LC-ESI-MS/MS characterization of strophanthin-K

A liquid chromatography-mass spectrometry (LC-MS) method was developed for the characterization of strophanthin-K, a mixture of cardiac glycosides extracted from the seeds of Strophanthus kombe. The method is based on the separation of the cardenolides using high performance liquid chromatography (HPLC) followed by detection with electrospray ionization mass-spectrometry (ESI-MS). Chromatographic separation of the analytes was achieved on a RP C-18 column using water: 1% formic acid in water (v/v):acetonitrile gradient mobile phase. Strophanthin-K glycosides studied in ESI-MS in negative ion mode formed abundant adduct ions [M+HCOO](-) while the pseudomolecular ions [M-H](-) are obtained in ESI-MS/MS experiments. Six different cardiac glycosides were identified and characterized: k-strophanthoside, k-strophanthin-beta, helveticoside (erysimin), erysimoside, cymarin and neoglucoerysimoside. Forced degradation investigations done with strophanthin-K showed that k-strophanthidin (the aglycone of strophanthin-K glycosides) was the main product of degradation in acidic conditions; however, in basic conditions, the hydrolysis of the unsaturated 17beta-lactones to the corresponding gamma-hydroxy acids was the predominant degradation pathway.     

Characterization of steroidal saponins in crude extracts from Dioscorea zingiberensis C. H. Wright by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Steroidal saponins are the major bioactive constituents of Dioscorea zingiberensis C. H. Wright (D. zingiberensis). In this work, ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was applied to the separation and characterization of steroidal saponins in crude extracts from D. zingiberensis. The results showed that fragment ions from glycosidic and cross-ring cleavages gave a wealth of structural information related to aglycone skeletons, sugar types and the sequence of sugar units. According to the summarized fragmentation patterns, identification of steroidal saponins from D. zingiberensis could be fulfilled, even when reference standards were unavailable. As a result, a total of thirty-one saponins with five aglycone skeletons, including fourteen new trace saponins, were identified or tentatively elucidated in crude extracts from D. zingiberensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data.     

Comparative concentrations of brevetoxins PbTx-2, PbTx-3, BTX-B1 and BTX-B5 in cockle, Austrovenus stutchburyi, greenshell mussel, Perna canaliculus, and Pacific oyster, Crassostrea gigas, involved neurotoxic shellfish poisoning in New Zealand.

Previously, we found brevetoxins PbTx-3, BTX-B5 and BTX-B1 in cockle, Austrovenus (A.) stutchburyi, PbTx-2, PbTx-3 and BTX-B1 in Pacific oyster, Crassostrea (C.) gigas and PbTx-3 and BTX-B1 in greenshell mussel, Perna (P.) canaliculus following outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand by isolation and/or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this study, procedures for quantitative determination of PbTx-2 and BTX-B5 were developed and those for PbTx-3 and BTX-B1 were further examined by LC-MS/MS. In mass spectrometry with an electrospray ionization interface operating in the positive or negative ion mode, the protonated ions [M+H]+ of PbTx-2 (m/z 895), [M+H]+ of PbTx-3 (m/z 897), [M-H]- of BTX-B5 (m/z 909), and [M-Na]- of BTX-B1 (m/z 1016) were generated abundantly, when 0.1% formic acid-acetonitrile was used as the mobile phase for column chromatography. The product ions of m/z 877, 725, 111 and 80 from PbTx-2, PbTx-3, BTX-B5 and BTX-B1 were identified, respectively, allowing unambiguous confirmation of these toxins by selective reaction monitoring LC-MS/MS analysis. High levels of PbTx-3 and BTX-B5 were detected in C. gigas, of PbTx-3, BTX-B1 and BTX-B5 in A. stutchburyi, and of PbTx-2, PbTx-3 and BTX-B5 in P. canaliculus by this LC-MS/MS method.     

Potent estrogenic metabolites of bisphenol A and bisphenol B formed by rat liver S9 fraction: their structures and estrogenic potency.

