Quantitative Analysis of Daporinad (FK866) and Its In Vitro and In Vivo Metabolite Identification Using Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry

Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography–quadrupole-time-of-flight–mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration²) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.     

Combined approach for high-throughput preparation and analysis of plasma samples from exposure studies

In drug discovery today, drug exposure is determined in preclinical efficacy and safety studies and drug effects are related to measured concentrations rather than to the administered dose. This leads to a strong increase in the number of bioanalytical samples, demanding the development of higher throughput methods to cope with the increased workload. Here, a combined approach is described for the high-throughput preparation and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis of drug levels in plasma samples from the preclinical efficacy and safety studies, i.e. exposure studies. Appropriate pharmacokinetic (PK) compartmental models were fitted to data from PK screening studies in the rat, which were subsequently used to simulate the expected plasma concentrations of the respective exposure studies. Information on the estimated drug concentrations was used to dilute the samples to appropriate concentration levels. A Tecan Genesis RSP liquid handling system was utilized to perform automated plasma sample preparation including serial dilution of standard solutions, dilution of plasma samples, addition of internal standard solution and precipitation with acetonitrile. This robotic sample preparation process permitted two studies of 1-96 samples each to be run simultaneously. To ensure the performance of this method the accuracy and precision for diazepam were examined. Two novel drugs were used to illustrate the suggested approach. In conclusion, our method for sample preparation of exposure samples, based on the combined use of PK simulations, a liquid handling system and a fast LC/MS/MS method, increased the throughput more than three times and minimized the errors, while maintaining the required accuracy and precision.     

Quantification and application of a liquid chromatography–tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid‐phase extraction

A liquid chromatographic–electrospray ionization–time‐of‐flight/mass spectrometric (LC‐ESI‐TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro‐elution solid‐phase extraction (SPE) for sample preparation and LC‐ESI‐TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro‐elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration²), with the equation y = ax² + bx + c was used to fit calibration curves over the concentration range of 3.02–2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within‐run and the between‐run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC‐ESI‐TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.     

Development of an ICP-IDMS method for accurate routine analyses of toxic heavy metals in polyolefins and comparison with results by TI-IDMS

An inductively coupled plasma isotope dilution mass spectrometric (ICP-IDMS) method was developed as a suitable method – with respect to its sensitivity, precision, accuracy, and time-consumption – for the analysis of toxic heavy metal traces (Pb, Cd, Cr, and Hg) in polyolefins. Results for Pb, Cd, and Cr were compared with those obtained by thermal ionization isotope dilution mass spectrometry (TI-IDMS), which was used as a reference method. Because of its high first ionization potential and its high volatility mercury could not be determined by TI-IDMS. A multi-element spike solution, containing isotopically enriched ²⁰⁶Pb, ¹¹⁶Cd, ⁵³Cr, and ²⁰¹Hg, was used for the isotope dilution step. Decomposition of the polyolefin samples was carried out with concentrated HNO₃ at temperatures of about 300?°C in a high pressure asher (HPA). This procedure decomposes polyolefins completely and allows isotopic equilibration between sample and spike isotopes. Detection limits of 16 ng/g, 5 ng/g, 164 ng/g, and 9 ng/g were obtained for Pb, Cd, Cr, and Hg by ICP-IDMS using only sample weights of 0.25 g. In different commercially available polyethylene samples heavy metal concentrations in the range of < 5 ng/g to 4 × 10³ ng/g were analyzed. Both mass spectrometric methods were applied within the EU project “Polymeric Elemental Reference Material (PERM)” for the certification of two polyethylene reference materials. The ICP-IDMS results agreed very well with those of TI-IDMS which demonstrates the accuracy of the ICP-IDMS method also suitable for routine analyses.     

Determination of Total Selenium in Human Urine and Serum by Graphite-Furnace Atomic-Absorption Spectrophotometry with Palladium-Nickel-Ammonium Nitrates as a Matrix Modifier

