| Common cnidium fruit is the dried ripe fruit of Cnidium monnieri(L.) Cusson, Apiaceae and, as one of the most popular traditional Chinese medicinal herbs, it has been used to treat impotence, frigidity and skin diseases. Phytochemical studies reveal that Common cnidium fruit contains many compounds such as coumarins, flavonoids, and volatile oil and their glycosides. Among these, coumarins and flavonoids are generally considered to be the major active components.To date, studies on quantitative determination of chemical constituents in Common cnidium fruit and their pharmacokinetics have been very few. In present study, we firstly developed an accurate and simple LC-ESI-MS/MS method for simultaneous determination of 16 bioactive constituents in Common cnidium fruit. The satisfactory results demonstrated that the LC-ESI-MS/MS method was a good option for routine analysis and could be applied as a reliable quality control method for Common cnidium fruit. In this study, we developed a rather sensitive and selective UPLC-ESI-MS/MS method to simultaneously determine xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and isoimperatorin in rat plasma. The method was applied to pharmacokinetics after oral administration of Common cnidium fruit extract to rats. A sensitive and selective UPLC-ESI-MS/MS method was developed for simultaneous determination of the 14 main active components in Bazibushen Capsules. The obtained results would be very helpful for evaluating the clinical application of this herb. Also, A sensitive and selective GC-MS-SIM method was developed for simultaneous determination of the 7 main coumarins in Common cnidium fruit. Lastly, We developed a UPLC-Q-TOF-MS/MS method for identify the metabolites of bergapten in rat urine and bile after oral administration of bergapten. The metabolic pathway of bergapten were summarized. The method provided an important reference for the clinical application of bergapten. Part one Simultaneous quantification of 16 bioactive constituents in Common Cnidium Fruit by liquid chromatography-electrospray ionization-mass spectrometryObjective: To develop a novel quantitative method using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry for the simultaneous determination of the 16 bioactive constituents, including nine coumarins(osthole, xanthotoxol, isoporalen, isopimpinelline, bergapten, xanthotoxin, columbianedin, imperatorin and isoimperatorin) and seven flavonoids(kaempferol, isorhamnetin, quercitrin, luteolin, quercetin, hesperidin and rutin) in Common cnidium fruit samples from different regions. In addition, 12 batches of Common cnidium fruit from different sources were compared using the developed method.Methods: The chromatographic separation was performed on a C18 column with linear gradient elution of acetonitrile and 0.1% acetic acid at a flow rate of 1.0 m L/min in 15 min. Quantification of the analytes was achieved by use of a hybrid quadrupole linear ion-trap mass spectrometer. Multiple-reaction monitoring scanning was employed with switching electrospray ion source polarity between positive and negative modes in a single run. The operating conditions for the ESI interface were as follows: the ion spray voltage was set to 4500 and-4500 V, respectively, the turbo spray temperature was maintained at 550 oC, nebulizer gas(gas1) and heater gas(gas2) was set at 50 and 60 arbitrary units, respectively. The curtain gas was kept at 25 arbitrary units and the interface heater was on. Entrance potential(EP) was set at 10/-10 V and collision cell exit potential(CXP) was set at 5/-5 V. Nitrogen was used in all cases. Multiple-reaction monitoring mode was employed for quantification. The dwell time of each ion pair was 40 ms. The full-scan mass covered the range from m/z 100 to 1000.Results: All the marker substances showed good linearity(r2>0.9987) in the test ranges. The LODs and LOQs for the compounds ranged from 1.71 to 7.29 ng/m L and from 5.10 to 20.01 ng/m L, respectively. The overall intra-day(n=6) and inter-day(n=3) RSDs were 0.5–3.6% and 0.6–3.5%, respectively, and