Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

Liquid chromatography-mass spectrometry assay for quantitation of ifosfamide and its N-deschloroethylated metabolites in rat microsomal medium

A specific and sensitive quantitative assay has been developed using high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) for the simultaneous quantitation of the antitumor drug ifosfamide (IFM) and its two metabolites, N2-deschloroethylifosfamide (N2-DCE-IFM) and N3-deschloroethylifosfamide (N3-DCE-IFM) in microsomal medium. The analytes and the internal standard (cyclophosphamide) were isolated by ethylacetate extraction from rat liver microsomes. They were analysed on a Nucleosil C18 HD column (125 mm x 4 mm, 5 microm) using a step gradient with the mobile phase (2 mM ammonium formate and methanol). The HPLC-ESI-MS method used selected ion monitoring of ions m/z 199.1 Th and m/z 261.1 Th and was validated in the concentrations ranges of 100-5000 ng/mL for IFM and 50-2500 ng/mL for its N-deschloroethylated metabolites (DCE-IFM) with good accuracy and precision (CV less than 15%). The low limits of quantitation (LLOQ) were found at 50 ng/mL for N-deschloroethylated metabolites and at 100 ng/mL for the parent drug (IFM). The method was applied for the determination of ifosfamide and its N-deschloroethylated metabolites in rat microsomal incubations.     

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC-MS/MS--application to a preclinical pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quanti?cation (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.     

Simultaneous determination of five main active bufadienolides of Chan Su in rat plasma by liquid chromatography tandem mass spectrometry

To study the pharmacokinetics of Chan Su, a sensitive and selective method was developed and validated for the determination of five main bufadienolides (cinobufagin, resibufogenin, bufalin, bufotalin and arenobufagin) in rat plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate after internal standard (IS, caudatin) spiked. The separation was performed by a ZORBAX SB-C18 column (3.5 microm, 2.1 mmx100 mm) and a C18 guard column (5 microm, 4.0 mmx2.0 mm) with an isocratic mobile phase consisted of acetonitrile-water-formic acid (50:50:0.05, v/v/v) at a flow rate of 0.3 mL/min. The Agilent G6410A triple quadrupole LC/MS system was operated under the multiple reaction monitoring mode (MRM) using the electrospray ionization technique in positive mode. The nominal retention times for cinobufagin, resibufogenin, bufalin, bufotalin, arenobufagin and caudatin were 3.07, 3.55, 2.30, 1.62, 1.22 and 3.43 min, respectively. All analytes showed good linearity in a wide concentration range (r>0.995) and their lower limits of quantification (LLOQ) were all 1.0 ng/mL. The method was linear for all analytes with correlation coefficients>0.995 for all analytes. The average extract recoveries of the five analytes from rat plasma were all over 85%, the precisions and accuracies determined were all within 15%. This method has been successfully applied to pharmacokinetic study of Chan Su in rats following oral administration.     

Simultaneous determination of two Amadori compounds in Korean red ginseng (Panax ginseng) extracts and rat plasma by high-performance anion-exchange chromatography with pulsed amperometric detection

A new simple, rapid and sensitive high-performance anion-exchange chromatography method with pulsed amperometric detection (HPAEC-PAD) was developed and validated for the simultaneous determination of two Amadori compounds, arginyl-fructose and arginyl-fructosyl-glucose in Korean red ginseng (Panax ginseng) extracts, rat plasma. Separation of the two target analytes was efficiently undertaken on CarboPac PA1 anion-exchange column with isocratic elution (400 mM sodium hydroxide and deionized water (90:10, v/v)) at flow rate 0.7 mL/min within 15 min of single chromatographic run. Under optimized conditions, the detection limits (signal-to-noise ratio equal to 3) were 20 and 25 ng/mL for arginyl-fructose and arginyl-fructosyl-glucose, respectively. Calibration curves of peak area for the two analytes were linear over three orders of magnitude with a correlation coefficients greater than 0.999. The accuracy of the method was tested by recovery measurement of the spiked samples which yielded good results of 94.15-102.62%. This method was successfully applied to the quantification of arginyl-fructose and arginyl-fructosyl-glucose in herbal extracts and in the plasma samples from rat.     