After human urine or serum was diluted (1 + 9) with HNO₃ (0.2%, v/v) and standard additions of Se solution (100 μ L^?1), the diluted sample (10 μL) was introduced into the graphite cuvette. The matrix modifier [10μL, containing Pd (0.6 μg) + Ni (25 μg) + NH₄NO₃ (80 μg) in HNO₃ (0.2%, v/v) for urine, or Pd (0.3 μg) + Ni (30 μg) + NH₄NO₃ (80 μg) + Triton X-100 (0.04%) in HNO₃ (0.2%, v/v) for serum, respectively] was added and the mixture was heated according to a temperature program. The matrix modifier containing NH₄NO₃ in a suitable amount and a small amount of Pd enhanced the sensitivity for Se. The method detection limits (3σ) after dilution were about 4.9 ± 0.8 and 2.36 ± 0.18 μg L^?1 for urine and serum, respectively. The accuracy of this method was tested with SRM #2670 human urine Se and Seronorm Trace Elements #116 human serum Se, respectively, and the results of 97.6 – 101% and 100 – 104% were obtained with precision ± 0.3% and ± 2%, respectively. This method can be applied easily and accurately to the determination of concentration of total Se in human urine and serum.     

Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of PM02734, a novel antineoplastic agent, in dog plasma

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) assay was developed and validated to quantify a novel antineoplastic agent, PM02734, in dog plasma. The method was validated to demonstrate the specificity, limit of quantification (LOQ), accuracy, and precision of measurements. The calibration range for PM02734 was established using PM02734 standards from 0.05 to 100 ng/mL in blank plasma. The dominating ions were doubly charged molecular ions [M+2H]2+ at m/z 740.0 instead of singly charged ones at m/z 1478.4. The selected reaction monitoring (SRM), based on the m/z 740.0 --> 212.2 transition, was specific for PM02734, and that based on the m/z 743.8 --> 212.2 transition was specific for deuterated PM02734 (the internal standard, IS); no endogenous materials interfered with the analysis of PM02734 and IS from blank plasma. The assay was linear over the concentration range 0.05-100 ng/mL. In terms of sensitivity of assay 0.05 ng/mL is a very low LLOQ, especially considering PM02734 is a peptide. The correlation coefficients for the calibration curves ranged from 0.9990 to 0.9999. The mean intraday and interday accuracies for all calibration standards (n = 9) ranged from 93 to 111% (< or =11% bias) in dog plasma, and the mean interday precision for all calibration standards was less than 6.4%. The mean intra- and interday assay accuracy for all quality control replicates in dog plasma (n = 9), determined at each QC level throughout the validated runs, ranged from 85-111% (< or =15% bias) and from 99-109% (< or =9% bias), respectively. The mean intra- and interday assay precision was less than 12.1 and 13.3% for all QC levels, respectively. The assay has been used to support preclinical pharmacokinetic (PK) and toxicokinetic studies. The results showed that preclinical samples could be monitored for PM02734 up to 168 h after dosing, which allowed us to identify multiple elimination phases and accurately estimate PK information.     

A reversed-phase ultra-fast liquid chromatography–photodiode array detector (RP-UFLC-PDA) method for simultaneous estimation of ayapanin and umbelliferone in Ayapana triplinervis Vahl

A rapid validated ultra-fast liquid chromatography–photodiode array detector (UFLC-PDA) method was developed to identify and quantify ayapanin (AY) and umbelliferone (UM) simultaneously in Ayapana triplinervis Vahl methanolic extract. The method was validated for linearity, limit of detection (LOD; 3:1σ/S), limit of quantification (LOQ; 10:1σ/S), precision, accuracy, specificity and robustness. The response was linear with a good correlation between concentration and mean peak area through a correlation coefficient of 0.9996, y = 7025.7x – 2269.8 and 0.9997, y = y = 16,262x – 946 with LOD of 6.256 ± 0.52 and 3.325 ± 0.36, and LOQ of 18.838 ± 0.18 and 8.870 ± 0.85 for AY (0.67% w/w) and UM (0.18% w/w), respectively. The relative standard deviation (%) of precision and recovery of AY and UM was <2.0%. The proposed method was simple, accurate, specific, precise and reproducible.     

