| Radix Glehniae (Beishashen), from the roots of Glehnia littoralis (G. littoralis) Fr. Schmidt ex Miq. (Umbelliferae), is one of the most commonly used traditional Chinese medicines (TCM). It has been recorded in Chinese Pharmacopoeia and is used to treat respiratory and gastrointestinal disorders in China. Previous study found that Radix Glehniae contained a large number of coumarins which are active constituents. LC-MS technology has demonstrated its value in analyzing complex mixtures and has become a powerful tool in the online structural characterization and quantitation of various natural compounds owing to its high sensitivity, good separation efficacy and our considerable knowledge regarding its structure. In the present study, HPLC-MS was performed to analyze chemical components in Radix Glehniae. First, coumarins and coumarin glycosides were characterized in crude extract of Radix Glehniae with the summarized fragmentation rules. Second, a novel sensitive and selective HPLC-ESI-MS/MS method was developed and validated to simultaneously determinate and identify constituents in Radix Glehniae samples. Third, a sensitive and selective LC-ESI-MS method for the simultaneous determination of xanthotoxin, psoralen, isoimpinellin, bergapten in rat plasma was firstly developed and validated to analyze plasma samples of the four analytes after oral administration of Radix Glehniae extract.Part one A practical strategy for the characterization of coumarins in Radix Glehniae by liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometryObjective: To summarize fragmentation rules and develop a high sensitive and efficient liquid chromatography-mass spectrometry (LC-MS) method for detection and characterization of the trace coumarins in Radix Glehniae.Methods: First, we studied the mass fragmentation patterns of 10 coumarin standards in the positive ion mode by full scan mass spectra, product ion scan (PI) and precursor ion scan(PREC). On the basis of the summarized new rules, coumarins in a crude extract of Radix Glehniae were characterized by the combined use of the MIM-IDA-EPI and PREC-IDA-EPI modes on a hybrid triple quadrupole-linear ion trap mass spectrometer, for the first time. Based on the simultaneous appearance of [M+H]+ and [M+NH4]+ in the spectrum of Prec, the molecular weight could be unambiguously identified. The chromatographic separation was performed on a C18 column with 1 mmol/L ammonium acetate and methanol as the mobile phase.Results: Six fragmentation rules were summarized and with the linear- type furocoumarins substituted with the same single oxysubstituent group, the retention times of linear-type furocoumarins substituted with an oxysubstituent group at C-8 were less than those with substitution at C-5. According to the summarized fragmentation rules, polarity rules of isomer, retention times, accurate molecular weights and characteristic fragment ions, 29 coumarins and 12 coumarin glycosides in crude extract of Radix Glehniae were characterized by combination use of the two modes.Conclusion: In this study, a high sensitive, accurate and effective LC- MS method for on-line qualitative analysis of the trace coumarins in Radix Glehniae has been developed. This study has demonstrated the unprecedented advantage of the combination use of the MIM-IDA-EPI and Prec-IDA-EPI mode. The MIM-IDA-EPI mode is sensitive and no pre-acquisition of MS/MS spectra of the parent ion due to the same precursor ion and product ion. Therefore, the characterization of the trace coumarins has become very easy and accurate by combination use of the two modes. It has played an important role in controlling the quality of medicinal herb and supplied a method for other trace compounds from natural and synthetic source in the future.Part two Simultaneous determination of 15 components in Radix Glehniae by high performance liquid chromatography -electrospray ionization tandem mass spectrometryObjective: To develop a novel qualitative and quantitative method using high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous analysis of 15 components including ten coumarins, four phenolic acids and adenosine in Radix Glehniae, an important traditional Chinese medicine.Methods: First, an information-dependent acquisition (IDA) method was employed to trigger product ion scans above the MRM signal threshold so that the 15 components could be identified through enhanced product ion (EPI) scans. Second, multiple-reaction monitoring (MRM) was employed in positive and negative mode at the same time in single analysis process and validated to simultaneously determinate and identify 15 constituents in 20 batches of Radix Glehniae. The instrument operated using electrospray ionization source in positive and negative mode simultaneously. The ion spray voltage was set to 5500V and -4500V, respectively. The turbo spray temperature was maintained at 500°C. Nebulizer gas (gas 1) and heater gas (gas 2) was set at 40 and 50 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. The