Study On Quality Control Method Of Peucedani Radix And Differentiation Of Furanocoumarin Isomers By Mass Spectrometry

Peucedani Radix is the dried roots of Peucedanum praeruptorum Dunn. It has been recorded in Chinese Pharmacopoeia and can be used to clear phlegm with pant, treat cough with thick sputum. Coumarins, the main chemical constituents of Peucedani Radix, including the angular-type pyranocoumarins, furanocoumarins and others, are an important indicator of the quality evaluation of Peucedani Radix. The study of Peucedani Radix concentrated on qualitative analysis of chemical compositions, or quantitative analysis of a few coumarins. There is still a lack of a comprehensive analysis method for identification and quantification of coumarins from Peucedani Radix. Two aspects in view of the existing problems in the quality evaluation of Peucedani Radix were researched. 1 Ultra-high performance liquid chromatography–quadrupole time of flight tandem mass spectrometry(UHPLC-Q-TOF-MS/MS) was used to establish a method to identify and quantify coumarins in Peucedani Radix simultaneously. The method was used to compare the different samples of Peucedani Radix and adulterants. 2 The quality standard of Peucedani Radix was reformulated based on the requirements recorded in European Pharmacopoeia, including characters, microscopic examination, thin layer chromatography, loss on drying, total ash, ash insoluble in hydrochloric acid and the determination of praeruptorin A and praeruptorin B in Peucedani Radix by HPLC.The basic framework of furanocoumarins is consisted of benzo-a pyranone and furan ring, divided into two types of linear and angular. Furanocoumarins are widely distributed in higher plants, and often co-exist in C-5 substituted and C-8 substituted isomeric forms. Because of the same molecular weight, structure and physicochemical properties of the isomers, it is often difficult to distinguish the isomers by chromatographic behavior and simple fragmentation pathways. In this study, a method based on the ratio of the relative abundance of characteristic fragment ions(RRA) to distinguish C-5 substituted and C-8 substituted furanocoumarin isomers was established by analyzing 4 pairs of furanocoumarin isomers by UHPLC-Q-TOF-MS/MS. The method was applied to recognize and identify the isomeric furanocoumarins in Angelicae dahuricae Radix, and also provided a new way for the research of isomers in complex system of TCM. Part one Simultaneous identification and quantification analysis of coumarins in Peucedani Radix and its application in the analysis of Peucedani Radix and adulterantsObjective: To establish a method for the simultaneous identification and quantification analysis of coumarins in Peucedani Radix by UHPLC-Q-TOF-MS/MS, and to evaluate different samples and adulterants.Methods:1 Detection conditions: UHPLC-Q-TOF-MS/MS was used for the detection. The chromatographic separation was performed on a Thermo Hypersil GOLD column(100 mm×2.1 mm, 3 ?m). The mobile phase was consisted of ammonium acetate aqueous solution(A) and acetonitrile(B) with a linear gradient elution. The flow rate was 0.5 mL/min and the injection volume was 5 ?L. The high-resolution mass spectrometer was operated in the positive ion Electrospray mode. A full scan was employed in MS1. Dynamic background subtract(DBS) was used to trigger the IDA function in the experiment. For the IDA criteria, the 12 most intense candidate ions of per cycle that exceed 100 cps counts were selected to a production scan.2 Sample preparation: Methanol was selected as the extraction solvent, and the ultrasonic treatment was used to prepare the sample.3 Compound identification and quantitative analysis: The known chemical formula was input into the data processing software MasterView, the chromatographic peak error in the range of ±5 ppm was selected. The acquired exact molecular weight and mass spectra were used to identify compounds coupled with cleavage law of the coumarins and the information of fragments of the compounds had been reported in the literature. In the same period of the identification, the 9 standards(including praeruptorin A, praeruptorin B, praeruptorin E, imperatorin, isoimperatorin, xanthotoxin, bergapten, psoralen and isoimpinellin) were used for absolute quantification, and all the peaks with exclusive separation were used for relative determination analysis.4 Sample analysis: The peak area of compounds in control sample was set as the standard, and the peak area of each compound in other samples was compared with that in control sample. Thus the peak area ratios were occurred. 23 batches of samples were analyzed based on the sum of peak area ratios and principal component analysis. On the basis of the identified compounds 13 batches of adulterants were analyzed.Results: 54 coumarins were identified by UHPLC-Q-TOF-MS/MS including 36 angular-type pyranocoumarins, 13 furanocoumarins and 5 other coumarins. The linear relationships, instrument repeatability, limit of detection, limit of quantification, precision, accuracy and stability were in line with the requirements of the 9 standards. The range of total content of 9 standards in all batches of samples was 694.57~26532.10 ?g/g, and the RSD value is 51%. 