Yuelin Song

Rapid determination of pesticide residues in herbs using selective pressurized liquid extraction and fast gas chromatography coupled with mass spectrometry

A selective pressurized liquid extraction and gas chromatography coupled with triple quadrupole mass spectrometer method was developed for simultaneous determination of 52 pesticide residues in medicine and food dual-purpose herbs. The developed extraction method integrated extraction and cleanup processes for sample preparation. The sorbents, 5 g Florisil and 100 mg graphitized carbon black, were placed inside the extraction cell to remove matrix interferences. Optimized conditions of selective pressurized liquid extraction were ethyl acetate as extraction solvent, 120°C of extraction temperature, 6 min of static extraction time, 50% of flush volume extracted for two cycles. An ultra inert capillary GC-MS HP-5 UI column (20 m × 0.18 mm id, 0.18 μm) and column backflush system were used for the analysis. Multiple-reaction monitoring was employed for the quantitative analysis with electron ionization mode. All calibration curves showed good linearity (r(2) > 0.995) within the test ranges. The average recoveries of most pesticides were from 81 to 118%. The validated method was successfully applied for the determination of pesticide residues in four herbs. The results indicate that selective pressurized liquid extraction and GC-MS/MS is a sensitive and reliable analytical method for the simultaneous determination of multiple pesticide residues in herbs.

Qualitative analysis and enantiospecific determination of angular-type pyranocoumarins in Peucedani Radix using achiral and chiral liquid chromatography coupled with tandem mass spectrometry

Angular-type pyranocoumarins (APs), the derivatives of khellactone, are widely documented as the main active constituents in Peucedani Radix (Chinese name: Qian-hu). Owing to the natural occurrence of chiral centers, enantiomers of APs are extensively distributed in the original plant, and enantioselective performances have been definitely demonstrated for these enantiomers. In current study, the chemical characterization of the major and minor APs in Peucedani Radix was performed using ultra high performance liquid chromatography coupled with diode array detector and hybrid ion trap-orbitrap mass spectrometry. On the other hand, a heart-cut two-dimensional achiral-chiral liquid chromatography combining triple quadropole-linear ion trap mass spectrometry system (2D LC-MS/MS) was developed for simultaneous enantiospecific quantification of eighteen coumarins, including seven pairs of enantiomers. Eleven APs (1-11) were recruited to propose UV absorption characteristics and electrospray ionization fragmentation patterns of APs. A total of 42 components were categorized into APs based on their UV spectral properties and identified according to the proposed mass fragmentation pathways, while two linear-type furanocoumarins (12-13) were unambiguously assigned by further purification. A Capcell core RP-C18 column was employed in the primary LC dimension to achieve efficient racemic separation for the main chemical constituents (1-9 and 12-13) in Peucedani Radix, while a Chiralpak AD-RH column was utilized in the secondary dimension to contribute enantioselective separation for seven enantiomerically enriched components (1, 3 and 5-9). Collectively, the results provided the chemical evidences for revealing the material basis of the therapeutic effects of Peucedani Radix, and the developed 2D LC-MS/MS system in the present study is expected to be an ideal tool for the quality control of Peucedani Radix as well as a reliable technique for complex matrices containing both achiral and chiral components.

Rapid simultaneous determination of isoflavones in Radix puerariae using high-performance liquid chromatography–triple quadrupole mass spectrometry with novel shell-type column

A high-performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/MS) method was developed for rapid determination of 13 isoflavones in Radix puerariae. A novel shell-type column, namely Kinetex core-shell C(18) column (50 mm×2.1 mm id, 2.6 μm), and gradient elution were used during the analysis. The chromatographic peaks of 13 investigated compounds were identified by comparing their retention time and MS data with the related reference compounds. Multiple-reaction monitoring (MRM) was employed for the quantitative analysis with negative ionization mode. All calibration curves showed good linearity (r(2)>0.9990) within test ranges. The LOD and LOQ were lower than 0.017 and 0.873 μg/mL on column, respectively. The intra- and inter-day precisions for 13 analytes were <1.17 and 2.17%, respectively, and the recoveries were 93.1-104.4%. The validated method was applied for quantitative analysis of 13 isoflavones in 7 species of Radix puerariae. The result demonstrated that HPLC-MS/MS system with Kinetex column could be a promising analytical tool for the determination of isoflavones in traditional Chinese medicines, which is helpful for comprehensive evaluation of quality of R. puerariae.

