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Ultra-performance LC-ESI/quadrupole-TOF MS for rapid analysis of chemical
constituents of Longdan-Xiegan decoction
*
YUJIA SUN,SHANSHAN WANG,HUI SUN, ZHIGANG WANG and XIJUN WANG
Department of Pharmaceutical Analysis, Heilongjiang University of Chinese Medicine, National TCM Key Lab of Serum Pharmacochemistry, Heping road 24, Harbin, China;
Abstracts: The traditional Chinese medicine,Londan-Xiegan decoction(LXD),is a classic formulae containing ten medicinal herbs,Gentianae Radix,ScutellariaeRadix,Gardeniae Fructus,AlismatisRhizoma,Akebia Trifoliata Varaustralis,AngelicaeSinensisRadix,Rehmanniae Radix,Bupleuri Radix,GlycyrrhizaeRadix et Rhizoma,PlantaginisSemen.It is commonly used to treat jaundice,conjunctival congestion,earache,scrotum and extremitas inferior eczema,etc.Recently,we conducted in depth discussion and research in the modern clinical,made more achievements,to achieve further development and application of LXD has an important guiding significance.In this study,a sensitive and specific rapid ultra-performance LC-ESI/quadrupole-TOF high-definition MS (UPLC-ESI-Q-TOF-MS) with automated MetaboLynx analysis in negative ion mode were established to characterize the chemical constituents of LXD.The analysis was performed on a Waters UPLCTM HSS T3 (2.1 x100 mm,1.8 |m) using gradient elution system.MS/MS fragmentation behavior was proposed for aiding the structural identification of the constituents.By comparing the retention time and mass spectrometry data and retrieving the reference literatures,a total of 66 peaks were provisionally characterized with the optimization of conditions.Of note,5 ingredients were identified from Gentianae Radix,24 were from ScutellariaeRadix,8 were from Gardeniae Fructus,2 were from AlismatisRhizoma,5 were from Akebia Trifoliata Varaustralis,3 were from AngelicaeSinensisRadix,4 were from Rehmanniae Radix,4 were from Bupleuri Radix,7 were from GlycyrrhizaeRadix et Rhizoma,4 were from PlantaginisSemen.To concluded,UPLC-ESI-Q-TOF-MS,as a reliable and efficient platform,was successfully developed for globally identifying multiple-gradient of Chinese medicine prescriptions.
Keywords: Constituents;Traditional Chinese medicine;Identification;Longdan-Xiegan decoction;UPLC-ESI-Q-TOF-MS
1. Introduction
Traditional Chinese medicine(TCM) is the crystallization of the wisdom of the Chinese nation for thouthands of years,it not only has made an indelible contribution to the reproduction of both Chinese nation and people's health,but also greatly promoted a lot of neighboring Asian countries' medical career development[1].Due to their long historical clinical use and reliable therapeutic efficacy,the international influence of TCM is increasing day by day.It is indispensable to develop a rapid and effective analytical method to identify the constituents of TCM,then ensure the reliability in clinical application and increase quality control[2-8].
Longdan-Xiegan decoction (LXD,a traditional Chinese formulae),a precious TCM,is a classic recipe in TCM containing Gentianae Radix,ScutellariaeRadix,Gardeniae Fructus,AlismatisRhizoma,Akebia trifoliata varaustralis,AngelicaeSinensisRadix,Rehmanniae Radix,Bupleuri Radix,GlycyrrhizaeRadix et Rhizoma,PlantaginisSemen which are formulated in specified ratio[9-10].In recent years,chemical analytical methods have been established for constituent analysis of LXD and the reported methods included HPLC.Despite this method made significant contributions to the studies of LXD,it cann't be detected the global gradients.The present study is aimed at developing an analysis method to identify the systemic phytochemical constituents of the LXD,conducted via ultra-performance LC-ESI/quadrupole-TOF-MS (UPLC-ESI-Q-TOF-MS)[11].Our screening results proved that the developed method was high throughput and efficient.