A sensitive liquid chromatography-mass spectrometry method for simultaneous determination of two active chromones from Saposhnikovia root in rat plasma and urine

A sensitive and efficient liquid chromatography-mass spectrometry method was developed and validated for the simultaneous determination of two active chromones (prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol) from Saposhnikovia root in rat plasma and urine. The plasma or urine samples were prepared by protein precipitation. Chromatographic separation of the two active chromones from matrix interferences was achieved on an Angilent TC-C(18) column with a mobile phase consisted of methanol, water and 0.1% formic acid. Puerarin was added as the internal standard. The method was validated with the concentration range 1.0-100 ng/mL in rat plasma and 10-1000 ng/mL in urine for prim-O-glucosylcimifugin, 1.5-150 ng/mL in plasma and 15-1500 ng/mL in urine for 4'-O-D-glucosyl-5-O-methylvisamminol. The lower limit of quantitation (LLOQ) of prim-O-glucosylcimifugin and 4'-O-D-glucosyl-5-O-methylvisamminol was 1.0 and 1.5 ng/mL in plasma, 10 and 15 ng/mL in urine, respectively. The intra- and inter-day precision across three validation days over the entire concentration range was lower than 9.0% as terms of relative standard deviation (R.S.D.). Accuracy determined at three quality control concentrations (2.0, 25 and 75 ng/mL for prim-O-glucosylcimifugin; 3.0, 37.5 and 112.5 ng/mL for 4'-O-D-glucosyl-5-O-methylvisamminol) ranged from -1.9 to 3.9% as terms of relative error (R.E.). The LC-ESI-MS method was further applied to assess pharmacokinetics and urine excretion of the two chromones after oral administration of Fangfeng extract to rats. Practical utility of this new LC-MS method was confirmed in pilot pharmacokinetic studies in rats following oral administration.     

Simultaneous UFLC-ESI-MS/MS determination of piperine and piperlonguminine in rat plasma after oral administration of alkaloids from Piper longum L.: application to pharmacokinetic studies in rats

The alkaloids from Piper longum L. showed protective effects on Parkinson's disease models in our previous study and piperine and piperlonguminine were the two main constituents in the alkaloids. The present study aimed at developing a rapid, sensitive, and accurate UFLC-ESI-MS/MS method and validating it for the simultaneous determination of piperine and piperlonguminine in rat plasma using terfenadine as the internal standard. The analytes and internal standard (IS) were extracted from rat plasma using a simple protein precipitation by adding methanol/acetonitrile (1:1, v/v). A Phenomenex Gemini 3 u C18 column (20 mm × 2.00 mm, 3 μm) was used to separate the analytes and IS using a gradient mode system with a mobile phase consisting of water with 0.1% formic acid (mobile phase A) and acetonitrile with 0.1% formic acid (mobile phase B) at a flow rate of 0.4 mL/min and an operating column temperature of 25°C. The total analytical run time was 4 min. The detection was performed using the positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode with transitions at m/z 286.1-201.1 for piperine, m/z 274.0-201.1 for piperlonguminine, and m/z 472.4-436.4 for the IS. The calibration curves were both linear (r>0.995) over a concentration range of 1.0 to 1000 ng/mL; the lower limit of quantification (LLOQ) was 1.0 ng/mL for both piperine and piperlonguminine. The intra-day and inter-day precisions (RSD %) were <12.1%, accuracies ranged from 86.6 to 120%, and recoveries ranged from 90.4 to 108%. The analytes were proven stable in the short-term, long-term, and after three freeze-thaw cycles. The method was successfully applied to pharmacokinetic studies of piperine and piperlonguminine in rats after oral administration of alkaloids from P. longum L.     

Development and validation of liquid chromatography-tandem mass spectrometric method for simultaneous determination of fosinopril and its active metabolite fosinoprilat in human plasma

A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for simultaneous determination of the prodrug fosinopril and its major active metabolite fosinoprilat for pharmacokinetic studies in healthy subjects. In order to get the lower limit of quantification (LLOQ), especially for analysis of fosinopril, key points of the method have been investigated including chromatographic conditions and selection of LC-MS/MS conditions. The analytes were extracted from plasma samples by liquid-liquid extraction, separated on a reversed-phase C(8) column using gradient procedure, and detected by tandem mass spectrometry with a triple quadrupole ionization interface. The analytes and internal standard zaleplon were detected using positive electrospray ionization (ESI) in the SRM mode. The LLOQ of the method down to 0.1 ng mL(-1) for fosinopril and 1.0 ng mL(-1) for fosinoprilat were identifiable and reproducible. The standard calibration curves for both fosinopril and fosinoprilat were linear over the ranges of 0.1-15.0 and 1.0-700 ng mL(-1) in human plasma, respectively. The within- and between-batch precisions (relative standard deviation (RSD)%) and the accuracy were acceptable. The validated method was successfully applied to reveal the pharmacokinetic properties of fosinopril and fosinoprilat after oral administration.     

