A Stability-Indicating LC Method for Assay of Topotecan Hydrochloride

This paper describes the development of a stability-indicating high-performance liquid chromatographic (HPLC) method for quantitative determination of topotecan hydrochloride, a semi-synthetic derivative of camptothecin and anti-tumor drug with topoisomerase I-inhibitory activity. Chromatographic separation was achieved on a C₁₈ column with a mixture of phosphate buffer and acetonitrile as mobile phase. The method was validated for linearity, accuracy, precision, and robustness. Forced degradation studies were performed by treating bulk samples of topotecan hydrochloride with acid (0.5 M hydrochloric acid), base (0.5 M sodium hydroxide), oxidizing agent (10% v/v hydrogen peroxide), heat (60 °C), and UV light (254 nm).     

Metabolic study of Angelica dahurica extracts using a reusable liver microsomal nanobioreactor by liquid chromatography–mass spectrometry

Highly active and recoverable nanobioreactors prepared by immobilizing rat liver microsomes on magnetic nanoparticles (LMMNPs) were utilized in metabolic study of Angelica dahurica extracts. Five metabolites were detected in the incubation solution of the extracts and LMMNPs, which were identified by means of HPLC‐MS as trans‐imperatorin hydroxylate (M1), cis‐imperatorin hydroxylate (M2), imperatorin epoxide (M3), trans‐isoimperatorin hydroxylate (M1′) and cis‐isoimperatorin hydroxylate (speculated M2′). Compared with the metabolisms of imperatorin and isoimperatorin, it was found that the five metabolites were all transformed from these two major compounds present in the plant. Since no study on isoimperatorin metabolism by liver microsomal enzyme system has been reported so far, its metabolites (M1′ and M3′) were isolated by preparative HPLC for structure elucidation by ¹H‐NMR and MS² analysis. M3′ was identified as isoimperatorin epoxide, which is a new compound as far as its chemical structure is concerned. However, interestingly, M3′ was not detected in the metabolism of the whole plant extract. In addition, a study with known chemical inhibitors on individual isozymes of the microsomal enzyme family revealed that CYP1A2 is involved in metabolisms of both isoimperatorin and imperatorin, and CYP3A4 only in that of isoimperatorin. Copyright © 2015 John Wiley & Sons, Ltd.     

Medicago sativa as a green corrosion inhibitor for 1018 carbon steel in 0.5 M H2SO4 solution

A study on the use of the methanol extract of Medicago sativa as a green corrosion inhibitor for 1018 carbon steel in 0.5?M of sulfuric acid has been carried out by using potentiodynamic polarization curves, electrochemical impedance spectroscopy and gravimetric tests. Testing temperatures were 25°C, 40°C and 60°C. Results showed that M. sativa is a good corrosion inhibitor, with its efficiency increasing with its concentration and with time, but decreasing with the temperature. M. sativa forms a passive film on top of the steel with a passive current density and pitting potential values lower than that for uninhibited solution, and remained on the steel for 8–12?h. This film formed by iron ions and heteroatoms present in OH^? and amine groups from the extract are adsorbed on the steel and form a protective film on to the steel.     

Quantitative determination of regorafenib and its two major metabolites in human plasma with high‐performance liquid chromatography and ultraviolet detection

A simple, highly sensitive and specific high‐performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N‐oxidemetabolite (M‐2) and the desmethyl N‐oxide metabolite (M‐5) in human plasma. Regorafenib, M‐2, M‐5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH₂PO₄ (pH 3.5)–acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M‐2 and M‐5 were 10 ng/mL for each analyte. A procedure using solid‐phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra‐ and inter‐day assays were <12.2% for regorafenib, <12.3% for M‐2 and <15.1% for M‐5. Accuracies for intra‐ and inter‐day assays were <9.4% for regorafenib, <8.0% for M‐2 and <12.8% for M‐5 over a linear range from 10 to 10,000 ng/mL. This HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry after solid-phase extraction

Coconut (Cocos nucifera L.) water, which contains many uncharacterized phytohormones is extensively used as a growth promoting supplement in plant tissue culture. In this paper, a high-performance liquid chromatography (HPLC) method was developed for the simultaneous determination of various classes phytohormones, including indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), abscisic acid (ABA), gibberellic acid (GA), zeatin (Z), N⁶-benzyladenine (BA), α-naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) in young coconut water (CW). The analysis was carried out using a reverse-phase HPLC gradient elution, with an aqueous mobile phase (containing 0.1% formic acid, pH adjusted to 3.2 with triethylamine (TEA)) modified by methanol, and solute detection made at 265 nm wavelength. The method was validated for specificity, quantification, accuracy and precision. After preconcentration of putative endogenous phytohormones in CW using C₁₈ solid-phase extraction (SPE) cartridges, the HPLC method was able to screen for putative endogenous phytohormones present in CW. Finally, the identities of the putative phytohormones present in CW were further confirmed using independent liquid chromatography–tandem mass spectrometry (LC–MS/MS) equipped with an electrospray ionization (ESI) interface.     