Half-life of plutonium-241: Past and present

Half-life of²⁴¹Pu is of great importance in nuclear technology. In view of large variation in the values (13–15 y) reported till 1974 in literature, efforts have been made in different laboratories to determine this half-life with high precision and accuracy. In our laboratory, it has been determined by different methods which may be classified in two categories, viz. (1) parent decay method, and (2) daughter growth method. In the parent decay method, change in isotope ratios²⁴¹Pu/²³⁹Pu,²⁴¹Pu/²⁴⁰Pu and²⁴¹Pu/²⁴²Pu was studied periodically by a thermal ionization mass spectrometer. Single as well as double ratio method was used to calculate the half-life. In the daughter growth method, the half-life was obtained in four independent ways. These were (1) alpha spectrometry taking²³⁹Pu and²⁴²Pu separately as reference isotopes and studying periodically the increase in alpha activity ratio, (2) alpha proportional counting for observing periodically the change in total alpha activity, (3) isotope dilution alpha spectrometry using²⁴³Am as a spike, (4) isotope dilution mass spectrometry using²⁴³Am as a spike. In all these methods, synthetic mixtures were prepared for achieving high precision and accuracy in different measurements. Based on the results obtained in this laboratory and the values reported by other laboratories, a half-life value of 14.4±0.1 y is recommended. The paper reviews the past history, puts forth the present status, highlights the current trends for studying the effect of chemical composition of plutonium on the half-life of²⁴¹Pu and presents the future requirements for achieving higher accuracy in the half-life of²⁴¹Pu.     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.     

Activation analysis of halogens in photographic emulsions using a252Cf neutron multiplier

A nondestructive neutron activation technique for the simultaneous direct determination of chlorine, bromine, and iodine in silver halide mixtures is described using a²⁵²Cf neutron multiplier (CFX). About 5–50 mg of sample are used. The analysis is semiroutine, without the need for a monitor included with each sample since the flux stability and reproducibility are within ±1%, decaying only with the 2.65 year half-life of²⁵²Cf. The precision and accuracy are counting-statistics controlled and are generally ±1% RSD or better. The method offers an attractive alternative to existing chemical and instrumental methods for these determinations in silver halide mixtures because it has the potential for providing reasonably rapid analyses with good precision and accuracy.     

The development and validation of a high‐throughput LC–MS/MS method for the analysis of endogenous β‐hydroxy‐β‐methylbutyrate in human plasma

A high‐throughput, sensitive, and rugged liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the rapid quantitation of β ‐hydroxy‐β ‐methylbutyrate (HMB) in human plasma has been developed and validated for routine use. The method uses 100 μL of plasma sample and employs protein precipitation with 0.1% formic acid in methanol for the extraction of HMB from plasma. Sample extracts were analyzed using LC–MS/MS technique under negative mode electrospray ionization conditions. A ¹³C–labeled stable isotope internal standard was used to achieve accurate quantitation. Multiday validation was conducted for precision, accuracy, linearity, selectivity, matrix effect, dilution integrity (2×), extraction recovery, freeze–thaw sample stability (three cycles), benchtop sample stability (6 h and 50 min), autosampler stability (27 h) and frozen storage sample stability (146 days). Linearity was demonstrated between 10 and 500 ng/mL. Inter‐day accuracies and coefficients of variation (CV) were 91.2–98.1 and 3.7–7.8%, respectively. The validated method was proven to be rugged for routine use to quantify endogenous levels of HMB in human plasma obtained from healthy volunteers.     

Revisiting type-A uncertainties relating to the measurement of mass fraction of lead using isotope-dilution inductively coupled plasma mass spectrometry: a way of improving measurement precision and expanded uncertainty

A quadrupole inductively coupled plasma mass spectrometer (Q-ICP-MS) has been used for determination of lead in plant materials using isotope-dilution inductively coupled plasma mass spectrometry. The accuracy of the method was demonstrated by analysis of a matrix certified reference material, NIST SRM 1547 Peach Leaves. Specific instrumental parameters of Q-ICP-MS, including isotope analysis mode, integration time per point, number of points per mass, and number of measurements, were optimized to obtain the best measurement precision. The precision (expressed as relative standard deviation) associated with replicate measurement of the ²⁰⁸Pb/²⁰⁶Pb isotope ratio and its mass-bias correction factor was <0.2%. Following “Example A7” of the Eurachem/CITAC Guide, the relative expanded uncertainty, U _rel, (coverage factor k = 2) was found to be ±1.1%, which fulfilled the target value of ±2% maximum and was lower than the uncertainty of ±3.4% reported by NIST based on isotope-dilution thermal ionization mass spectrometry. Sample recovery of 99% was obtained.     

