites in human plasma and urine

A sensitive and specific method was developed and validated for the quantitation of quercetin in human plasma and urine. The application of liquid chromatography-tandem mass spectrometry (LC/MS/MS) with a TurboIonspray (TIS) interface in negative mode under multiple reactions monitoring was investigated. Chromatographic separation was achieved on a C12 column using a mobile phase of acetonitrile/water with 0.2% formic acid (pH 2.4) (40/60, v/v). The detection limit was 100 pg/ml and the lower limit of quantification was 500 pg/ml for plasma samples; the detection limit was 500 pg/ml and the lower limit of quantification was 1 ng/ml for urine samples. The calibration curve was linear from 1 to 800 ng/ml for plasma samples and was linear from 1 to 200 and 50 to 2000 ng/ml for urine samples. All the intra- and inter-day coefficients of variation were less than 11% and intra- and inter-day accuracies were within +/-15% of the known concentrations. This represents a LC/MS/MS assay with the sensitivity and specificity necessary to determine quercetin in human plasma and urine. This assay was used to determine both parent quercetin and the quercetin after enzymatic hydrolysis with beta-glucuronidase/sulfatase in human plasma and urine samples following the ingestion of quercetin 500 mg capsules.     

Determination of tiropramide in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of tiropramide in human plasma was developed. Tiropramide and internal standard, cisapride were extracted from human plasma by liquid-liquid extraction and analyzed on a Luna C8 column with the mobile phase of acetonitrile-ammonium formate (10mM, pH 4.5) (50:50, v/v). The analytes was detected using an electrospray ionization tandem mass spectrometry in the multiple-reaction-monitoring mode. The standard curve was linear (r=0.998) over the concentration range of 2.0-200 ng/ml. The intra- and inter-assay coefficients of variation ranged from 2.8 to 7.8 and 6.7 to 8.9%, respectively. The recoveries of tiropramide ranged from 50.2 to 53.1%, with that of cisapride (internal standard) being 60.9+/-5.3%. The lower limit of quantification for tiropramide was 2.0 ng/ml using 100 microl plasma sample. This method was applied to the pharmacokinetic study of tiropramide in human.     

Development and validation of a liquid chromatography/tandem mass spectrometry assay for the simultaneous determination of nateglinide, cilostazol and its active metabolite 3,4-dehydro-cilostazol in Wistar rat plasma and its application to pharmacokinetic study

Nateglinide (NTG), an insulin secretogogue, has been studied in rats for drug-drug interaction with cilostazol (CLZ), an antiplatelet agent commonly used in diabetics. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) based method that is capable of simultaneous monitoring plasma levels of nateglinide, cilostazol, and its active metabolite 3,4-dehydro-cilostazol (DCLZ). All analytes including the internal standard (Repaglinide) were chromatographed on reverse phase C(18) column (50 mm x 4.6mm i.d., 5 microm) using acetonitrile: 2mM ammonium acetate buffer, pH 3.4 (90:10, v/v) as mobile phase at a flow rate 0.4 ml/min in an isocratic mode. The detection of analyte was performed on LC-MS/MS system in the multiple reaction monitoring (MRM) mode. The quantitations for analytes were based on relative concentration. The method was validated over the concentration range of 20-2000 ng/ml and the lower limit of quantitation was 20 ng/ml. The recoveries from spiked control samples were >79% for all analytes and internal standard. Intra- and inter-day accuracy and precision of validated method were with in the acceptable limits of <15% at all concentration. The quantitation method was successfully applied for simultaneous estimation of NTG, CLZ and DCLZ in a pharmacokinetic drug-drug interaction study in Wistar rats.     

Rapid and sensitive LC–MS/MS assay for the quantitation of 20(S)-protopanaxadiol in human plasma

This paper describes a rapid and sensitive method for the quantitation of 20(S)-protopanaxadiol (PPD) in human plasma based on high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and internal standard (I.S.), ginsenoside Rh₂, were extracted from plasma by liquid–liquid extraction and separated on a Zorbax extend C₁₈ analytical column using methanol–acetonitrile-10 mM ammonium acetate (47.5:47.5:5, v/v/v) as mobile phase. Detection was by tandem mass spectrometry using electrospray ionization in the positive ion mode and multiple reaction monitoring (MRM). The assay was linear over the concentration range 0.1–100.0 ng/ml with a limit of detection of 0.05 ng/ml. The method was successfully applied to a clinical pharmacokinetic study in healthy volunteers after a single oral administration of a PPD 25 mg capsule.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Quantitative determination of apigenin and its metabolism in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