the overall stability and repeatability variations were 0.5–3.9% and 0.5–3.2%. The overall recoveries were between 96.89 and 101.6% for all compounds.Conclusion: A efficient, rapid and sensitive LC-ESI-MS/MS method operating both positive and negative scanning modes in a single analysis process was first established for the qualitation and quantification of 16 major components in Common cnidium fruit. Validation of the assay showed appropriate sensitivity and specificity and was successfully utilized to analyze 12 batches of Common cnidium fruit samples from different sources. The satisfactory results demonstrated that the LC-ESI-MS/MS method was a good option for routine analysis and could be applied as a reliable quality control method for Common cnidium fruit. The results demonstrated that the amounts of the 16 marker compounds within the plant material are very variable and this may be due to a number of causes including place of origin, plant source, growth conditions, cultivation methods, harvesting time, processing and storage conditions. Part two Simultaneous determination and pharmacokinetic study of six main active components from Common Cnidium Fruit extract in rat plasma by UPLC-ESI-MS/MSObjective: To establish a sensitive, specific and rapid liquid chromatography-mass spectrometry(UPLC-ESI-MS/MS) method for the determination of the six main active components including xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and isoimperatorin in rat plasma after the orally administrating of Common cnidium fruit extract, and this method was used and validated to study the pharmacokinetics.Methods: Six rats were given single doses of Common cnidium fruit extract(6 m L/kg) and blood samples were collected into heparinized centrifuge tubes from the vein of the eye ground at 0.08, 0.17, 0.33, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 9, 12, 18, 24 and 30 h after a single oral administration. 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 and extracted by a simple protein precipitation method with methanol. Sulfamethoxazole(SMZ) was used as internal standard. Chromatographic conditions: Waters ACQUITY BEH C18 column(50mm×2.1mm,1.7mm) with the column temperature set at 40℃. A linear gradient elution of eluents A(acetonitrile) and B(0.1% acetic acid; v/v) was used for the separation. The following gradient condition was used: initial 0–1.3 min, linear change from 30%A to 50%A; 1.3–2.2 min, linear change from 50%A to 80%A; 2.2–3.5 min, isocratic elution 80%A; 3.5–4.0 min, linear change from 80%A to 30%A; 4.0–5.0 min, isocratic elution 30%A. The flow rate was set at 0.3 m L/min. Mass spectrometry: The mass spectrometer was operated by ESI source in positive modes for a single run. The source temp was set at 150℃. The capillary voltage was set at 2.0 k V. The source offset voltage was kept at 50 V. The desolvation temp was set at 500 ℃. The desolvation flow was 800 L/Hr. The cone flow was 150 L/Hr. The nebuliser pressure was 7.0 bar. For structural identification of each analyte, the information-dependent acquisition(IDA) method was used to trigger the enhanced product ion(EPI) scans by analyzing MRM signals. The optimized mass transition ion-pairs(m/z) for quantitation were 216.97/201.95 for xanthotoxin, 246.97/231.91 for isopimpinelline, 216.97/201.96 for bergapten, 271.03/202.95 for imperatorin, 244.97/188.97 for osthole, 270.97/202.96 for isoimperatorin, and 254.12/156.08 for IS. The total run time was 5.0 min between injections.Results: The calibration curves were linear over the investigated concentration range: 0.20~100.16 ng/m L(xanthotoxin), 0.20~99.61 ng/m L(isopimpinelline), 0.20 ~ 100.22 ng/m L(bergapten), 0.20 ~ 98.03 ng/m L(imperatorin), 0.20 ~ 99.50 ng/m L(osthole) and 0.20 ~ 100.13 ng/m L(isoimperatorin), with all correlation coefficients higher than 0.9983. The lower limits of quantitation(LLOQ) of these analytes were less than 0.21 ng/m L. The intra-day and inter-day RSD were no more than 6.2% and the relative errors were within the range of-7.5% to 5.1%. The average extraction