Quantitative determination of rupestonic acid in rat plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A sensitive and specific high‐performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC‐ESI‐MS/MS) method was developed and validated for determination of rupestonic acid in rat plasma. Protein precipitation method was used to extract rupestonic acid and the internal standard (IS) warfarin sodium from rats plasma. The chromatographic separation was performed on an Agela Venusil XBP Phenyl column with an isocratic mobile phase consisting of methanol–0.1% formic acid in water (40:60, v/v), pumped at 0.4 mL/min. Rupestonic acid and the internal standard (IS) warfarin sodium were detected at m/z 247.2 → 203.1 and 307.1 → 161.3 in positive ion and multiple reaction monitoring mode respectively. The standard curves were linear over the concentration range of 2.5–5000 ng/mL (r² > 0.99). The within‐day and between‐day precision values for rupestonic acid at four concentrations were 4.7–5.7 and 4.4–8.7%, respectively. The method described herein was fully validated and successfully applied to the pharmacokinetic study after an intravenous administration of rupestonic acid in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Characterization and identification of iridoid glucosides,flavonoids and anthraquinones in Hedyotis diffusa by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry

The multiple bioactive constituents in Hedyotis diffusa Willd. (H. diffusa) were extracted and characterized by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC‐ESI‐MSⁿ). The optimized separation condition was obtained using an Agilent ZorBax SB‐C₁₈ column (4.6×150 mm, 5 μm) and gradient elution with water (containing 0.1% formic acid) and acetonitrile (containing 0.1% formic acid), under which baseline separation for the majority of compounds was achieved. Among the compounds detected, 14 iridoid glucosides, 10 flavonoids, 7 anthraquinones, 1 coumarin and 1 triterpene were unambiguously identified or tentatively characterized based on their retention times and mass spectra in comparison with the data from standards or references. The fragmentation behavior for different types of constituents was also investigated, which could contribute to the elucidation of these constituents in H. diffusa. The present study reveals that even more iridoid glycosides were found in H. diffusa than hitherto assumed. The occurrence of two iridoid glucosides and five flavonoids in particular has not yet been described. This paper marks the first report on the structural characterization of chemical compounds in H. diffusa by a developed HPLC‐ESI‐MSⁿ method.     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Essential oil composition of aerial parts of Micromeria persica Boiss. from Western of Shiraz,Iran

Micromeria persica Boiss. is medicinal and aromatic plant, belonging to the Lamiaceae family. The chemical composition of the essential oils (EOs) from aerial parts of M. persica were extracted using hydro-distillation method and analysed using GC and GC–MS. Fifty-two compounds were identified in the EOs of aerial parts of M. persica. The main chemical compositions were n-hexadecanoic acid (14.9%), thymol (9.5%), linoleic acid (8.0%), carvacrol (5.6%), (E)-nerolidol (5.5%), linolenic acid (5.5%), α-cadinol (2.7%), linalool (2.7%), borneol (2.6%), caryophyllene oxide (2.3%) and pulegone (2.0%). Presence of borneol, thymol, carvacrol and pulegone suggests the potential of this plant as a flavouring source in the food industry, being used in perfumery and cosmetics industry, vitamin E synthesis and exhibit strong fungicidal, antibacterial and antimicrobial activities.     

Evaluation of tramadol and its main metabolites in horse plasma by high‐performance liquid chromatography/fluorescence and liquid chromatography/electrospray ionization tandem mass spectrometry techniques