Ganoderiol A-enriched extract suppresses migration and adhesion of MDA-MB-231 cells by inhibiting FAK-SRC-paxillin cascade pathway.

Cell adhesion, migration and invasion are critical steps for carcinogenesis and cancer metastasis. Ganoderma lucidum, also called Lingzhi in China, is a traditional Chinese medicine, which exhibits anti-proliferation, anti-inflammation and anti-metastasis properties. Herein, GAEE, G. lucidum extract mainly contains ganoderiol A (GA), dihydrogenated GA and GA isomer, was shown to inhibit the abilities of adhesion and migration, while have a slight influence on that of invasion in highly metastatic breast cancer MDA-MB-231 cells at non-toxic doses. Further investigation revealed that GAEE decreased the active forms of focal adhesion kinase (FAK) and disrupted the interaction between FAK and SRC, which lead to deactivating of paxillin. Moreover, GAEE treatment downregulated the expressions of RhoA, Rac1, and Cdc42, and decreased the interaction between neural Wiskott-Aldrich Syndrome protein (N-WASP) and Cdc42, which impair cell migration and actin assembly. To our knowledge, this is the first report to show that G.lucidum triterpenoids could suppress cell migration and adhesion through FAK-SRC-paxillin signaling pathway. Our study also suggests that GAEE may be a potential agent for treatment of breast cancer.

Simultaneously enantiospecific determination of (+)-trans-khellactone, (+/−)-praeruptorin A, (+/−)-praeruptorin B, (+)-praeruptorin E, and their metabolites, (+/−)-cis-khellactone, in rat plasma using online solid phase extraction-chiral LC-MS/MS

Many chiral drugs are used as the racemic mixtures in clinical practice. The occurrence of enantioselectively pharmacological activities calls for the development of enantiospecific analytical approaches during pharmacokinetic studies of enantiomers. Sample preparation plays a key role during quantitative analysis of biological samples. In current study, a rapid and reliable online solid phase extraction-chiral high performance liquid chromatography-tandem mass spectrometry (online SPE-chiral LC-MS/MS) method was developed for the simultaneously enantiospecific quantitation of (+)-trans-khellactone (dTK), (+/-)-cis-khellactone (d/lCK), (+/-)-praeruptorin A (d/lPA), (+/-)-praeruptorin B (d/lPB) and (+)-praeruptorin E (dPE), the main active angular-type pyranocoumarins (APs) in Peucedani Radix (Chinese name: Qian-hu) or the major metabolites of those APs, in rat plasma. The validation assay results described here show good selectivity and enantiospecificity, extraction efficiency, accuracy and precision with quantification limits (LOQs) of 2.57, 1.28, 1.28, 1.88, 4.16, 4.16 and 4.18ngmL(-1) for dTK, lCK, dCK, dPA, dPB, lPB and dPE, respectively, while lPA was not detected in rat plasma due to the carboxylesterase(s)-mediated hydrolysis. In addition, the validated system was satisfactorily applied to characterize the pharmacokinetic properties of those components in normal and chronic obstructive pulmonary disease (COPD) rats following oral administration of Qian-hu extract. dCK and lCK were observed as the main herb-related compounds in plasma. Enantioselectively pharmacokinetic profiles occurred for dCK vs lCK, dPA vs lPA, and dPB vs lPB in either normal or COPD rats. The proposed whole system is expected to be a preferable analytical tool for in vivo study of chiral drugs, in particular for the characterization of enantioselectively pharmacokinetic profiles.