2. Materials and methods
2.1 Chemicals and materials.
HPLC grade acetonitrile was purchased from Merck (Darm-stadt,Germany).Acetonitrile and methanol was of chromatographic grade (Fisher,USA).Distilled water used in all experients was purchased from Watson's Food&Beverage Co. (Guangzhou,China).Leucine enkephalin from Sigma-Aldrich (MO,USA) was used.Formic acid was purchased from Tianjin Kermel (Tianjin, China).Gentianae Radix,ScutellariaeRadix, Gardeniae Fructus, AlismatisRhizoma, Akebiatrifoliata varaustralis,AngelicaeSinensisRadix,Rehmanniae Radix,Bupleuri Radix, GlycyrrhizaeRadix et Rhizoma,PlantaginisSemen were purchased from Harbin Tongrentang Drug Store (Harbin,China), which were authenticated by Prof. Xijun Wang,Department of Pharmacognosy of Heilongjiang University of Chinese Medicine.
2.2 Preparation of LXD samples for LC/MS analysis.
Ten crude herbs of the mixture of Gentianae Radix (6g),ScutellariaeRadix (9g),Gardeniae Fructus (9g),AlismatisRhizoma (12g),Akebia Trifoliata Varaustralis (6g),AngelicaeSinensisRadix (3g),Rehmanniae Radix (9g),Bupleuri Radix (6g),GlycyrrhizaeRadix et Rhizoma(6g),PlantaginisSemen(9g) were immersed in 450 mL of ditilled water for 1 h and then decocted to boil for 1 h.The supernatant of the extractive solution was filtered through six layer gauzes and cooled,finally the decoction was transformed into the freeze-dried power.Freeze-dried power of the single herb extracts was prepared as the same of LXD. Freeze-dried power of LXD was accurately weighed 0.1g,soluted in 50 mL of volumetric flask with 75% methanol and ultrasonically extract for 30 min,after cooling to room temperature to constant volume.The solution was centrifuged at 13 000 rpm for 15 min at 4°C and the upper pellucid liquid was taken out,which was filtered through a 0.22-|m filter,the filtrate was subjected to UPLC analysis.
2.3 Chromatographic separation.
Chromatographic was performed on a AcquityTM Ultra Performance LC systems (Waters,Milford,USA) controlled with Masslynx(V4.1).ACQUITY UPLC HSS T3 Column (2.1 x 100 mm,1.8 |m, Waters) was used for Seperation,and the column temperature held at 45°C,and the flow rate was 0.5 mL/min.The optimal mobile phase consisted of A (HCOOH/CH3CN, 0.1:100, v/v) and B (HCOOH/H2O, 0.1:100, v/v).The optimized UPLC elution conditions were as follows:0.0-1.0 min,1-15% B;1.0-4.0 min,15-20% B;4.0-7.0 min,20-25% B;7.0-11.0 min,25-30% B;11.0-14.0 min,30-55% B;14.0-16.0 min,55-70% B;16.0-17.0 min,70-99% B;17.0-18.0 min,99% B;18.0-19.0 min,99-1% B;19.0-20.0 min,1% B.The injection volume was 5 |L.
2.4 Mass spectrometry.
A Waters SynaptTM high definition TOF mass (HD MS) system(Waters) equipped with an electrospray ion source operating in positive and negative ESI mode was equipped with the UPLC.The optimal conditions were as follows:the nebulizer gas flow rate was set to 800 L/h at a temperature of 300°C in positive ion mode, the cone gas was 40 L/h, the source temperature was 110°C, the capillary and cone voltages were 3000 and 40 V, respectively, the capillary and cone voltages were set at 2400 and 40 V in negative ion mode,respectively and the Q-TOF acquisition rate was 0.1s, the full-scan MS data were produced across the mass range of 100-1500 Da and the collision energy was of 10-55 eV, scans were of 0.30 s duration.In order to achieve an accurate
mass, leucine-enkephalin via a lockspray interface at a concentration of 0.2 ng/mL corrected mass during acquisition,andgenerate a reference ion at 556.2771 Da ([M+H]+) for positive ESI mode and,while at m/z 554.2615 Da ([M-H]-) in negative ion mode to ensure accuracy during the MS analysis. The accurate mass and composition for the precursor ions and for the fragment ions were calculated using the Masslynx 4.1 software (Waters) that was incorporated with the instrument.