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat pain in humans. The clinical response of tramadol is strictly correlated to its metabolism, because of the different analgesic activity of its metabolites. O‐Desmethyltramadol (M1), its major active metabolite, is 200 times more potent at the µ‐receptor than the parent drug. In recent years tramadol has been widely introduced in veterinary medicine but its use has been questioned in some species. The aim of the present study was to develop a new sensible method to detect the whole metabolic profile of the drug in horses, through plasma analyses by high‐performance liquid chromatography (HPLC) coupled with fluorimetric (FL) and photodiode array electrospray ionization mass spectrometric (PDA‐ESI‐MS) detection, after its sustained release by oral administration (5 mg/kg). In HPLC/FL experiments the comparison of the horse plasma chromatogram profile with that of a standard mixture suggested the identification of the major peaks as tramadol and its metabolites M1 and N,O‐desmethyltramadol (M5). LC/PDA‐ESI‐MS/MS analysis confirmed the results obtained by HPLC/FL and also provided the identification of two more metabolites, N‐desmethyltramadol (M2), and N,N‐didesmethyltramadol (M3). Another metabolite, M6, was also detected and identified. The present findings demonstrate the usefulness and the advantage of LC/ESI‐MS/MS techniques in a search for tramadol metabolites in horse plasma samples. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Simultaneous Determination of Monoamines,Their Precursor Amino Acids,and Related Metabolites in Mice Brain by High‐Performance Liquid Chromatography with a Coulometric Electrode Array System

Abstract

A method based on high performance liquid chromatography with a coulometric electrode array system (HPLC‐coulometric electrode array) using C₁₈ column has been developed for the simultaneous determination of norepinephrine (NE), epinephrine (E), l‐3,4‐dihydroxyphenylalanine (l‐DOPA), p‐tyrosine (p‐TYR), dopamine (DA), m‐tyrosine (m‐TYR), 5‐hydroxytryptamine (5‐HT), homovanillic acid (HVA), and 5‐hydroxyindoleacetic acid (5‐HIAA) in mice brain. The chromatography was performed using a C₁₈ column (250 mm×4.6 mm i.d. and 5 µm) with sodium acetate buffer (pH 5.0, 0.05 M) and methanol as the mobile phase. Elution of analytes was carried out at a flow rate of 1.0 ml/min. The nine compounds were monitored using an ESA electrochemical detector. Potentials of three electrodes in series were set at 200, 500, and 700 mV, respectively. Optimization of the pH of the mobile phase and the proportion of methanol were also considered. The minimal detection limits were 2–8 ng/ml. Linear (r=0.99) detector performances were observed within a range of 10～2000 ng/ml. Recoveries for the nine compounds in spiked samples were over 90% and the relative standard deviations (RSD) were less than 4.0%.     

Autoradiolysis of the 2-deoxy-2-[<Superscript>18</Superscript>F]fluoro-D-glucose radiopharmaceutical

Summary To control virtually the toxic compounds and to improve quality control of the solution of 2-deoxy-2-[¹⁸F]fluoro-d-glucose (2-[¹⁸F]FDG), the products of its autoradiolysis were analyzed by high-performance liquid chromatography with electrospray mass spectrometric and radiometric detectors (HPLC/MS/RAD), thin layer chromatography on TLC silica plate and HPTLC on amino modified silica plate. Except Kryptofix^™ 2.2.2, glucose and fluoride anion, no by-products and impurities were observed by LC/MS analysis of fresh 2-[¹⁸F]FDG samples. The analysis performed in the time interval of 6 to 48 hours after the end of 2-[¹⁸F]FDG synthesis indicated that the activity of the autoradiolysis products separated by HPLC did not exceed 1.3%. As the main autoradiolysis products of 3.3 ^. 10^-5 to 4.4 ^. 10^-5M 2-[¹⁸F]FDG solution of original specific activity 0.5-1.5 GBq ^. cm^-3 were established: arabinose - 2.8 μM (G= 0.07/100 eV), gluconic and glucuronic acids 1.8-0.5 μM (G =0.01-0.05/100 eV), arabinose and araburonic acids occurred under 0.5 μM concentration at residual glucose contents about 0.14 mM. Radiation chemical yields of active products were calculated from molar activity of 2-[¹⁸F]FDG and the percentage of their activity: 0.5% radiochemical yield of 2-[¹⁸F]fluoroglucuronic acid corresponds to the G = 0.004/100 eV and 0.3% yield of 2-[¹⁸F]fluorogluconic acid issues G = 0.003/100 eV.     