We previously demonstrated that the estrogenicity of either bisphenol A [BPA; 2,2-bis(4-hydroxyphenyl)propane] or bisphenol B [BPB; 2,2-bis(4-hydroxyphenyl)butane] was increased several times after incubation with rat liver S9 fraction (Yoshihara et al., 2001). This metabolic activation, requiring both microsomal and cytosolic fractions, was observed with not only rat liver, but also human, monkey, and mouse liver S9 fractions. To characterize the active metabolites of BPA and BPB, we investigated the structures of the isolated active metabolites by negative mode LC/MS/MS and GC/MS. The active metabolite of BPA gave a negative mass peak at [M-H](-) 267 on LC/MS and a single daughter ion at m/z 133 on MS/MS analysis, suggesting an isopropenylphenol dimer structure. Finally, this active metabolite was confirmed to be identical with authentic 4-methyl-2,4-bis(p-hydroxyphenyl)pent-1-ene (MBP) by means of various instrumental analyses. The corresponding peaks of the BPB metabolite were [M-H](-) 295 and m/z 147, respectively, suggesting an isobutenylphenol dimer structure. Further, coincubation of BPA and BPB with rat liver S9 afforded an additional active metabolite(s), which gave a negative mass peak at [M-H](-) 281 and two daughter ion peaks at m/z 133 and m/z 147 on MS/MS analysis. These results strongly suggest that the active metabolite of either BPA or BPB might be formed by recombination of a radical fragment, a one-electron oxidation product of carbon-phenyl bond cleavage. It is noteworthy that the estrogenic activity of MBP, the active metabolite of BPA, is much more potent than that of the parent BPA in several assays, including two reporter assays using a recombinant yeast expressing human estrogen receptor alpha and an MCF-7-transfected firefly luciferase plasmid.     

Sensitive and selective liquid chromatography-electrospray ionization mass spectrometry analysis of ginkgolide B in dog plasma

As an important active constituent of Ginkgo biloba extract, ginkoglide B is a highly selective and competitive PAF receptor antagonist which has been widely used in clinical applications. A novel high-performance liquid chromatography-electrospray ionization mass spectromentry (LC-ESI-MS) method was developed for the determination of ginkgolide B in dog plasma. After liquid/liquid extraction with ether and high-performance liquid chromatography (HPLC) gradient separation with 0.01% of ammonia water (v/v)-methanol as the mobile phase, the deprotonized anions [M-H](-1) at m/z 423 of ginkoglide B, and [M-H](-1) at m/z 492 of internal standard (IS) glibenclamide were analyzed by LC-ESI-MS in selected ion monitoring (SIM) mode. Chromatographic separation was achieved in less than 9 min and calibration curve was linear over a concentration range of 0.1-20 ng/ml. The described assay method was successfully applied to the pre-clinical pharmacokinetic study of ginkoglide B. After intragastric administration of ginkgolide B to beagle dogs, C(max) and T(max) of ginkgolide B were 43.8 +/- 6.24 ng/ml and 0.5 h, respectively, and the elimination half-life (t(1/2)) was 2.85 +/- 0.54 h.     

Identification of O-diglycosyl flavanones in Fructus aurantii by liquid chromatography with electrospray ionization and collision-induced dissociation mass spectrometry

This study reported the application of LC-ESI/MS method to characterize O-diglycosyl flavanones of traditional Chinese medicine Fructus aurantii(Zhiqiao) and UPLC retention parameters method to expatiate the structure-retention relationship of these O-diglycosyl flavanones. The extract of F. aurantii was found containing neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin and neoponcirin. Tandem mass spectrometric method has been utilized to elucidate structure and differentiate the interglycosidic linkage of isomeric O-diglycosyl flavanones. Based on the relative abundance of fragments formed by fragmentation at glycosidic bonds in positive ion mode and CID MS spectra of deprotonated molecule [M-H](-) in negative ion mode, the interglycosidic linkage of O-diglycosyl flavanones (flavonoid O-neohesperidosides and O-rutinosides, 1,2- and 1,6-) can be unambiguously differentiated. UPLC and a CSASS software were performed to obtain the retention parameters a, c and k values of these compounds. The Deltaa, Deltac and alpha values within compound pair naringin to neoriocitrin, neoponcirin to neohesperidin, naringin to isonaringin, neohesperidin to hesperidin, hesperidin to isonaringin, neohesperidin to naringin were calculated. We found there were some relationship between structure and retention parameters.     