2.5 Data processing.
UPLC-ESI-Q-TOF-MS data of all determined samples were further processed by Markerlynx V4.1 software (Waters) for peak detection and peak alignment.The method parameters for data processing were set as follows:retention time range 0.1-20.0 min,mass range 100-1000 Da,retention time tolerance 0.2 min,mass tolerance 0.05 Da,noise elimination level 6;peak intensity threshold 50.The peak width at 5% height and the peak-to-peak baseline noise were automatically determined.For further confirmed the structure and the source of the metabolites,the raw data were processed by a Metabolynx XS version 4.1 (Waters).
3. Results and discussion
3.1Chromatographic conditions and Q-TOF-MS/MS method development.
In order to shorten analytical time and improve sensitivity and efficiency of analysis,the following Chromatographic conditions were optimized,including the column,column temprature,flow rate and mobile phase.The Waters UPLCTM HSS T3 was finnaly selected for the analysis as it gave a stronger retention peak and better resolution.So the optimized conditions were AcQuITY UPLC HSS T3 Column,column temprature at 45°C,flow rate at 0.5 mL min-1 and acetonitrile-formic acid aqueous solution.Under the optimized constituent elution,over 60 peaks were detected within 18 min. Fig.1 shows clearly the representative base peak intensity chromatograms of LXD in positive mode and negative.In this paper,a 18min UPLC-Q-TOF-MS tryptic profiling significantly shortens the time of sample analysis without compromising chromatographic peak resolution.This method could reduce the severe ion suppression,although the complexity of LXD makes seperation difficult.
3.2UPLC-MS characterization of chemical constituents from LXD
All information of MS data obtained from the robust UPLC-ESI-Q-TOF-MS analysis that was performed using the aforementioned protocol indicated the retention time and precisemolecular mass and provided the MS/MS data that wasnecessary for the structural identification. Using MarkerLynx,their element compositions were also calculated on basisof the mass accuracy (ppm) and i-FIT(Norm). With the mass accuracy less than 5.0 ppm and the i-FIT value<1.0,it can be relatively easy for the elemental composition to be examined.The precise molecular mass was determined withina reasonable degree of measurement error (<5 ppm) usingQ-TOF, and the potential element composition, degree ofunsaturation were also obtained. Global profiling in bothpositive and negative ion modes were analyzed by UPLC-ESI-Q-TOF-MS.Using the optimal UPLC-MS conditions describedabove, the total ion current chromatograms at bothESI modes are shown in Fig. 1. Compounds inthe UPLC chromatogram were rapidly identified with theMasslynx MassFragment. By scrutinizing their elemental compositions, a total of 66 peaks were obtained,and all of which were identified or tentatively characterizedby matching the in-house formula database within an errorof 5 ppm, and their information is shown in Table 1. In addition,5 ingredients were identified from Gentianae Radix,24 were from ScutellariaeRadix,8 were from Gardeniae Fructus,2 were from AlismatisRhizoma,5 were from Akebia Trifoliata Varaustralis,3 were from AngelicaeSinensisRadix,4 were from Rehmanniae Radix,4 were from Bupleuri Radix,7 were from GlycyrrhizaeRadix et Rhizoma,4 were from PlantaginisSemen.
4. Conclusions
As an advanced quatitative tool and an efficiency and sensitive method, UPLC-ESI-Q-TOF-MS was successfully established for quality assessments and the separation of LXD.Based on our present analysis result, 66 gradients in the complex herbal mixture,LXD were simultaneously examined and identified by UPLC-ESI-Q-TOF-MS profiling method.Furthermore,with the development of the UPLC-ESI-Q-TOF-MS into the TCM,it will become a powerful tool for the
analysis of pharmaceuticals and herbal products as the hyphenated technique.We expected that the method is not only used for the TCMs,but also our food,teas and other substance for human needs in future. References
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Fig.1. UPLC-MS base peak intensity chromatograms of Longdan-Xiegan Decoction in negative
mode (A) and positive mode
(B).