Apigenin is a flavone and is being developed for treatment of cardiovascular disease. A sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the measurement of apigenin and luteolin levels in rat plasma is described. Analytes were separated on a separation by a Luna C(18) (5 microm, 100 mm x 2.0 mm) column with acetonitrile:methanol:water (35:40:60, v/v/v) as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. Good linearity (R(2)>0.9997) was observed for both analytes over the range of 2.5-5000 ng/mL in 0.1mL of rat plasma. The overall accuracy of this method was 93-105% for apigenin and 95-112% for luteolin in rat plasma. Intra-assay and inter-assay variabilities were less than 11% in plasma. The lowest quantitation limit for both apigenin and luteolin was 2.5 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC/MS/MS method was demonstrated in a pilot pharmacokinetic study in rats following intravenous administration of apigenin. Metabolism of apigenin to luteolin in vivo was established.     

Quantitative determination of the anticancer agent tubeimoside I in rat plasma by liquid chromatography coupled with mass spectrometry

Tubeimoside I is an important component isolated from Bolbostemma paniculatum. Tubeimoside I has been demonstrated to possess many pharmacological activities, including anti-inflammatory, antitumor, and antitumor-promoting effects. The purpose of the present study was to examine in vivo pharmacokinetics and bioavailability of tubeimoside I in rats by using a liquid chromatography coupled with mass spectrometry quantitative detection method (LC/MS). The plasma samples were deproteinated, evaporated and reconstituted in 100 microl methanol prior to analysis. The separation was performed by Waters Symmetry C18 reversed-phase column (3.5 microm, 150 mm x 2.1mm, Waters Inc., USA) and a SB-C18 guard column (5 microm, 20 mm x 4.0mm). The mobile phase was a mixture of acetonitrile and water containing 5 microM NaAc (60:40, v/v). The method was validated within the concentration range 20-5000 ng/ml, and the calibration curves were linear with correlation coefficients >0.999. The lowest limit of quantitation (LLOQ) for tubeimoside I was 20 ng/ml in 0.1 ml rat plasma. The intra-assay accuracy and precision ranged from 92.4 to 104.9% and from 5.8 to 10.5%, respectively, while inter-assay accuracy and precision ranged from 94.2 to 95.0% and from 5.1 to 8.8%, respectively. The method was further applied to assess pharmacokinetics and oral bioavailability of tubeimoside I after intravenous and oral administration to rats. The oral bioavailability of tubeimoside I is only 0.23%, which indicates that tubeimoside I has poor absorption or undergoes acid-induced degradation. Practical utility of this new LC/MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Determination of ragaglitazar,a novel dual acting peroxisome proliferator-activated receptor (PPAR) alpha and -gamma agonist,in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry

A sensitive and specific LC/MS/MS method has been developed and validated for determination of ragaglitazar (NNC 61-0029 or DRF 2725) in human plasma. After solid-phase extraction (SPEC((R)) PLUS C(8)) of plasma, separation was performed on a Symmetry Shield RP8 column (mobile phase: acetonitrile: 10 mM ammonium acetate, pH 5.6 (40:60 v/v)). Two ranges were validated having LLOQs of either 0.500 or 100 ng/ml and linearity up to either 500 or 50000 ng/ml. The intra-assay precision and accuracy were 1.1% to 15.7% and 85.8% to 118.2% (range 0.500-500 ng/ml) and 2.0% to 8.8% and 92.9% to 104.8% (range 100-50000 ng/ml). The method was applied for determination of ragaglitazar in plasma from phase 1 and 2 clinical studies.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

High-throughput liquid chromatography-tandem mass spectrometry determination of bupropion and its metabolites in human, mouse and rat plasma using a monolithic column