Characterization of in vitro and in vivo metabolites of carnosic acid, a natural antioxidant, by high performance liquid chromatography coupled with tandem mass spectrometry.

Carnosic acid (CA) is a widely employed antioxidant and the main active component in rosemary and sage, but its metabolism remains largely unknown. The present study investigated the metabolism of CA in vitro and in vivo for the first time, using high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (HPLC-Q-trap-MS). A couple of scan modes were adopted in mass spectrometer domain, including Q1 full scan, neutral loss scan-information dependent acquisition-enhanced product ion (NL-IDA-EPI) and precursor ion scan-information dependent acquisition-enhanced product ion (PI-IDA-EPI). In particular, a prediction was carried out on the basis of in vitro metabolism results, and gave birth to a multiple ion monitoring-information dependent acquisition-enhanced product ion (MIM-IDA-EPI) mode aiming to detect the trace metabolites in CA-treated biological samples. A total of ten metabolites (M4-13), along with three degradative products (M1-3), were identified for CA from in vitro metabolism models, including liver microsomes of human and rats (HLMs and RLMs), human intestinal microsomes (HIMs) and two species of Cunninghamella elegans. Twelve (U1-12) and six (F1-6) metabolites were detected from CA-treated urine and feces, respectively. In addition, five metabolites (SM2-6) in vivo were purified and definitely identified using NMR spectroscopy. The results of both in vitro and in vivo metabolism studies indicated poor metabolic stability for CA, and the glucuronidation and oxidation metabolisms extensively occurred for CA in vitro, while oxidation, glucuronidation and methylation were the main metabolic pathways observed in vivo.

Stereoselective metabolism of (±)-praeruptorin A, a calcium channel blocker from Peucedani Radix, in pooled liver microsomes of rats and humans

(±)-Praeruptorin A (PA) is the major component of Peucedani Radix. The present study investigated stereoselectivity in PA metabolism in liver microsomes of rats (RLMs) and humans (HLMs), for the first time. PA was enantioseparated by semi-preparative chiral HPLC. Metabolic profiles of the dextrorotatory (dPA) and the levorotatory (lPA) forms in HLMs and RLMs were determined using LC-MS/MS. (-)-cis-Khellactone (D1) prepared from basic hydrolysis of dPA, and (3′R, 4′R)-4′-angeloyl-khellactone (L8) and (3′R, 4′R)-3′-angeloyl-khellactone (L9) isolated from a scale-up incubation of lPA with rat plasma were unambiguously identified by LC-MS/MS and NMR analysis. Other metabolites were tentatively identified using LC-MS/MS. In the absence of NADPH-regenerating system, dPA remained intact, however, lPA yielded L8 and L9 via a carboxylesterase(s)-mediated process. In the presence of NADPH-regenerating system, lPA produced 9 (L1-9) metabolites in both species, while dPA generated 12 (D1-12) and 6 (D1-3, 6, 9 and 10) metabolites in RLMs and HLMs, respectively. Hydrolysis, oxidation and acyl migration were demonstrated to be the predominant pathways for both enantiomers. Both enantiomers were eliminated faster in RLMs than in HLMs, while lPA showed greater species difference. PA enantiomers exhibited stereoselective metabolism in RLMs and HLMs, implying chiral discrimination in their actions.

Metabolic differentiations of Pueraria lobata and Pueraria thomsonii using 1H NMR spectroscopy and multivariate statistical analysis

Puerariae Radix was a widely used herbal medicine. Pueraria lobata (PL) and Pueraria thomsonii (PT) were the two authorized sources of Puerariae Radix (gegen) in China. In this study, metabolic differentiations between these two species were investigated using NMR spectroscopy followed by principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA). The content of puerarin in PL and PT was also determined using quantitative (1)H NMR spectroscopy. Thirteen isoflavones were tentatively identified based on 1D and 2D NMR spectroscopic data in two species. The (1)H NMR spectra of PL and PT were obviously different. PL and PT could also be markedly discriminated from (1)H NMR spectroscopic data by PCA and PLS-DA. For the crude drug resources, isoflavones, in which puerarin is the most important one, were regarded as the reasonable markers for the discrimination of the two species. The contents of puerarin and total isoflavones in PL were quantitated much higher than those in PT. Above all, (1)H NMR spectroscopy, which can provide comprehensive profiles of the metabolites and achieve convenient determinations of puerarin and total isoflavones in a single run, is an efficient means for evaluating the medicinal samples and achieving a better quality control of Puerariae Radix.