Pharmacokinetics of methyl salicylate‐2‐O‐β‐D‐lactoside,a novel salicylic acid analog isolated from Gaultheria yunnanensis,in dogs

Methyl salicylate‐2‐O‐β‐D‐lactoside (MSL), a natural salicylate derivative of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis), has been shown to provide a beneficial anti‐inflammatory effect in animal models. Studies on the pharmacokinetics and bioavailability of MSL can provide both a substantial foundation for understanding its mechanism and empirical evidence to support its use in clinical practice. A simple and sensitive high‐performance liquid chromatography (HPLC) method, coupled with ultraviolet analyte detection, was developed for determining the concentration of MSL and its metabolite in beagle plasma. Chromatographic separation was achieved on a Agilent Zorbax SB‐C₁₈ column (5 μm ,4.6 × 250 mm). The mobile phase consisted of aqueous solution containing 0.1% phosphoric acid and acetonitrile (82:90, v/v), at a flow rate of 1 mL/min. Validation of the assay demonstrated that the developed HPLC method was sensitive, accurate and selective for the determination of MSL and its metabolite in dog plasma. After orally administering three doses of MSL, it could no longer be detected in dog plasma and its metabolite, salicylic acid, was detected. Salicylic acid showed a single peak in the plasma concentration–time curves and linear pharmacokinetics following the three oral doses (r² > 0.99). In contrast, only MSL was detected in plasma following intravenous administration. These results will aid in understanding the pharmacological significance of MSL. The developed method was successfully used for evaluation of the oral and intravenous pharmacokinetic profile of MSL in dogs. Copyright © 2013 John Wiley & Sons, Ltd.     

Preparation of single-enantiomer semiochemicals using 2-methoxy-2-(1-naphthyl)propionic acid and 2-methoxy-2-(9-phenanthryl)propionic acid

Enantioresolution of 3-octanol, 6-methyl-5-hepten-2-ol (sulcatol), and 1-octen-3-ol was conducted using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid (MαNP acid) and (S)-(+)-2-methoxy-2-(9-phenanthryl)propionic acid (M9PP acid). In each case, the diastereomeric esters obtained were readily separated by HPLC. The stereochemistry of the esters could be assigned from their respective ¹H NMR analyses. Solvolyses of the esters gave enantiopure alcohols and acids. MαNP and M9PP acids displayed almost equivalent properties in ¹H NMR anisotropy. The chiral resolving ability of M9PP acid was slightly superior to that of MαNP acid in HPLC.     

Determination of asymmetric and symmetric dimethylarginines in human plasma by HPLC with electrochemical detection

A new HPLC method was developed and validated for the determination of asymmetric and symmetric dimethylarginines and l ‐arginine in human plasma. After SPE and evaporation of the eluate, the samples were derivatised with an o‐phthaldialdehyde reagent containing 3‐mercaptopropionic acid. The derivatives formed were analysed by isocratic RP‐HPLC with electrochemical detection at +320 mV. The mobile phase consisted of 50 mM phosphate buffer (pH 6.1) containing 10% v/v acetonitrile, the flow rate was 1 mL/min. The retention times of all compounds including monomethylarginine (internal standard) were <24 min. The LODs (S/N 3:1) were 0.012 μM for both dimethylarginines and 0.013 μM for l ‐arginine; the linearity of the method was from 0.1 to 20 μM for both dimethylarginines and from 1 to 200 μM for l ‐arginine. Absolute extraction recoveries measured for all analytes ranged from 85 to 88%.     

Chemical Fingerprint and Determination of Triterpenoids in Cultured Cyclocarya paliurus Cells by High-Performance Liquid Chromatography

Cyclocarya paliurus is a medicinal plant containing various bioactive components with significant health benefits. Cell cultures of C. paliurus have been used to produce these bioactive metabolites. A chemical fingerprint was obtained by high-performance liquid chromatography (HPLC) to monitor the synthesis of major triterpenoids in cultured C. paliurus cells and provide a reliable quality assessment for the cell strain screening. The determination of five triterpenoids, namely, maslinic acid, corosolic acid, betulinic acid, oleanic acid, and ursolic acid, was also performed. The HPLC method for the determination of triterpenoids in the cultured cells was accurate, stable, and reliable, and therefore suitable for chemical fingerprint analysis. Sixteen C. paliurus cell strains varied dramatically in their triterpenoid accumulations. The concentrations of the triterpenoids were 0.45–2.19 (maslinic acid), 0.92–5.34 (corosolic acid), 2.58–4.70 (betulinic acid), 4.07–12.47 (oleanic acid), and 12.64–40.98 (ursolic acid) mg/g. A high yield cell strain had a total triterpenoid concentration of 66.34?mg/g. Ten peaks in the HPLC chromatogram with reasonable height and high resolution were assigned as characteristic for fingerprint and analysis. A reference fingerprint was also obtained for cell strain assessment.     