A sensitive and specific liquid chromatography-mass spectrometry method for determination of metacavir in rat plasma

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the quantification of metacavir in rat plasma using tinidazole as an internal standard (I.S.). Following ethyl acetate extraction, the analytes were separated on a Shim-pack ODS (4.6 microm, 150 mm x 2.0 mm I.D.) column and analyzed in selected ion monitoring (SIM) mode with a positive ESI interface using the respective [M+H](+) ions, 266 for metacavir and 248 for tinidazole. The method was validated over the concentration range of 1-600 ng/mL for metacavir. Between and within-batch precisions (R.S.D.%) were all within 15% and accuracy (%) ranged from 92.2 to 105.8%. The lower limit of quantification (LLOQ) was 1 ng/mL. The extraction recovery was on average 89.8%. The validated method was used for the pharmacokinetic study of metacavir in rats.     

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.     

Simultaneous determination of tectorigenin, irigenin and irisflorentin in rat plasma and urine by UHPLC-MS/MS: application to pharmacokinetics

A sensitive and reliable ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS) has been developed and validated for the simultaneous determination of three active components, i.e., tectorigenin, irigenin and irisflorentin, in rat plasma and urine after oral administration of Rhizoma Belamcandae extract. Chromatographic separation was achieved on a Zorbax SB-C(18) column (50 mm × 2.1 mm, 1.8 μm; Agilent, USA) with gradient elution using a mobile phase that consisted of acetonitrile - 0.1% formic acid in water at a flow rate of 0.4 mL/min. Detection was performed by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between the negative (for tectorigenin and irigenin) and positive (for irisflorentin) ionization modes. The calibration curve was linear over a range of 50-50,000 ng/mL for tectorigenin, 10-5000 ng/mL for irigenin and 0.1-200 ng/mL for irisflorentin, respectively. The intra- and inter-day precisions (RSD %) were within 11.3% for all analytes, whereas the deviation of assay accuracies ranged from -8.7 to +11.1%. All analytes were proven to be stable during all sample storage and analysis procedures. This method was successfully applied to a pharmacokinetic study of the three isoflavones after oral administration of Rhizoma Belamcandae extract to rats.     

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.     

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.     

Simultaneous determination of vitexin-4″-O-glucoside,vitexin-2″-O-rhamnoside,rutin and vitexin from hawthorn leaves flavonoids in rat plasma by UPLC–ESI-MS/MS

A sensitive and accurate ultra-performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC–ESI-MS/MS) method was developed and validated for the simultaneous determination of vitexin-4″-O-glucoside (VGL), vitexin-2″-O-rhamnoside (VRH), rutin (RUT) and vitexin (VIT) in rat plasma after intravenous administration of hawthorn leaves flavonoids (HLF). Following protein precipitation by methanol, the analytes were separated on an ACQUITY UPLC BEH C₁₈ column packed with 1.7 μm particles by gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. The analytes and diphenhydramine (internal standard, IS) were detected in the multiple reaction monitoring (MRM) mode by means of an electrospray ionization (ESI) interface (m/z 292.96 for vitexin-4″-O-glucoside, m/z 293.10 for vitexin-2″-O-rhamnoside, m/z 299.92 for rutin, m/z 310.94 for vitexin and m/z 166.96 for IS). The calibration curve was linear over the range 10–40,000 ng/mL for vitexin-4″-O-glucoside, 10–50,000 ng/mL for vitexin-2″-O-rhamnoside, 8–1000 ng/mL for rutin and 16–2000 ng/mL for vitexin. The intra- and inter-run precisions (relative standard deviation, RSD) of these analytes were all within 15% and the accuracy (the relative error, RE) ranged from −10% to 10%. The stability experiment indicated that the four analytes in rat plasma samples and plasma extracts under anticipated conditions were stable. The developed method was applied for the first time to pharmacokinetic studies of the four bioactive compounds of hawthorn leaves flavonoids following a single intravenous administration of 20 mg/kg in rats.     