Table 1. Characterization of chemical constituents of Longdan-Xiegan DecoctionbyUPLC-ESI-Q-TOF-
MS.a:GentianaeRadixb:GardeniaeFructusc:BupleuriRadixd:AlismatisRhizomae:ScutellariaeRadixf: PlantaginisSemeng:AngelicaeSinensisRadixh:GlycyrrhizaeRadixetRhizomai:AkebiaTrifoliataVarau stralisj :RehmanniaeRadix
N Rt Identification Negative ion (m/z) Positive ion (m/z) Element composition mv(Da) Sour
Indicated ppm Indicated ppm
1 0.5 4-Carboxyphenyl 4-hexyloxybenzoate 341.1218 -3.0 343.1278 -1.4 C20H22O5 342.1389 g
2 0.8 Scupontin E 665.3036 -4.5 - - C34H50O13 666.3173 e
3 0.8 Gentianose 503.1648 -4.1 505.1730 3.6 C18H32O16 504.1612 a
4 1.4 Shanzhiside 391.1147 -1.2 393.1318 1.9 C16H24O11 392.1240 b
5 1.6 Loganic acid 375.1171 -4.6 - - C16H24O10 376.1291 a
6 1.8 Hederagenin 471.7026 -3.7 495.7184 [M+Na]+ 2.1 C30H48O4 472.7103 i
7 2.1 Wogonoside 459.3978 -2.9 461.4002 -1.4 C22H20O11 460.4015 e
8 2.2 3,4,5- Trimethoxyphenylacetic acid 225.0682 -1.1 227.0641 -3.6 C11H14O5 226.0763 b
9 2.5 4'-O--D-Glucopyranoside 549.1590 -2.8 551.1568 -4.2 C26H30O13 550.1608 h
10 3.0 Not identified 565.1412 2.5 567.1302 -4.3 C22H30O17 566.1405 i
11 3.2 Aucubin 345.3321 -1.4 - - C15H22O9 346.3453 f
12 3.3 Plantagoside 465.0987 -0.6 467.1183 3.3 C21H22O12 466.1033 f
13 3.6 8-Arabinopyranosyl -6 glucopyranosyl-5,7 -dihydroxyflavone 547.1391 -1.6 549.1560 3.7 C26H28O13 548.1452 e
14 3.8 Not identified 547.1791 3.8 549.1715 2.1 C30H27O10 548.1604 b
15 3.9 Isoacteoside 623.1861 -3.0 625.1938 -0.7 C29H36O15 624.1976 j
16 4.0 Calceolarioside B 477.1263 -1.5 479.1265 -2.5 C23H26O11 478.1397 i
17 4.1 Galericulin 547.2579 -1.1 549.2547 -3.2 C29H40O10 548.2621 e
18 4.3 Scutellarioside II 507.1505 -0.4 531.1521 [M+Na]+ -1.3 C24H28O12 508.1580 e
19 4.4 Globularin 491.1600 -1.1 493.1738 2.0 C24H28O11 492.1631 e
20 4.5 Acteoside 623.1961 -1.0 625.1870 -3.7 C29H36O15 624.1976 j
21 4.6 Not identified 303.0521 -3.1 305.0642 -1.1 C19H12O4 304.0657 g
22 4.9 Liquiritin 415.1184 -0.2 417.1130 2.3 C21H22O9 416.1029 h
23 5.1 2',3',5,7- Tetrahydroxyflavone 285.0274 -2.7 287.0271 -3.1 C15H10O6 286.0399 e
24 5.5 6''-O-p-cis- Coumaroylgenipin gentiobioside 695.2301 2.4 C32H40O17 696.2261 b
25 5.7 5-O-(3-Hydroxy-3 -methylglutaroyl) 659.1548 -2.0 683.1597 [M+Na]+ -2.8 C31H32O16 660.1612 b
26 5.8 Gardenin 417.4101 4.1 419.4132 3.4 C21H22O9 418.4054 b
27 6.1 Viscidulin III 345.0566 -1.1 347.0782 1.9 C17H14O8 346.0610 e
28 6.3 7-O--D- Glucuronopyranoside 445.0667 -3.9 447.0628 -2.2 C21H18O11 446.0771 e
29 6.4 Dihydrobaicalein 271.0707 2.0 273.0705 2.5 C15H12O5 272.0606 e
30 6.6 3-O-Caffeoyl-4-O-sinapoylquinic acid 559.1480 1.8 - - C27H28O13 560.1452 b