Quantification of urapidil, α-1-adrenoreceptor antagonist, in plasma by LC-MS/MS: validation and application to pharmacokinetic studies

A sensitive high‐performance liquid chromatography–positive ion electrospray tandem mass spectrometry method was developed and validated for the quantification of urapidil in plasma. Following liquid–liquid extraction, the analyte was separated using an isocratic mobile phase on a reverse‐phase column and analyzed by MS/MS in the multiple reaction monitoring mode using the respective [M + H]⁺ ions, m/z 388 to 205 for urapidil and m/z 452 to 344 for the internal standard. The assay exhibited a linear dynamic range of 0.1–500 ng/mL for urapidil in plasma. Acceptable precision (<7%) and accuracy (100 ± 8%) were obtained for concentrations over the standard curve range. The method was successfully applied to quantify urapidil concentrations in a preclinical pharmacokinetic study after a single oral administration of urapidil at 3 mg/kg to rats. Following oral administration the maximum mean concentration in plasma (C_max; 616 ± 73 ng/mL) was achieved at 0.5 h (T_max) and area under curve (AUC_0–24) was 1841 ± 308 ng h/mL. The half‐life (t_1/2) and clearance (Cl) were 2.47 ± 0.4 h and 1660 ± 276 mL/h/kg, respectively. Moreover, it is plausible that the assay method in rat plasma would facilitate the adaptability of urapidil quantification in human plasma for clinical trials. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of theophylline in serum by high performance liquid chromatography/mass spectrometry with atmospheric pressure chemical-ionization (APCI HPLC/MS)

 A method for the determination of theophylline (TH), without derivatization, in serum by isotope dilution mass spectrometry using labelled [1, 3-¹⁵N₂-2-¹³C]theophylline (LTH) as internal standard is described. After deproteinization, the analyte is directly injected into a high performance liquid chromatography – mass spectrometer operating with atmospheric-pressure chemical-ionization (APCI HPLC/MS). The concentrations of TH in sera measured by APCI HPLC/MS are compared with results from gas chromatography – isotope dilution mass spectrometry (GC-ID/MS), high performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA). The accuracy, precision and recovery of the APCI HPLC/MS and GC-ID/MS methods are discussed. The coefficient of variation (CV) determined from duplicate samples was less than 2%. The detection limit was 10 ng/ml at a signal-to-noise ratio of 3:1. Received: 17 January 1996/Revised: 26 March 1996/Accepted: 5 April 1996     

Sensitive and Selective LC–ESI–MS Analysis of Aesculin in Rat Plasma

A rapid and sensitive liquid chromatography–electrospray ionization mass spectrometry method was developed for the determination of aesculin in rat plasma. The analyses were chromatographed on a Zorbax Extend-C18 analytical column (150 × 2.1 mm I.D., 5 µm) with 30:70 (v/v) methanol–0.1% formic acid as mobile phase. Detection was performed by triple-quadrupole tandem mass spectrometry in multi-reaction-monitoring mode with an electrospray ionization source. The method was validated for accuracy and precision, and linearity in the two matrices was good. The assay was linear in the range 12.5–1,800 ng mL^?1. The lower limit of quantification of aesculin (LLOQ) was 12.5 ng mL^?1. The recovery of aesculin and tinidazole (IS) were well above 85%. The within- and between-batch accuracy was 100–104% and 97–109%, respectively. There were no stability-related problems in the procedure for the analysis of aesculin. The method was successfully used in a preclinical study of the pharmacokinetics of aesculin in rats.     

Development and validation of a quantification method for ziyuglycoside I and II in rat plasma: Application to their pharmacokinetic studies

This study provided a novel and generally applicable method to determine ziyuglycoside I and ziyuglycoside II in rat plasma based on liquid chromatography with tandem mass spectrometry. A single step of liquid–liquid extraction with n‐butanol was utilized, and ginsenoside Rg3 was chosen as internal standard. Final extracts were analyzed based on liquid chromatography with tandem mass spectrometry. Chromatographic separation was achieved using a Thermo Golden C₁₈ column, and the applied gradient elution program allowed for the simultaneous determination of two ziyuglycosides in a one‐step chromatographic separation with a total run time of 10 min. The fully validated methodology for both analytes demonstrated high sensitivity (the lower limit of quantitation was 2.0 ng/mL), good accuracy (% RE ≤ ± 15) and precision (% RSD ≤ 15). The average recoveries of both ziyuglycosides and internal standard were all above 75% and no obvious matrix effect was found. This method was then successfully applied to the preclinical pharmacokinetic studies of ziyuglycoside I and ziyuglycoside II. The presently developed methodology would be useful for the preclinical and clinical pharmacokinetic studies for ziyuglycoside I and ziyuglycoside II.     