Mass spectral characterization of three anthracycline antibiotics: a comparison of thermospray mass spectrometry to particle beam mass spectrometry.

Mass spectral results for three anthracyclines, doxorubicin, daunorubicin, and carminomycin are compared by using thermospray (TS) or particle beam (PB) [electron ionization (EI) and chemical ionization (CI)] instruments. Typically, positive ion TS mass spectrometry (MS) provided intense [MH]+ ions and some fragment ions, while PBMS in the EI mode provided only fragment ions. Significant [MH]+ ions were observed for carminomycin and daunorubicin when analyzed using PBMS in the positive ion CI mode. Under negative ion detection, TSMS yielded intense [M-H]- ions for these compounds while PBCIMS resulted in significant M- ions. Fragment ions observed in all three anthracyclines under positive and negative ion detection by TSMS and PBCIMS are due mainly to the cleavage of glycosidic bond, loss of H2O, and the loss of the side chain (COCH2R2) from the aglycone.     

Metabolic profiling of roots of liquorice (Glycyrrhiza glabra) from different geographical areas by ESI/MS/MS and determination of major metabolites by LC-ESI/MS and LC-ESI/MS/MS

Liquid chromatography electrospray mass spectrometry (LC-ESI/MS) has been applied to the full characterization of saponins and phenolics in hydroalcoholic extracts of roots of liquorice (Glycyrrhiza glabra). Relative quantitative analyses of the samples with respect to the phenolic constituents and to a group of saponins related to glycyrrhizic acid were performed using LC-ESI/MS. For the saponin constituents, full scan LC-MS/MS fragmentation of the protonated (positive ion mode) or deprotonated (negative ion mode) molecular species generated diagnostic fragment ions that provided information concerning the triterpene skeleton and the number and nature of the substituents. On the basis of the specific fragmentation of glycyrrhizic acid, an LC-MS/MS method was developed in order to quantify the analyte in the liquorice root samples. Chinese G. glabra roots contained the highest levels of glycyrrhizic acid, followed by those from Italy (Calabria).     

Separation, identification and rapid determination of liensine, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera Gaertn. by liquid chromatography coupled to diode array detector and tandem mass spectrometry

An application of mass spectrometric methods has been developed to characterize, prepare and quantitatively analyze three bisbenzylisoquinoline alkaloids (liensinine, isoliensinine and neferine) from embryo of the seed of Nelumbo nucifera Gaertn. Initially, an analytical method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) with positive ionization mode using a MonoChrom C18 column (4.6 mm x 250 mm i.d. 10 microm) has been developed to characterize liensinine, isoliensinine and neferine, and then scaled up to purify them on a 21.4 mm x 250 mm preparative column. The structures of liensinine, isoliensinine and neferine were elucidated by NMR. Finally, a LC-MS/MS determination method, successfully applied to separation within 3 min, was developed for high throughput simultaneous measurement of liensinine, isoliensinine, and neferine in the extract samples. Multiple reaction monitoring (MRM) was used to monitor the transition of the protonated molecules m/z 611, 611, 625 [M+H]+ to the product ions m/z 206, 192, 206 for analysis of liensinine, isoliensinine and neferine. The LC-MS/MS system was linear in the concentration range of 0.0247-6.02 microg/ml with correlation coefficients of r2>0.992. The quantitative method was validated, with an S/N=3 detection limit of 0.15 ng for liensinine, 0.19 ng for isoliensinine and 0.1 2ng for neferine. The mass fractions of liensinine, isoliensinine, and neferine in the crude extract and the phenolic alkaloid sample of embryo of the seed of N. nucifera Gaertn. were 16.5+/-1.1 and 228.6+/-11.9 for liensinine (mg/g), 45.7+/-1.8 and 640.7+/-15.2 for isoliensinine (mg/g), 59.7+/-6.4 and 58.8+/-9.8 for neferine (mg/g).     

Implication of brevetoxin B1 and PbTx-3 in neurotoxic shellfish poisoning in New Zealand by isolation and quantitative determination with liquid chromatography-tandem mass spectrometry.