31 6.7 Orientalol F 237.1908 -0.8 261.1877 [M+Na]+ -3.9 C15H26O2 238.1932 d
32 6.9 Dihydrobaicalein 271.0589 -1.3 - - C15H12O5 272.0606 E
33 7.1 Decussatin 301.0893 2.8 303.0645 -1.7 C16H14O6 302.0790 a
34 7.3 Scutellaprostin F 479.1051 -0.7 481.1130 1.6 C25H20O10 480.1056 e
35 7.5 Oroxylin A 283.0593 -3.7 285.0551 -1.2 C16H12O5 284.0606 e
36 7.7 2',5,7-Trihydroxy-6'-methoxyflavone 299.0457 -3.5 301.0681 1.3 C16H12O6 300.0556 e
37 7.9 Not identified 429.0605 -0.9 431.0764 -2.6 C14H22O15 430.0880 j
38 8.1 Oroxyloside 459.1062 1.3 461.0876 -3.7 C22H20O11 460.0927 e
39 8.3 Lateriflorin 475.0604 -3.0 477.0739 -4.3 C22H20O12 476.0877 e
40 8.4 Scutellaprostin A 447.1069 -1.8 449.1207 3.8 C25H20O8 448.1158 e
41 8.8 6-Glucosyl-5,7-dihydroxyflavone 415.1262 2.3 - - C21H20O9 416.1107 e
42 9.3 5,7-Dihydroxy-2',8-dimethoxyflavone 313.0665 -4.7 - - C17H14O6 314.0712 e
43 10. Bisabolangelone 247.1528 3.9 271.1398 [M+Na]+ -1.9 C15H20O3 248.1412 g
44 10. 2',5,7-Trihydroxyflavone 269.0305 -4.1 271.0356 -2.3 C15H10O5 270.0450 e
45 10. Not identified 659.5760 -4.4 - - C31H32O16 660.5986 i
46 10. Gardendiol 197.8481 1.3 199.8562 3.3 C10H14O4 198.8476 b
47 11. 5,6,7-Trihydroxyflavone 269.0326 -0.7 271.0328 -2.9 C15H10O5 270.0450 e
48 12. Trifloroside 781.2057 -3.8 783.2035 -4.0 C35H42O20 782.2191 a
49 12. Narcissin 623.5541 -3.2 625.5591 -1.6 C28H32O16 624.5638 h
50 12. 9,12,13-Trihydroxy-10-octadecenoic acid 329.2249 -1.6 331.2349 1.3 C18H34O5 330.2328 c
51 12. Norarjunolic acid 471.3051 -4.3 473.3279 3.1 C29H44O5 472.3188 i
52 13. Glycyrrhizic acid 821.3901 -2.9 823.4019 -0.9 C42H62O16 822.4037 h
53 13. Oroxylin A 283.0690 2.3 285.0728 2.7 C16H12O5 284.0606 e
54 136 Verbascose 827.7231 -2.2 - - C30H52O26 828.7301 j
55 13. 2',5-Dihydroxy-3',6,7,8-tetramethoxyflavone 373.0889 -2.3 C19H18O8 374.0923 e
56 13. 2''-O-Feruloylisoorientin 623.1420 -3.1 625.1306 -3.2 C31H28O14 624.1479 a
57 14. Gancaonin W 367.1174 -0.9 391.1093 [M+Na]+ -1.6 C21H20O6 368.1182 h
58 14. Plantaginin 447.0933 -1.4 449.1131 2.0 C21H20O11 448.1005 f
59 14. Gancaonin C 353.1192 0.6 355.1001 -1.1 C20H18O6 354.1025 h
60 15. Calceolarioside A 477.1309 -2.9 479.1326 -4.1 C23H26O11 478.1475 f
61 15. Not identified 265.1105 -3.0 - - C11H22O7 266.1287 e
62 15. Gancaonin A 351.0860 -1.8 353.0761 -3.1 C20H16O6 352.0869 h
63 15. Hexudecanoic acid 255.4301 -0.7 - - C16H32O2 256.4381 c
64 15 Orientalol E 253.3607 -3.6 255.3803 3.9 C15H26O3 254.3707 d
65 16. Not identified 311.1619 -4.8 - - C13H28O8 312.1706 c
16. Longispinogenin 457.7271 -3.7 459.7451 2.6 C30H50O3 458.7324 c