recoveries for all compounds were between 72.3 % and 88.7%. Imperatorin could achieve the maximum plasma concentration at 1 h, isoimperatorin could achieve the maximum plasma concentration at 1.5 h, xanthotoxin could achieve the maximum plasma concentration at 2 h, while isopimpinelline and bergapten could achieve the maximum plasma concentration at 3 h after oral administration. But osthole need 6 h to achieve the maximum plasma concentration after oral administration. The six analytes have distinctive pharmacokinetic parameters in vivo. All of the six analytes were absorbed rapidly and have the similar elimination rate.Conclusion: A selective UPLC-ESI-MS/MS method was developed and validated for the simultaneous determination of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole and isoimperatorin in rat plasma after the orally administrating of Common cnidium fruit extract. The results showed that this method is robust, specific and sensitive and it can successfully fulfill the requirements of pharmacokinetic study. The results provided a meaningful basis for the clinical application of this herb. Part three Determination of 14 components in Bazibushen Capsule by UPLC-ESI-MS/MSObjective: To develop a method for the determination of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole, isoimperatorin, deoxyschizandrin, schisandrin, hyperoside, quercitrin, luteolin, kaempferol, isorhamnetin and ursolic acid in Bazibushen Capsule by UPLC-ESI-MS/MS.Methods: Chromatographic conditions: Waters ACQUITY BEH C18 column(50mm×2.1mm,1.7mm) with the column temperature set at 40℃. A linear gradient elution of eluents A(acetonitrile) and B(0.1% acetic acid; v/v) was used for the separation. The following gradient condition was used: initial 0–1.3 min, linear change from 30%A to 50%A; 1.3–2.2 min, linear change from 50%A to 80%A; 2.2–3.5 min, isocratic elution 80%A; 3.5–4.0 min, linear change from 80%A to 30%A; 4.0–5.0 min, isocratic elution 30%A. The flow rate was set at 0.3 m L/min and the injection volume was 2 μL. Mass spectrometry: The source temp was set at 150℃. The capillary voltage was set at 2.0 k V. The source offset voltage was kept at 50 V. The desolvation temp was set at 500 ℃. The desolvation flow was 800 L/Hr. The cone flow was 150 L/Hr. The nebuliser pressure was 7.0 bar. For structural identification of each analyte, the information-dependent acquisition(IDA) method was used to trigger the enhanced product ion(EPI) scans by analyzing MRM signals. Xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole, isoimperatorin, deoxyschizandrin and schisandrin were determined under positive ESI conditions. While, hyperoside, quercitrin, luteolin, kaempferol, isorhamnetin and ursolic acid under negative ESI conditions.Results: The complete separation was obtained within 5 min for the 14 compounds. The regression equations showed linear relationships between the peak area and content of each compound(r2≥0.9989). The average recoveries of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole, isoimperatorin, deoxyschizandrin, schisandrin, hyperoside, quercitrin, luteolin, kaempferol, isorhamnetin and ursolic acid were 99.56%, 99.47%, 99.69%, 101.2%, 99.89%, 100.2%, 99.86%, 101.2%, 98.69%, 101.1%, 98.65%, 99.75%, 101.9% and 97.89%(n=3), and the RSDs were 1.1%, 0.9%, 1.3%, 1.1%, 1.5%, 1.4%, 1.4%, 0.8%, 1.7%, 1.7%, 1.9%, 1.5%, 1.6% and 2.1%, respectively.Conclusion: The method is simple, accurate and highly reproducible, and can be used for the determination of xanthotoxin, isopimpinelline, bergapten, imperatorin, osthole, isoimperatorin, deoxyschizandrin, schisandrin, hyperoside, quercitrin, luteolin, kaempferol, isorhamnetin and ursolic acid in Bazibushen capsule. Part four Simultaneous quantification of 7 coumarins in Common Cnidium Fruit by GC-MS-SIM Objective: To develop a novel quantitative GC-MS-SIM method for simultaneous determination of the 7 coumarins in Common cnidium fruit samples from different regions. In addition, 12 batches of Common cnidium fruit from different sources were compared using the developed method.Methods: The 7 bioactive constituents were separated on DB-1 capillary column( 30 m ×0. 