Wanghui Jing

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.

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.

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.

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.

Simultaneous determination of caffeine, gallic acid, theanine, (−)-epigallocatechin and (−)-epigallocatechin-3-gallate in green tea using quantitative 1H-NMR spectroscopy

Green tea consists of the dried leaves of Camellia sinensis, and enjoys great popularity all over the world due to its pleasant taste and positive impact on human health. It has also been regarded as a natural medicine containing great amounts of caffeine (CA), gallic acid (GA), theanine (TH), and tea polyphenols, mainly including epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG). In the present study, 1H-NMR spectroscopy is utilized for chemical characterization along with simultaneous determination of CA, GA, TH, EGC and EGCG in commercial green tea. Signal assignment for representative samples was facilitated by reference compounds and comparing with information in the literature. On the other side, the diagnostic singlet signals at δ 7.68, 7.14, 6.59 and 6.62 in the 1H-NMR spectra were selected as quantitative peaks for CA, GA, EGC and EGCG, respectively, while the triplet signal at δ 1.12 with a coupling constant (J) of 7.26 Hz was chosen for the determination of TH. TSP-d4 was adopted as the internal standard (IS) and the reference chemical shift of δ 0.00. The limits of detection (LODs) and limits of quantitation (LOQs) were measured as 28.9 and 57.8 μg mL−1 for CA, 18.7 and 37.4 μg mL−1 for GA, 23.4 and 46.8 μg mL−1 for TH, 28.1 and 56.2 μg mL−1 for EGC, and 28.1 and 56.2 μg mL−1 for EGCG, respectively. The relative standard deviation of both precision and repeatability assays were lower than 4.5%. The mean recoveries of high, medium and low concentration levels for each analyte were among the range of 93.1–106.3%. The contents of CA, GA, TH, EGC and EGCG were measured among the ranges of 3.72–8.38 mg g−1, 0.34–1.88 mg g−1, 1.56–4.48 mg g−1, 2.96–8.50 mg g−1 and 2.78–10.60 mg g−1, respectively, in 9 batches of green tea. Above all, 1H-NMR spectroscopy is proven as a reliable tool not only for metabolic characterization, but also for simultaneous determination of effective components in green tea.

Metabolic characterization of (±)-praeruptorin A in vitro and in vivo by high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry

(±)-Praeruptorin A (PA) is the major bioactive component in Peucedani Radix (Chinese name: Qian-hu), and exhibits dramatically anti-hypertensive effect typically through acting as a calcium channel blocker. The current study aims on the characterization of the metabolic profiles of PA in vitro and in vivo using high performance liquid chromatography (HPLC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). A total of 12 phase I metabolites (M1-12) in rat liver microsomes (RLMs), 9 phase I metabolites (M1-3, M5-6 and M9-12) in human liver microsomes (HLMs), 2 hydrolyzed products in rat plasma (M11 and M12), none metabolite in human plasma, none metabolite in rat intestinal bacteria, 7 metabolites (M1, M4-7, M13 and M15) in PA-treated rat urine and 6 metabolites (M1, M4-7 and M15) in PA-treated feces were detected and tentatively identified using predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (predictive MRM-IDA-EPI) mode in combination with enhanced mass spectrum-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode in the mass spectrometer domain, respectively, while TOF-MS was adopted to confirm the identification. Further, 2 glucuronidated metabolites (M13-14) in RLMs and none metabolite in HLMs of cis-khellactone (CKL), which was the main actual form of PA in vivo, were generated, while its sulfated product was not observed in either rat liver S9 fractions (RS9) or human liver S9 fractions (HS9). Oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation were demonstrated to be the predominant metabolic types for PA in vitro and in vivo. Judging from the decrement of peak areas, PA was metabolized quickly in both RLMs and HLMs, indicating extensively hepatic first-pass elimination. Taken together, the metabolic fates of (±)-praeruptorin A in vitro and in vivo were elucidated in current study, and Q-trap-MS coupled with LightSight™ software can be adopted as a useful tool for quick detection and identification of metabolites in complex biological matrices.