High throughput LC-MS/MS method for simultaneous quantification of lamivudine, stavudine and nevirapine in human plasma

A selective and high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and validated to separate, detect and simultaneously quantify lamivudine (3TC), stavudine (d4T) and nevirapine (NVP) in human plasma using metaxalone as internal standard (IS). After solid phase extraction (SPE), the analytes and the IS were chromatographed on a Symmetry C18 (150 mmx3.9 mm i.d., 5 microm particle size) column using 5 microL injection volume with a run time of 4.5 min. An isocratic mobile phase consisting of 0.5% glacial acetic acid in water:acetonitrile (20:80, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The method was validated over the range of 25-3000 ng/mL for 3TC, 20-2000 ng/mL for d4T and 50-5000 ng/mL for NVP. The absolute recoveries for analytes (>or=86%) and IS (98.12%) achieved from spiked plasma samples were consistent and reproducible. Inter-batch and intra-batch precision (%CV) across four validation runs (LLOQ, LQC, MQC and HQC) was less than 10. The accuracy determined at these levels was within +/-8% in terms of relative error. The method was successfully applied to a pivotal bioequivalence study of [60 (3TC)+12 (d4T)+100 (NVP)] mg dispersible tablets in 60 healthy human subjects under fasting condition.     

Pharmacokinetic Study of 14-(3-Methylbenzyl)matrine and 14-(4-Methylbenzyl)matrine in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

A rapid, sensitive and selective high-performance liquid chromatography-tandem mass spectrometric method (HPLC-MS) was developed and validated to determine the 14-(3-methylbenzyl)matrine (3MBM) and 14-(4-methylbenzyl)matrine (4MBM) levels in rat plasma in the present study. The analytes were separated using a C18 column (1.9 μm, 2.1 mm × 100 mm) equipped with a Security Guard C18 column (5 μm, 2.1 mm × 10 mm), followed by detection via triple-quadrupole mass spectrometry using an electrospray ionization (ESI) source. Sample pretreatment involved one-step protein precipitation with isopropanol:ethyl acetate (v/v, 25:75), and pseudoephedrine hydrochloride was used as an internal standard. The method was linear in the concentration range of 5–2000 ng/ml for both compounds. The intra-day and inter-day relative standard deviations (RSDs) were less than 15%, and all relative errors (REs) were within 15%. The proposed method enables the unambiguous identification and quantification of these two compounds in vivo. This study is the first to determine the 3MBM and 4MBM levels in rat plasma after oral administration of these compounds. These results provide a meaningful basis for evaluating the clinical applications of these medicines.     

Determination of schizandrin in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.     

Simultaneous determination of hydrochlorothiazide, quinapril and quinaprilat in human plasma by liquid chromatography-tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous estimation of hydrochlorothiazide, quinapril and its metabolite quinaprilat in human plasma. After solid phase extraction (SPE), the analytes and IS were chromatographed on a hypurity C8 (100mmx2.1mm i.d., 5mum particle size) column using 2muL injection volume with a run time of 2.8min. An isocratic mobile phase consisting of 0.5% (v/v) formic acid:acetonitrile (25:75, v/v) was used to separate all these drugs. The precursor and product ions of these drugs were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring mode (MRM) without polarity switch. The proposed method was validated over the range of 5-500ng/mL for hydrochlorothiazide method and 5-1500ng/mL for quinapril and quinaprilat. Inter-batch and intra-batch precision (coefficient of variation - % CV) across five validation runs lower limit of quantitation (LLOQ), lower quality control (LQC), middle quality control (MQC), higher quality control (HQC) and upper limit of quantitation (ULOQ) was less than 15. The accuracy determined at these levels was within +/-13% in terms of relative percentage error.     

A rapid and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of sibiricaxanthone F in rats

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantifying sibiricaxanthone F (SF) in rat plasma following oral and intravenous dosings. After addition of the internal standard (IS) kaempferol and the antioxidant, 20% ascorbic acid, plasma samples were precipitated with acetonitrile and separated on an Aglient Zorbax XDB-C(18) column (50 mm × 4.6mm I.D., 2.1 μm) with gradient acetonitrile and water (both containing 0.01% formic acid) as the mobile phase. The detection was performed on a Sciex API 4000 LC-MS/MS with electrospray ionization (ESI) inlet in the negative multiple reaction monitoring (MRM) mode. Good linearity was achieved over the concentration range of 0.5-500.0ng/mL (r>0.996). Intra- and inter-day precisions were less than 7.60%, and accuracy ranged from 97.18% to 99.84%. The lower limit of quantification for SF was 0.5 ng/mL, and analytes were stable under various conditions (during freeze-thaw, at room temperature and under deep-freeze conditions). This validated method was successfully applied to the preliminary pharmacokinetic study of SF in rats for the first time, and the absolute bioavailability of SF was found to be only 0.22 ± 0.15%.