Characterization of in vitro metabolism of capsazepine,a vanilloid transient receptor potential channel antagonist,by liquid chromatography quadrupole ion trap mass spectrometry

Capsazepine is an antagonist of the transient receptor potential channel vanilloid 1 (TRPV1), which is known to play an important role in the regulation of pain and inflammation. A selective and sensitive quantitative method for the determination of capsazepine by HPLC‐ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray quadrupole ion trap mass spectrometry. The chromatographic separation was achieved using a 100 × 2 mm C₁₈ Waters Symmetry column combined with a gradient mobile phase composed of acetonitrile and 0.1% formic acid aqueous solution at a flow rate of 220 µL/min. The mass spectrometer was operating in full‐scan MS/MS mode using two‐segment analysis. An analytical range of 10–5000 ng/mL was used in the calibration curve constructed in rat plasma. The inter‐batch precision and accuracy observed were 10.1, 6.4 and 6.1% and 100.8, 98.5 and 106.2% at 50, 500 and 5 000 ng/mL, respectively. An in vitro metabolic stability using rat, dog or mouse liver microsomes was performed to determine the intrinsic clearance of capsazepine. The results suggest a very rapid degradation with T_1/2 ranging from 2.6 to 4.3 min and a high clearance, suggesting that drug bioavailability is considerably reduced following extravascular administrations, consequently affecting drug response. Three metabolites were identified by HPLC‐MS/MS. S‐hydroxylation (M + 16), oxidative desulfuration (M − 16) and desulfuration (M − 32) metabolites of capsazepine were observed following exposure to rat, dog and mouse microsomes. Copyright © 2010 John Wiley & Sons, Ltd.     

Fusaricidins from Paenibacillus polymyxa M‐1, a family of lipohexapeptides of unusual complexity—a mass spectrometric study

Paenibacillus polymyxa are rhizobacteria with a high potential to produce natural compounds of biotechnological and medical interest. Main products of P . polymyxa are fusaricidins, a large family of antifungal lipopeptides with a 15‐guanidino‐3‐hydroxypentadecanoic acid (GHPD) as fatty acid side chain. We use the P . polymyxa strain M‐1 as a model organism for the exploration of the biosynthetic potential of these rhizobacteria. Using matrix‐assisted laser‐desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) about 40 new fusaricidins were detected which were fractionated by reversed‐phase (rp) HPLC. Their structure was determined by MALDI‐LIFT‐TOF/TOF fragment analysis. The dominant fragment in the product ion spectra of fusaricidins appeared at m /z 256.3, 284.3 and 312.4, respectively, indicating variations in their fatty acid part. Two new subfamilies of fusaricidins were introduced which contain guanidino‐3‐hydroxyhepta‐ and nonadecanoic acid as fatty acid constituents. Apparently, the end‐standing guanidine group is not modified as shown by direct infusion nano‐electrospray ionization mass spectrometry (nano‐ESI MS). The results of this study suggest that advanced mass spectrometry is the method of choice for investigating natural compounds of unusual diversity, like fusaricidins. Copyright © 2016 John Wiley & Sons, Ltd.     

Simultaneous determination of 20(S)‐protopanaxadiol and its three metabolites in rat plasma by LC–MS/MS: application to their pharmacokinetic studies

The aim of this study was to develop an LC–MS/MS method for simultaneous determination of 20(S) protopanaxadiol (PPD) and its three metabolites, PPD‐glucuronide (M1), (20S,24S)‐epoxy‐dammarane‐3,12,25‐triol (M2) and (20S,24R)‐epoxydammarane‐3,12,25‐triol (M3), in rat plasma. Precipitation with acetonitrile was employed for sample preparation and chromatographic separations were achieved on a C₁₈ column. The sample was detected using triple quadrupole tandem mass spectrometer with selected reaction monitoring mode. The monitored precursor‐to‐product ion transitions were m/z 459.4 → 375.3 for PPD, m/z 635.4 → 113.0 for M1, m/z 477.4 → 441.4 for M2 and M3 and m/z 475.4 → 391.3 for IS. The developed assay was validated according to the guidelines of the US Food and Drug Administration. The calibration curves showed good linearity over the tested concentration ranges (r > 0.9993), with the LLOQ being 1 ng/mL for all analytes. The intra‐ and inter‐day precisions (RSD) were < 9.51% while the accuracy (RE) ranged from −8.91 to 12.84%. The extraction recovery was >80% and no obvious matrix effect was detected. The analytes were stable in rat plasma with the RE ranging from −12.34 to 9.77%. The validated assay has been successfully applied to the pharmacokinetic study of PPD as well as its metabolites in rat plasma. According to the pharmacokinetic parameters, the in vivo exposures of M1, M2 and M3 were 11.91, 47.95 and 22.62% of that of PPD, respectively.