In the present work, a high-throughput LC/MS/MS method using a Chromolith RP-18 (50 mm x 4.6 mm) monolithic column was developed and partially validated for the determination of bupropion (BUP), an anti-depressant drug, and its metabolites, hydroxybupropion and threo-hydrobupropion (TB), in human, mouse, and rat plasma. A modern integrated liquid chromatograph and an LC/MS/MS system with a TurboIonSpray (TIS) interface were used for the positive electrospray selected reaction monitoring (SRM) LC/MS analyses. Spiked control plasma calibration standards and quality control (QC) samples were extracted by semi-automated 96-well liquid-liquid extraction (LLE) using ethyl acetate. A mobile phase consisting of 8mM ammonium acetate-acetonitrile (55:45, v/v) delivered isocratically at 5 ml/min, and split post-column to 2 ml/min directed to the TIS, provided the optimum conditions for the chromatographic separation of bupropion and its metabolites within 23s. The isotope-labeled D(6)-bupropion and D(6)-hydroxybupropion were used as internal standards. The method was linear over a concentration range of 0.25-200 ng/ml (bupropion and threo-hydrobupropion), and 1.25-1000 ng/ml (hydroxybupropion). The intra- and inter-day assay accuracy and precision were within 15% for all analytes in each of the biological matrices. The monolithic column performance as a function of column backpressure, peak asymmetry, and retention time reproducibility was adequately maintained over 864 extracted plasma injections.     

Determination of the retinobenzoic acid derivative Am580 in rat plasma by high-performance liquid chromatography

A specific liquid chromatographic method for the determination of 4-[[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbonyl]amino]benzoic acid (Am580) in rat plasma is described. The procedure includes one-step isolation of the compound and the internal standard (naphtol AS) from protein precipitated with acetonitrile, resolution on a reversed-phase column (Supelcosil LC18-DB, 5 μm) with water-acetonitrile-methanol-n-butanol (45:40:14:1, v/v) containing 65 mM ammonium acetate as elution system and UV absorbance detection at 280 nm. The assay was linear over a wide range (25–5000 ng ml⁻¹) and the limit of quantitation was 25 ng ml⁻¹ using 0.2 ml of plasma. It was precise and reproducible enough for pharmacokinetic studies. Application to a preliminary disposition study in the rat indicated that Am580 was characterized by a relatively large apparent volume of distribution (1.1–1.5 l kg⁻¹) and small clearance (8.8–9.7 ml min⁻¹ kg⁻¹). Its pharmacokinetic behaviour was linear within the dose range considered (2 and 10 mg kg⁻¹, i.p.).     

Application of liquid chromatography–turbo ion spray tandem mass spectrometry for quantitative analysis of a potent motilin receptor agonist,EM574, and its metabolites in human plasma

A liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the simultaneous determination of a new potent motilin receptor agonist as erythromycin derivative, EM574 (erythromycin derivative), and its three metabolites, M-IV, M-V and M-VI, in human plasma was developed. The internal standards (I.S.s) used were deuterated EM574, M-IV and M-V. For the quantitation of M-VI, deuterated M-V was used. The analytes and I.S. were extracted from plasma samples with diethyl ether at neutral pH. A turbo ion spray interface was used as the ion source of LC–MS–MS, and the analysis was performed in the selected reaction monitoring mode. The lower quantitation limits for all the analytes were 0.05 ng/ml when 0.2 ml of plasma was used, and the standard curves were linear in the range 0.05 to 20 ng/ml. The method was precise; the intra- and inter-day precisions of the method were not more than 19.8% for all the analytes. The accuracy of the method was good with the deviations between added and calculated concentrations of each analyte being typically within ±11.2%.     

Quantitation of iptakalim in human plasma by high-performance liquid chromatography-tandem mass spectrometry

This paper describes a rapid and sensitive analytical method for the quantitation of iptakalim, a novel antihypertensive drug, in human plasma. The method is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) using sildenafil as internal standard. Sample preparation involved liquid-liquid extraction with dichloromethane-diethyl ether (2:3, v/v) in a basic environment. Chromatography was carried out on an amino column with a mobile phase consisting of acetonitrile-water (55:45, v/v, water containing 0.5% formic acid). Detection employed electrospray ionization (ESI) tandem mass spectrometry in the multiple-reaction-monitoring (MRM) mode. The assay was linear in the concentration range of 0.5-100 ng/ml with a lower limit of quantitation (LLOQ) of 0.5 ng/ml. Intra- and inter-day precision (R.S.D.) were <4.5% and <12.0%, respectively and the accuracy (R.E.) was in the range +/-5%. The method was successfully applied to a single oral dose pharmacokinetic study in human volunteers.     