A practical strategy for chemical profiling of herbal medicines using ultra-high performance liquid chromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry: a case study of Mori Cortex

Herbal medicines (HMs) are believed to produce holistic action through the synergistic effects of many major and minor components. In the current study, a practical strategy was designed to comprehensively characterize the chemical constituents in HMs. Mori Cortex (MC), which contains diverse chemical constituents and consequently exhibits a broad spectrum of pharmacological activities, was chosen as a model case. The workflow included three steps: first, a thorough literature review was performed to summarize the available information about the phytochemistry and the biosynthetic pathways for the genus Morus; second, seven compounds, namely morin (polyhydroxyflavonoid), morusin (prenylflavone), mulberroside A and oxyresveratrol (stilbenes), mulberroside C (2-arylbenzofuran derivative), sanggenon C and kuwanon G (DA-type adducts), were selected to propose mass fragmentation pathways for the primary chemical types in MC; and third, a set of parent-to-parent ion transitions was constructed using quasi-molecular and sodium adduct ions of the identified compounds and their potential derivatives, and the multiple ion monitoring-information dependent acquiring-enhanced product ion (MIM-IDA-EPI) method was thereby used to detect and identify the constituents. As a result, a total of 140 components were detected with 133 identified in the MC extract, including 10 polyhydroxyflavonoids, 4 stilbenes, 16 2-arylbenzofuran derivatives, 60 prenylflavones, and 43 DA type adducts, while the identities of 7 components could not be elucidated due to insufficient structural information. Collectively, the strategy was demonstrated to be efficient, reliable and sensitive for global chemical profiling of HMs.

Enantiomeric separation of angular-type pyranocoumarins from Peucedani Radix using AD-RH chiral column

Enantiomers and diastereoisomers of angular-type pyranocoumarins (APs) are abundant in Peucedani Radix (Chinese name: Qian-hu), eliciting distinct activities in vitro and in vivo. Our ongoing investigation on APs yielded eight pairs of enantiomers (1a and 1b, 2a and 2b, 3a and 3b, 4a and 4b, 5a and 5b, 6a and 6b, 7a and 7b and 8a and 8b) via enantiomeric separation of trans-3′-angeloylkhellactone (1), trans-3′-acetyl-4′-isobutyrylkhellactone (2), trans-3′-acetyl-4′-angeloyl-khellactone (3), 3′-angeloyloxy-4′-oxo-3′,4′-dihydroseselin (4), cis-3′-acetyl-4′-angeloylkhellactone (5), cis-3′-isovaleryl-4′-acetylkhellactone (6), cis-3′-angeloyl-4′-isovalerylkhellactone (7) and cis-3′,4′-diisovalerylkhellactone (8), respectively, using semi-preparative AD-RH chiral column. All the compounds (1–8) were enantioseparated for the first time, while the absolute configurations of 2a, 2b, 6a and 8b were reported first.

A pretreatment free method for the determination of seven natural products in a high-salt matrix by online guard column extraction coupled with tandem mass spectrometry

In vitro cell models are easy, reproducible, and cost-effective tools for tracking drug absorption rates and for elucidating related mechanisms. Traditionally, before introduction into an analysis system, samples crossing an in vitro cell membrane usually undergo complicated processes including precipitation, centrifugation, and even filtering. In the current study, a generic, sensitive and rapid method was developed for the determination of natural products in the matrix of in vitro drug absorption systems (HBSS, Hanks balanced salt solution). A guard RP-C18 column was used to retain the target compounds, while a tandem mass spectrometry system in the multiple reaction monitoring mode was used to specifically detect the target compounds. In addition, a six-way valve was used to connect the LC and MS systems, and to automatically switch the flow directions between the mass spectrometer and the waste. The method was validated by determining the accuracy, precision and sensitivity using seven natural products including (+)-praeruptorin A (dPA), (−)-praeruptorin A (lPA), (+)-praeruptorin B (dPB), (−)-praeruptorin B (lPB) from Peucedani Radix (Chinese name: Qian-hu), morusin (Mo), sanggenon C (SC), and kuwanon G (KG) from Mori Cortex (Chinese name: Sang-Bai-Pi). The developed method is proposed to be applied for in vitro absorption and transport studies.