8 angular pyranocoumarins and 2 furanocoumarins had larger peak area in 29 compounds. PCA analysis showed that the contents of qianhucoumarin E, praeruptorin A and 3'-(isovaleryloxy/2-methybutyroyl)-4'-(isovaleryloxy/2-methybutyroyl) khellactone were the key factors to evaluate the quality of Peucedani Radix. The analysis of the 13 batches of adulterants showed that the same material basis existed in Peucedanum praeruptorum Dunn, Peucedanum Decursivum(Miq.) Maxim, Peucedanum harry-smithii var. subglabrum, Ligusticum brachylobum Franch. and Peucedanum terebinthaceum(Fisch.) Fisch.ex Turcz.. Peucedanum praeruptorum Dunn can be distinguished with Peucedanum Decursivum(Miq.) Maxim, Peucedanum harry-smithii var. subglabrum, Ligusticum brachylobum Franch. and Peucedanum terebinthaceum(Fisch.) Fisch.ex Turcz.. by 15, 13, 14, and 31 coumarins, respectively. However, Osterium citriodorum did not have the same material basis with Peucedanum praeruptorum Dunn. There were eight compounds(six angular-type pyranocoumarins, one furanocoumarins and one other) among the identified 54 compounds did not existed in the 13 batches of adulterants.Conclusion: This study established a UHPLC-Q-TOF-MS/MS method for the simultaneous identification and quantification analysis of coumarins in Peucedani Radix in one period for the first time. The method is simple, sensitive and specific. Angular pyranocoumarins are the key to evaluate the quality of Peucedani Radix and can be used to distinguish the adulterants. Part two Study on quality standard of Peucedani RadixObjective: To establish a method for the quality evaluation of Peucedani Radix, to provide references for formulating the quality standard in European Pharmacopoeia.Methods: 9 batches of samples were selected as the analysis object to explore the evaluation methods of Peucedani Radix.1 Identification: Identified by characters, microscopic identification and TLC identification methods. The external characters of Peucedani Radix were observed. The characteristics of the powder of Peucedani Radix were observed under microscope. Methanol was selected as the extraction solvent and ultrasonic treatment was used to prepare the samples in the TLC identification. Silica gel G thin-layer plate was used in the TLC with cyclohexane-ethyl acetate(2:1) as developing agent. The plate was detected under UV 365 nm.2 Test: Loss on drying, total ash and ash insoluble in hydrochloric acid were tested according to the requirements recorded in European Pharmacopoeia.3 Content determination: The content was determined by HPLC for the determination of praeruptorin A and praeruptorin B. Methanol was used as the extraction solvent and ultrasonic extraction time was 30 min. The extract was analyzed directly. The separation was performed on a Thermo ODS HYPERSIL column(4.6 mm×250 mm, 5 ?m) using methanol and water(68:32) as mobile phase. The flow rate was 0.8 mL/min, the injection was 10 ?L, and the detection UV was 322 nm.Results: 1 Identification: The external characteristics of the Peucedani Radix were outstanding. Longitudinal grooves, longitudinal striations and horizontal lenticels like protrusions were obvious. Characteristic cork cells, vessels and lignified fibres were outstanding in the powder of Peucedani Radix under microscope. TLC method is much more stable with 7 clear fluorescent spots. 2 Test: The water content of the 9 batches of samples was less than 8.9%, the total ash content was less than 7.8%, acid insoluble ash was less than 3.4%. 3 Content determination: The methodology validation presented that praeruptorin A and praeruptorin B were in good correlation in the range of 5.00-400.00 ?g/mL and 0.50-40.00 ?g/mL with the regression equations of Y=28.4782224X+6.484094( r=0.99999) and Y=25.5893187X-0.3288223(r=0.99999), respectively. The results of the precision(RSD) and recoveries were in the range of 0.6%-2.0% and 90.7%-104.8%, respectively. The content range of praeruptorin A was 0.73%-2.24%, and the content range of praeruptorin B was 0.12%-0.33%.Conclusion: The method is more stable and convenient for the quality control of Peucedani Radix. Part three Differentiation furanocoumarin isomers by UHPLC-Q-TOF-MS/MS and its application in Angelicae dahuricae RadixObjective: To establish a fast and stable method to distinguish C-5 substituted and C-8 substituted furanocoumarin isomers by UHPLC-Q-TOF-MS/MS, and be applied in the identification of furanocoumarin isomers in Angelicae dahuricae Radix.Methods: 4 pairs of furanocoumarin isomers, xanthotoxin and bergapten, imperatorin and isoimperatorin, psoralen and isopsoralen, impinellin and isoimpinellin were acquired by the MS-IDA-12MS/MS scan mode and product ion scan mode. The fragmentation rules and information of the fragments were summarized and analyzed by the software MasterView, and the isomeric furanocoumarins in Angelicae dahuricae Radix were differentiated by the established method. The chromatographic separation was performed on an Epic C18 column(15 cm×2.1 mm, 3 ?m). The mobile phase was consisted of ammonium acetate aqueous solution(A) and methanol(B) with a linear gradient elution.Results: C-5 substituted and C-8 substituted furanocoumarin isomers were distinguished by the method of the ratio of the relative abundance of characteristic fragment ions(RRA). The main fragmentation pathway for furanocoumarins was successively loss of CO(substituents would be lost firstly). According to value of ratio of the relative abundance of characteristic fragment ions the position of the substituent can be distinguished. The method with good repeatability was applied to identify 5 pairs of isomeric furanocoumarins in Angelicae dahuricae Radix.Conclusion: A stable and rapid mass spectrometric method to distinguish furanocoumarin isomers was established based on the fragmentation pattern of furanocoumarins. The method was applied to distinguish this class of isomers in a complex system of TCM successfully.