Brevetoxin B1 (BTX-B1) was isolated from Austrovenus stutchburyi following the 1992-1993 outbreak of neurotoxic shellfish poisoning (NSP) in New Zealand. We report here the first isolation of PbTx-3 from the same shellfish and the development of a procedure for quantitative determination of PbTx-3 and BTX-B1. PbTx-3 was isolated by chromatography on columns of SiO2, ODS, and LH-20, followed by reverse-phase HPLCs. In mass spectrometry (MS) with an electrospray ionization (ESI) interface operating in the positive or negative ion mode, the abundant protonated ion [M+H]+ of PbTx-3 (m/z 897) and the de-sodiated ion [M-Na]- of BTX-B1 (m/z 1016) were generated, respectively. These served as precursor ions for collision-induced dissociation, and the product ions of m/z 725 from PbTx-3 and m/z 80 from BTX-B1 were identified, allowing unambiguous confirmation of these toxins by selected reaction monitoring liquid chromatography-tandem mass spectrometry (SRM LC-MS/MS) analysis. The determination limits were 0.4 and 2 ng/g for BTX-B1 and PbTx-3 at a signal-to-noise ratio of five, respectively. This LC-MS/MS method was successfully applied to determine BTX-B1 and PbTx-3 in the NSP-associated toxic shellfish. BTX-B1 was found in both A. stutchburyi and Perna canaliculus, but not in Crassostrea gigas, while PbTx-3 was found in all three.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

蟛蜞菊内酯的电喷雾离子阱质谱分析

目的：应用电喷雾离子阱质谱（ESI-MSn）技术对蟛蜞菊内酯进行质谱裂解分析,通过主要特征碎片离子研究其裂解规律。方法：样品溶液进样后,采用ESI-MS1～3碰撞裂解方式,解析蟛蜞菊内酯的特征碎片离子。结果：蟛蜞菊内酯在正离子模式下,采用ESI-MS1获得准分子离子峰[M＋H]＋m/z 315;采用ESI-MS2获得了离子碎片m/z 297、m/z 287;采用ESI-MS3获得m/z259、m/z231等离子碎片。负离子模式下采用ESI-MS1获得[M-H]-m/z313,采用ESI-MS2获得了离子碎片m/z298;采用ESI-MS3获得m/z270、m/z254等离子碎片。结论：蟛蜞菊内酯在软电离模式下,正负离子模式对质谱裂解方式产生差异,其中主要产生的正负离子碎片分别为m/z287和m/z298。本研究为进一步研究蟛蜞菊内酯化合物的体内代谢过程与结构修饰提供实验依据。     

Qualitative and quantitative determination of ten alkaloids in traditional Chinese medicine Corydalis yanhusuo W.T. Wang by LC-MS/MS and LC-DAD

An analytical method incorporating high-performance liquid chromatography (HPLC) with MS and UV-detection was developed for the qualitative and quantitative determination of alkaloids in Corydalis yanhusuo. Ten alkaloids, including seven tertiary alkaloids and three quaternary alkaloids, were identified by comparing their retention times, UV and MS spectra with those of authentic compounds. Furthermore, the collision-induced dissociations of the [M+H](+) and [M](+) ions were studied to clarify the MS behavior of the different types of alkaloids. In positive ion electrospray ionization tandem mass spectrometry (ESI-MS) all the tertiary alkaloids yielded prominent [M+H](+) ions and quaternary alkaloids yielded prominent [M](+) ions in the first order mass spectra. Fragments involving losses of H, CH(3), CO, H(2)O and OCH(3) were observed in the MS/MS spectra. In addition, quantification of the 10 alkaloids in Corydalis yanhusuo from methanol and ethyl acetate extract of different origins were performed by this method, which provides a new tool for the assessment of quality of Corydalis yanhusuo preparations. The method provides the best sensitivity and specificity for characterization and quantitative determination of the alkaloids in Corydalis yanhusuo so far.     

In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC-MS studies

The metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000xg, microsomes, cytosol). Samples were extracted with CH3CN (2 vol.) containing 1% H3PO4 (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25-50 microg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000xg fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC-MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M-H]- and protonated [M+H]+ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed.     