Quantitative determination of formononetin and its metabolite in rat plasma after intravenous bolus administration by HPLC coupled with tandem mass spectrometry

A new simple, rapid, sensitive and accurate quantitative detection method using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for the measurement of formononetin (FMN) and daidzein (DZN) levels in rat plasma is described. Analytes were separated on a Supelco Discovery C18 (4.6 × 50 mm, 5.0 μm) column with acetonitrile: methanol (50:50, v/v) and 0.1% acetic acid in the ratio of 90:10 (v/v) as a mobile phase. The method was proved to be accurate and precise at linearity range of 5–100 ng/mL with a correlation coefficient (r) of ≥0.996. The intra- and inter-day assay precision ranged from 1.66–6.82% and 1.87–6.75%, respectively; and intra- and inter-day assay accuracy was between 89.98–107.56% and 90.54–105.63%, respectively for both the analytes. The lowest quantitation limit for FMN and DZN was 5.0 ng/mL in 0.1 mL of rat plasma. Practical utility of this new LC–MS/MS method was demonstrated in a pharmacokinetic study in rats following intravenous administration of FMN.     

Single quadrupole mass spectrometry for pre-clinical pharmacokinetic analysis: quantitation of carvedilol in dog plasma

The pharmaceutical industry standard for bioanalysis is LC/MS/MS. There are, however, many instances where a single quadrupole detector could successfully be used to provide adequate sensitivity and selectivity for quantitation of drug substances in biological matrices. This paper presents one example of how a single quadrupole detector can be employed in a sensitive and selective analytical method for quantitation of carvedilol. A Synergi Hydro-RP (50 mm x 2 mm i.d.; 4 microm) column was used with acetonitile:water:formic acid mobile phase (32:68:0.01, v/v) at a flow rate of 200 microL/min into a single quadrupole mass spectrometer with an electrospray interface in the positive SIM mode. Using a 300 microL plasma aliquot and a liquid-liquid extraction procedure the limit of quantitation for the assay was 1 ng/mL. The assay utility was demonstrated in the analysis of carvedilol pharmacokinetic profiles in beagle dogs following oral carvedilol administration.     

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS

An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm x 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 microl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.     

Determination of dioscin in rat plasma by liquid chromatography-tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for dioscin in rat plasma was developed. Ginsenoside Rh2 was employed as an internal standard. Dioscin is a naturally occurring saponin present in many traditional Chinese medicinal plants. Dioscin was determined after the acetonitrile-mediated plasma protein precipitation. The mobile phase consisted of acetonitrile:10 mmol/l aqueous ammonium acetate (95:5, v:v), which was pumped at 0.8 ml/min. The analytical column (100 mm x 4.6 mm i.d.) was packed with Hypersil ODS material (5 microm). The standard curve was linear from 1 to 100 ng/ml. The assay was specific, accurate (percentage deviations from nominal concentrations were <10%), precise and reproducible (within- and between-day coefficients of variation <10%). Dioscin in rat plasma was stable over three freeze-thaw cycles and at ambient temperatures for 24 h. The utility of the assay was demonstrated by determining dioscin plasma concentrations in five rats for 120 h following a single oral gavage dose of 90 mg/kg.     

Validation and implementation of a liquid chromatography/tandem mass spectrometry assay for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma

A reversed-phased liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of the total and unbound RO4929097, a γ-secretase inhibitor targeting Notch signaling, in human plasma. Sample preparation involved a liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved on a Waters X-Terra? MS C(18) column with an isocratic mobile phase consisting of methanol/0.45% formic acid in water (60:40, v/v) running at a flow rate of 0.2 ml/min for 6 min. The lower limits of quantitation (LLOQs) were 5 ng/ml for the total RO4929097 in plasma and 0.5 ng/ml for the unbound drug in phosphate buffer solution (PBS). Calibration curves were linear over RO4929097 concentration range of 5-2000 ng/ml in plasma for the total drug and 0.5-200 ng/ml in PBS for the unbound drug. The intra-day and inter-day accuracy and precision were within the generally accepted criteria for bioanalytical method (<15%). The method has been successfully employed to characterize the total and unbound plasma pharmacokinetics of RO4929097 after its oral administration in cancer patients.