precursor-to-product ion pairs of 15 analytes were adenosine (268.3/136.1), scopoletin (193.1/133.0), xanthotoxol(203.1/147.1), xanthotoxin (217.1/202.0), psoralen (187.2/131.1), isoimpinellin(247.1/232.1), bergapten (217.1/174.0), oxypeucedanin (287.2/203.1), imperatorin (271.1/203.2), cnidilin (301.1/233.1), isoimperatorin (271,1/147.1), chlorogenic acid (353.2/191.2), caffeic acid (179.3/135.3), vanillic acid (167.3/108.1), ferulic acid (193.3/134.2). The chromatographic separation was performed on an Agilent Zorbax Eclipse XDB-C18 column (150 mm×4.6 mm, 5μm), and the column temperature set at 25°C. The mobile phase consisted of (A) methanol and (B) 0.1% aqueous formic acid. Elution was performed by means of an isocratic gradient 30:70 (A: B, v/v) for 11 min. The flow rate was 1.0 ml/min.Results: The linear relationships, linearity, precision, accuracy, limit of detection and limit of quantification of the method were good for the 15 components. And we successfully applied it to analyze 20 Radix Glehniae samples from different sources. The results demonstrated that a number of reasons such as plant origin, growth circumstance and storage time might contribute to the differences in the level of active constituents among various Radix Glehniae samples and storage time was the principal reason. Meanwhile, the result indicated that many active components were in the cortex of the root and the peeling could lead to reduction of active components.Conclusion: A novel sensitive and selective HPLC-ESI-MS/MS method operating both negative and positive scanning modes in single analysis process was developed and validated to simultaneously determinate and identify 15 constituents in Radix Glehniae samples. Among these constituents, scopoletin, xanthotoxol, isoimpinellin and oxypeucedanin in Radix Glehniae were quantitated for the first time. The method plays an important role in the quanlity control of Radix Glehniae.Part three Simultaneous determination of psoralen, xanthotoxin, isoimpinellin and bergapten in rat plasma by liquid chromatography-electrospray ionization mass spectrometric method and its application to pharmacokinetic study of Radix Glehniae extractObjective: To develop a sensitive, specific and rapid liquid chromatography-mass spectrometry (LC-MS) method and validate for the simultaneous determination of xanthotoxin (8-methoxypsoralen), psoralen, isoimpinellin (5, 8-dimethoxypsoralen) and bergapten (5-methoxypsoralen) in rat plasma, using pimpinellin as an internal standard (IS).Methods: Blood samples were collected into heparinized centrifuge tubes from the fossa orbitalis vein at 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 360, 480, 600 min after single oral administration of Radix Glehniae decoction (10 mL/kg). Within 30 min after blood withdrawal, the samples were centrifuged at 4,000 rpm for 10 min and the separated plasma samples were frozen in polypropylene tubes at ?20°C prior to analysis. The plasma samples were pretreated by protein precipitation with methanol and chromatographic separation was performed on a C18 column with a mobile phase consisted of 1 mmol/L ammonium acetate and methanol (30:70, v/v). The detection was accomplished by multiple-reaction monitoring (MRM) scanning via electrospray ionization (ESI) source operating in the positive ionization mode. The optimized mass transition ion-pairs (m/z) for quantitation were 217.1/202.1 for xanthotoxin, 187.1/131.1 for psoralen, 247.1/217.0 for isoimpinellin, 217.1/202.1 for bergapten, and 247.1/231.1 for IS. The total run time was 6 min between injections.Results: The calibration curves were linear over the investigated concentration range: 0.680-680 ng/mL, (xanthotoxin), 1.21-1210 ng/mL (psoralen), 0.800-800 ng/mL (isoimpinellin) and 0.464-464 ng/mL (bergapten), with all correlation coefficients higher than 0.998. The lower limits of quantitation (LLOQ) of these analytes were less than 1.21 ng/mL. The intra- and inter-day RSD were no more than 9.7% and the relative errors were within the range of -8.1% to 4.5%. The average extraction recoveries for all compounds were between 90.7%-106.2%. The four analytes have parallel pharmacokinetic parameters in vivo, such as t1/2α, t1/2βand MRT. All of the four analytes were absorbed rapidly and eliminated quickly with the similar rate. But the Tmax of xanthotoxin, isoimpinellin, bergapten was longer than that of psoralen.Conclusion: A sensitive and selective LC-ESI-MS method for the simultaneous determination of xanthotoxin, psoralen, isoimpinellin, bergapten in rat plasma was firstly developed and validated. The proposed method showed appropriate accuracy and repeatability and was successfully applied to a plasma samples analysis of the four analytes after oral administration of Radix Glehniae extract, which maybe provide some references to the apprehension of the action mechanism and clinical application of Radix Glehniae.
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