Transport and metabolism of (±)-praeruptorin A in Caco-2 cell monolayers

(±)-Praeruptorin A (dl-PA) is one of the main pyranocoumarins of Peucedani Radix and the chemical marker for quality control of the herb in China. This study investigated the transport and metabolism of dl-PA, for the first time, in Caco-2 cell monolayers. PA enantiomers of dl-PA in the transport study were simultaneously determined using a simple and rapid enantio-selective high performance liquid chromatography-UV method. Both dextrorotatory (d–PA) and levorotatory (l–PA) enantiomers traversed Caco-2 monolayer rapidly in both directions (absorptive Papp: 2.01–3.03 × 10−5 cm/s; secretory Papp: 1.58–1.96 × 10−5 cm/s) mainly via passive diffusion. Higher transport rates of dPA were observed in both directions. PA enantiomers were incompletely recovered after the transport study. Nonspecific binding to the Transwell inserts, irreversible binding to cellular components and metabolism within the cells accounted for the loss. dl-PA was partially hydrolyzed in Caco-2 monolayers and yielded two stereoisomers via removal of the acetyl group from C-4′ position. Both phenylmethylsulphonyl fluoride (a nonspecific esterase inhibitor) and bis(p-nitrophenyl) phosphate sodium salt (a specific inhibitor of carboxylesterases) completely abolished dl-PA hydrolysis. In summary, PA enantiomers were rapidly transported across Caco-2 cells and partially hydrolyzed by carboxylesterases during permeation. These findings provide mechanistic understanding of in vivo pharmacokinetic properties of dl-PA.

1H nuclear magnetic resonance based-metabolomic characterization of Peucedani Radix and simultaneous determination of praeruptorin A and praeruptorin B

As a widely used traditional herbal medicine, it is crucial to characterize the holistic metabolic profile of Peucedani Radix (Chinese name: Qian-hu). However, it is quite arduous to obtain the whole picture of chemical constituents appropriately with the existing analytical techniques that were based on HPLC-UV or LC-MS/MS system. In present investigation, nuclear magnetic resonance (NMR) spectroscopy coupled with principal components analysis (PCA) was introduced to metabolomic characterization of Qian-hu crude extracts without any chromatographic separation. In addition, the contents of praeruptorin A (PA) and proaeruptorin B (PB) in Qian-hu were simultaneously determined using quantitative (1)H NMR (q(1)H NMR) spectroscopy. Eighteen reference compounds (1-18), which were purified from this herbal drug extract previously, were recruited for the assignment of the protonic signals in the (1)H NMR spectra. Following PCA, 15 batches of Peucedani Radix were divided into two groups (I and II), and angular-type pyranocoumarins, in particular PA and PB, as well as 5-methoxycoumarin were demonstrated as the predominant markers being responsible for the distinguishment of Qian-hu from different districts. The contents of the two analytes (PA & PB) were calculated by the relative ratio of the integral values of the target peak for each compound to the known amount of the internal standard, formononetin (IS). The lower limits of quantitation were determined as 19.5μg/mL for both PA and PB. The quantitative results indicated that the contents of PA and PB showed quite variable qualities among different extract samples. Above all, (1)H NMR spectroscopy, that could not only provide comprehensive profiles of the metabolites but also achieve convenient determination of praeruptorin A and praeruptorin B, is a promising means for evaluating the medicinal samples of Peucedani Radix.