25 mm×0. 25 μm) using temperature programming. The temperature programming was as follows: keep 50℃ for 2 min; Rise to 250℃, at the rate of 20℃/min, stayed for 10 min; Rise to 280℃, at the rate of 10℃/min, stayed for 10 min. The injection port temperature was set at 250℃; Split ratio: 40:1; The carrier gas(He) flow rate was set at 1.54 m L/min; The interface temperature was set at 280 ℃; Ion source temperature: 250℃; Solvent cutting time: 4 min. Quadrupole temperature 150℃; EI mode: 70 e V; The mass spectrometer detector was in SIM mode; Scan range: 50 ~ 350 amu.Results: All the 7 marker substances showed good linearity(r2>0.9986) in the test ranges. The LODs and LOQs for the compounds ranged from 1.06 to 10.11 ng/m L and from 3.21 to 29.88 ng/m L, respectively. The overall intra-day(n=6) and inter-day(n=3) RSDs were 0.7–2.5% and 1.2–3.3%, respectively, and the overall stability and repeatability variations were 0.5–2.5% and 0.8–3.0%. The overall recoveries were between 92.38 and 100.53% for all compounds.Conclusion: A efficient, rapid and sensitive GC-MS-SIM method was firstly established for the qualitation and quantification of 7 major components in Common cnidium fruit. Validation of the assay showed appropriate sensitivity and specificity and was successfully utilized to analyze 12 batches of Common cnidium fruit samples from different sources. The satisfactory results demonstrated that the GC-MS-SIM method was a good option for routine analysis and could be applied as a reliable quality control method for Common cnidium fruit. The results demonstrate that the amounts of the 7 marker compounds within the plant material are very variable and this may be due to a number of causes, including place of origin, plant source, growth conditions, cultivation methods, harvesting time, processing and storage conditions. Part five Identification of bergapten metabolites in rats by UPLC-Q-TOF-MS/MSObjective: To identify the metabolites of bergapten in rat urine and bile through using ultra high performance liquid chromatography combined with triple TOF mass spectrometry(UPLC-Q-TOF-MS/MS) after oral administration of bergapten. To summarize the metabolic pathway of bergapten.Methods: Rats were given a gavage with bergapten.The urine and bile samples were prepared by use of Waters Ostro. Chromatographic conditions: Reverse-phase waters HSS T3 C18(100mm×2.1mm,1.8mm) with the column temperature set at 40℃. A linear gradient elution of eluents A(0.1% formic acid in water; v/v) and B(0.1% formic acid in acetonitrile; v/v) was used for the separation. The flow rate was set at 0.5 m L/min and the injection volume was 2 μL. Mass spectrometry: The source temp was set at 120℃. The capillary voltage was set at 2.5 k V. The source offset voltage was kept at 50 V. The desolvation temp was set at 500 ℃. The desolvation flow was 800 L/Hr. The cone flow was 50 L/Hr. Bergapten and its metabolites were determined under positive ESI conditions.Results: There was no significant difference between the metabolites of bergapten in urine and bile. But the content is different. Seventeen metabolites(17 in urine, 16 in bile) were identified from the urine and bile samples of rats, as well as the prototype of bergapten. As for as the metabolic type was concerned, six metabolites belonged to phase I metabolism and eleven metabolites belonged to phase Ⅱmetabolism. The results indicated that double-bond oxidation, demethylation and hydrolysis might be the main phase I metabolic pathways of bergapten, while, glucuronidation and sulfated modification might be the main phase Ⅱ metabolic pathways of bergapten.Conclusion: We firstly developed a UPLC-Q-TOF-MS/MS method for identify the metabolites of bergapten in rat urine and bile after oral administration of bergapten. The metabolic pathway of bergapten were summarized. The method is simple, accurate and highly reproducible, and can be used for the screening and identifying trace constituents in vivo. |
PDF Full Text Request