Panax vietnamensis protects mice against carbon tetrachloride-induced hepatotoxicity without any modification of CYP2E1 gene expression

In order to explore the effect of Panax vietnamensis on carbon tetrachloride-induced hepatotoxicity, mice were pretreated for 7 days with either crude extract or total saponins. Crude extract and total saponins dramatically decreased carbon tetrachloride-induced increase of serum GST alpha level (-50.0%, -49.5% respectively). Serum AST level was significantly decreased only with total saponins (-52.2%) and ALT level was slightly modified. In vitro experiments shown that both preparations at high concentrations (> 2000 micrograms/ml) are able to inhibit CYP2E1 enzymatic activity in mouse and human microsomes. However, we did not observe any modification of Cyp2e1 gene expression (enzymatic activity, protein and mRNA levels) in mice treated with either crude extract or total saponins. Taken together, these data demonstrated that Panax vietnamensis could be used as an hepatoprotectant. However, the mechanism of action is not associated with CYP2E1 expression, as previously suggested in vitro in rat for total saponins from Panax ginseng.     

LC/ESI/MS method development for the analysis of hepatotoxic cyclic peptide microcystins in animal tissues.

Microcystins (MCYSTs) are a family of related cyclic heptapeptides produced by several genera and species of blue-green algae (cyanobacteria). MCYSTs are potent and specific inhibitors of the serine threonine family of protein phosphatases, especially PP1 and PP2A. MCYSTs inhibit a liver's protein phosphatase by forming a covalent linkage between MCYSTs' Mdha residue and the phosphatase's cysteine residue. Due to the covalent linkage, analysis of MCYSTs in animal tissues has been limited to determination of unbound MCYST concentration. The MMPB (2-methyl-3-methoxy-4-phenylbutyric acid) oxidation procedure allows for the detection of total MCYST burden by releasing the carboxylic acid MMPB from MCYST's Adda amino acid. An internal standard 4-phenylbutyric acid (4PB) accounts for losses during the method. LC/MS conditions were developed using a ThermoFinnigan LCQDuo ion trap in negative electrospray ionization (ESI). Since both compounds produce the [M-H](-) ion, analysis occurs in selected ion monitoring (SIM) mode for both MMPB (m/z 207.1) and 4PB (m/z 163.1). Complete oxidation of MCYST-LR in liver tissues occurs in 3h. A solid phase extraction (SPE) cartridge removes MMPB and 4PB from the oxidant solution. The process efficiency for the SPE procedure is only 51.3%; however, suppression experiments indicate a 41.8% loss in signal strength due to matrix interferences. Therefore, the extraction efficiency for the SDB-XC cartridge procedure is 93.1%. This research has been successful in developing an LC/MS method for the analysis of total MCYST burden in animal tissues.     

Sub-chronic Hepatotoxicity of Anacardium occidentale (Anacardiaceae) Inner Stem Bark Extract in Rats

The extracts of Anacardium occidentale have been used in the management of different cardiovascular disorders in Nigeria. These have necessitated the assessment of the toxicity of this plant extract in sub-chronic administration. The inner stem bark of Anacardium occidentale was extracted with 80 % methanol and quantitatively analysed for antinutrients and some heavy metals. The phytochemical compositions and acute toxicity of the extract were determined also. Toxicity profiles of the extract on some liver function parameters were evaluated following a sub-chronic oral administration at doses of 1.44 and 2.87 g/kg. The phytochemical screening of extract revealed the presence of high amount of tannins, moderate saponins and trace of free reducing sugars. The antinutrient levels were 5.75 % (tannins), 2.50 % (oxalates), 2.00 % (saponins), 0.25 % (phytate) and 0.03 % (cyanide). The quantity of iron detected from dried crude was 8.92 mg/100 g, while lead and cadmium were non-detectable. The extract had LD(50)of 2.154g/kg p.o. in mice. Sub-chronic administration of the extract significantly increased the serum levels of alanine aminotransaminase and aspartate aminotransaminase, which are indicative of liver damage. The serum levels of alkaline phosphatase and total protein of the treated animals were not significantly increased. The effects of sub-chronically administered extract on hepatocytes were minimal as the serum alkaline phosphatase; total bilirubin and total protein levels in treated animals were not significant (p< 0.05). Thus, sub-chronic administrations of Anacardium occidentale inner stem bark extract did not significantly (p< 0.05) depress the function of hepatocytes in Wistar rats.     