Isotope dilution gas chromatography/mass spectrometry for platinum determination in urine

The therapeutic importance of platinum (Pt) compounds, the growing accessibility of gas chromatography/mass spectrometry (GC/MS) systems in clinical laboratories, and the lack of a mass spectrometric method for the determination of Pt in biological samples motivated us to develop an isotope dilution GC/MS assay for Pt. The method is based on the use of lithium bis(trifluoroethyl) dithiocarbamate, Li(FDEDTC), as a chelating agent and enriched ¹⁹²Pt for isotope dilution. Conditions were optimized for the precise and accurate determination of isotope ratios of Pt by using a 10-m DB-l fused silica capillary column and a reverse-geometry double-focusing mass spectrometer with selected ion monitoring. An overall precision of 1% was obtained by combining within-run precision and between-run precision at the 10-ng level. No appreciable memory effect was observed when samples with different isotope ratios were analyzed sequentially. The method was validated by the quantitation of Pt in National Institute of Standards and Technology freeze-dried urine sample SRM 2670. A concentration value of 125 ± 6 /Lg/L (n = 6) was obtained by using four different sets of isotope ratios in the molecular ion and supports the National Institute of Standards and Technology recommended value of 120 ± ? μg/L. Limits-of-quantitation, estimated at 3 μg/L, are made possible by the high sensitivity of the method and the low blank value for Pt.     

Verification of the long term stability of uranium reference solutions of low concentration

The evolution of trace concentrations (sub ng·g^–1) of uranium in aqueous reference solutions was followed over time by isotope dilution inductively coupled plasma mass spectrometry (ID ICP-MS). Blank levels and detection limits needed to be determined prior to the actual investigations. Accuracy and precision of the method were determined on the basis of results obtained for uranium reference materials under similar experimental conditions. Reference solutions, certified for uranium concentration were prepared on gravimetrical basis. Analyses were performed over a period of 2 years. Taking into account the accuracy and the precision achieved, no significant change of the concentration is observed with time. Therefore, the mid and long term stability of the concentration of such reference materials was successfully demonstrated.     

Development and validation of an ultra‐high‐performance liquid chromatography–tandem mass spectrometry method to determine the bioavailability of warfarin and its major metabolite 7‐hydroxy warfarin in rats dosed with oral formulations containing different polymorphic forms

A simple, sensitive and rapid ultra‐high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry method was developed and validated for the quantification of warfarin and 7‐hydroxy warfarin in Sprague Dawley (SD) rats. Animals were administered a single dose of warfarin sodium formulations (crystalline and amorphous) at 12 mg/kg via oral gavage and blood was drawn over a 96‐h time course. Sample process recoveries, matrix effect and analyte stability were determined. The linearity for warfarin and 7‐hydroxy warfarin was from 5 to 2000 ng/mL in blank SD rat plasma. Correlation coefficients (r²) for standard calibration curves were >.98 and analytes quantified within ±15% of target at all calibrator concentrations. The average percent accuracy and precision for intra‐ and inter‐day were 93.7%–113.8% and ≤12.1%, respectively, for warfarin and 7‐hydroxy warfarin, across the quality control standards (5, 10, 500, 1800 and 2000 ng/mL). Acceptable analytical recovery (>55%) was achieved with process efficiencies >41.5% and matrix effects <139.9% over the analytical range. Both analytes were stable in stock solution, autosampler, benchtop and three cycles of freeze–thaw with percent accuracy ≥90.2% and precision (percent relative standard deviation) ≤14%. The validated method was successfully applied to a pre‐clinical bioavailability study of crystalline and amorphous warfarin sodium formulations in SD rats.     

Validation and application of a novel LC/MS/MS method for the determination of isoginkgetin in rat plasma

Isoginkgetin is a biflavonoid compound isolated from the leaf extracts of Ginkgo biloba. In this study, an liquid chromatography–tandem mass spectrometry (LC/MS/MS) with liquid–liquid extraction was developed and validated for the analysis of isoginkgetin in rat plasma. In the process of chromatographic separation, selected reaction monitoring transitions for isoginkgetin and IS were m/z 566.8 → 134.7 and m/z 430.8 → 269.3, respectively. The validation parameters including selectivity, linearity, LLOQ, accuracy, precision, matrix effect, stability and recovery were satisfactory. The intra‐ and inter‐batch precision (RSD) were <12.1% in plasma, while the accuracy (RE) was within ±14.3%. This method was employed in a pharmacokinetic study on rats after the intravenous administration of isoginkgetin.