Liquid chromatographic/mass spectrometry assay of triptolide in dog plasma and its application to pharmacokinetic study

A rapid and sensitive method for the determination of triptolide in dog plasma was developed and validated, using high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI/MS). Sample preparation consisted of liquid-liquid extraction with ethyl acetate from dog plasma. The analytes and internal standard prednisolone were well separated on a Zorbax Extend-C18 analytical column. Detection was performed on a triple quadrupole mass spectrometer using selected-ion monitoring (SIM) mode on the deprotonated ions [M-H]- at m/z 359. Calibration curves were linear over the concentration range of 0.5-200 ng/mL of triptolide with the intra- and inter-day precision (the relative standard deviation values) were being less than 7%. Triptolide was stable under different conditions. The intra-day and inter-day accuracy were 99.3-105.2% and 101.3-107.0%, respectively. The lower limit of quantification was 0.5 ng/mL. The method was successfully applied to a pharmacokinetic study after an intragastric administration (i.g.) of triptolide to dogs with a dose of 0.05 mg/kg. The results confirm that the assay is suitable for the pharmacokinetic study of triptolide.     

Detection and identification of glycoyessotoxin A in a culture of the dinoflagellate Protoceratium reticulatum.

The toxin composition of a culture of the dinoflagellate Protoceratium reticulatum was investigated using LC-FLD, after derivatization with DMEQ-TAD (4-(2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalimylethyl)-1,2,4-triazoline-3,5-dione)). Besides yessotoxin (YTX), the new YTX analogue, glycoyessotoxin A (G-YTXA) was detected in culture medium as well as in cells. The conditions for extraction were optimized and the production profile established. Retention time of the resulting fluorescent G-YTXA adduct was identified by comparison of the appropriate standard. Additionally, both G-YTXA and the DMEQ-TAD-G-YTXA adduct were confirmed by LC-MS showing ion peaks at m/z 1273 [M-2Na+H](-) and m/z 1618 [M-2Na+H](-), respectively. The LC-MS(n) displayed a fragmentation pattern similar to that of the YTX series.     

LC-MS determination and pharmacokinetic studies of ursolic acid in rat plasma after administration of the traditional chinese medicinal preparation Lu-Ying extract

Sambucus chinensis L. is a native perennial herb distributed throughout China. In traditional Chinese medicine (TCM), this herb is known as Lu-Ying. Ursolic acid is the major effective constituent of Lu-Ying. A rapid, sensitive, and accurate liquid chromatography-mass spectrometry (LC-MS) method for the determination of ursolic acid in rat plasma was developed and validated. Plasma samples taken from rats that had received Lu-Ying extract orally were acidified with acetic acid and then extracted with a mixture of hexane-dichloromethane-2-propanol (20:10:1, v/v/v). Separation of ursolic acid was accomplished on a C(18) column interfaced with a single quadrupole mass spectrometer. The mobile phase consisting of methanol and water (95:5, v/v) was delivered at a flow rate of 1.0 ml/min. Atmospheric pressure chemical ionization was operated in negative-ion mode. Using selected ion-monitoring mode, the deprotonated molecules [M-H](-) at m/z 455 and 469 were used to quantify ursolic acid and glycyrrhetic acid (internal standard), respectively. The assay was shown to be linear over the range of 10-1000 ng/ml (r> or =0.9960) with a lower limit of quantification of 10 ng/ml. The method was shown to be reproducible and reliable with intraday precision below 7.8%, interday precision below 8.1%, accuracy within +/-4.3%, and mean extraction recovery excess of 83.6%, which were all calculated from the blank plasma sample spiked with ursolic acid at three concentrations of 20, 200, and 800 ng/ml. The LC-MS method has been successfully applied to pharmacokinetic studies of ursolic acid after oral administration of Lu-Ying ethanolic extract (at a dose containing 80.32 mg/kg ursolic acid) to rats. The main pharmacokinetic parameters were: t(1/2), 4.3 h; K(e), 0.16 1/h; t(max), 1.0 h; C(max), 294.8 ng/ml; AUC(0-t) and AUC(0-infinity), 1007.1 ng.h/ml and 1175.3 ng.h/ml, respectively.