CN101153866A - Novel analysis method for complex composition of traditional Chinese medicine injection based on LC/MS-IT-TOF - Google Patents
Novel analysis method for complex composition of traditional Chinese medicine injection based on LC/MS-IT-TOF Download PDFInfo
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Abstract
The invention belonging to the pharmaceutical field relates to component analysis and quality control of traditional Chinese medicine injection and provides a novel component analysis method of complicated composition in traditional Chinese medicine injection based on LC/MS-IT-TOF. The novel method includes the following steps: (1) total components of traditional Chinese medicine injection are extracted through adopting a certain method; (2) through adopting LC/MS-IT-TOF and under a certain chromatogram massspectrum condition, total ion current chromatogram (TIC) is obtained through positive and negative ion full scan; (3) main ion peaks (i.e. essential ingredients) with appropriate intensity are searched and confirmed from the TIC; (4) multilevel massspectrum data of each essential ingredient is obtained through collision induced dissociation; (5) on the basis of accurate molecular weight, chemical formulas of molecular ion and fragment ion are predicted within a certain range of error; (6) all possible structures (i.e. to-be-selected structure) of each predicted chemical formula are obtained through searching a database; (7) the structure (i.e. confirmation structure) which can generate all corresponding fragment ion is selected from the to-be-selected structure.
Description
Technical field
The invention belongs to pharmaceutical field, relate to the constituent analysis and the quality control of traditional Chinese medicine, particularly relate to the analysis method for complex composition of MAILUONING ZHUSHEYE.
Background technology
Traditional Chinese medicine is the breakthrough innovation of contemporary form of Chinese drug, have the bioavilability height, act on characteristics such as rapid, can bring into play the effect of treatment by Chinese herbs acute disease severe preferably, in clinical practice, demonstrate good even the chemicals result of treatment that is beyond one's reach.The research and development of traditional Chinese medicine has extremely important meaning for the development of China's traditional Chinese medicine.But because the composition of Chinese medicine is very complicated, and be subjected to the not deep enough influence of fundamental research for a long time, it not is very clear that the one-tenth of present most traditional Chinese medicines is grouped into, when setting up quality control standard, part kind only adopts the wherein total content of certain constituents of determined by ultraviolet spectrophotometry, and the analytical approach specificity is poor; And another part kind is also just carried out quantitative measurement to indivedual index compositions or effective constituent.Obviously, this quality control method does not meet the effect characteristics of many components of Chinese medicine multi-target effect, quality control standard can not be reacted security, the validity of traditional Chinese medicine conscientiously, cause traditional Chinese medicine having a lot of problems aspect quality, clinical efficacy and the safety in utilization, so that the part medical personnel suspect to using traditional Chinese medicine.
It is the outstanding problem of present traditional Chinese medicine that quality standard requires low.Because most of product is the kind of research and development at the seventies in last century to the beginning of the eighties in the traditional Chinese medicine of clinical use, is subjected to the restriction of historical conditions at that time, material base research is weak, and detection means falls behind, and quality standard requires low.Though through rectifying and improving, some kinds had been set up the higher quality standard really afterwards, but the quality standard of most of kind still required low.2000, former National Drug Administration issued " strengthening the traditional Chinese medicine quality management ", proposed traditional Chinese medicine and will set up the finger-print examination criteria.Issued " traditional Chinese medicine fingerprint investigative technique governing principle (trying) " in August, 2000.Traditional Chinese medicine fingerprint is a traditional Chinese medicine quality standard important component part, can reflect the kind and the quantity of contained chemical constitution in Chinese medicine and the preparation thereof comparatively all sidedly, and then drug quality is carried out integral body describe and estimate.But because traditional Chinese medicine complicated component, and much be principal component not, and traditional Chinese medicine fingerprint just carries out similarity relatively, seldom its chemical constitution being carried out structure identifies, " yet know that its does not know its reason ", set up the traditional Chinese medicine quality control standard with existing traditional Chinese medicine fingerprint technology, can not be reflected the security and the validity of traditional Chinese medicine conscientiously.The main chemical compositions formation of illustrating traditional Chinese medicine is the inexorable trend and the requirement of research and development from now on.
In recent years, all begin to pay attention to the structure evaluation and material base research of traditional Chinese medicine ingredients both at home and abroad, the analytical technology means of being used mainly comprise HPLC-UV and multiple liquid/gas matter coupling technique etc.But on the whole, present research method also comes with some shortcomings: (1) most researchs are a certain key component group (as the water-soluble phenolic constituents) or the principal ingredient of a certain medicinal material at a certain medicinal material, rather than are conceived to full side.(2) most researchs are just carried out the selectivity evaluation to the chemical constitution that standard reference material is arranged, rather than overall the evaluation, to lacking the chemical constitution of standard items, are difficult to draw the qualification result of relatively determining.(3) present most liquid/gas matter coupling technique is difficult to obtain simultaneously multistage cataclastic texture information and accurate mass measurement (being accurate to 0.0001Da).(4) still lack a kind of high sensitivity, high-resolution Chinese medicine analysis of complex ingredient and structure authentication method at present with general applicability.
High performance liquid chromatogram-ion-trap time-of-flight mass spectrometer (LC/MS-IT-TOF) is the high-end mass spectrometer of the up-to-date release of Tianjin, island company, be the present unique connection ion trap and the tandem mass spectrometry technology of flight time mass spectrum, possessed characteristics such as high resolving power, high-quality degree of accuracy, high speed detection simultaneously, abundant, accurate compound structure information can be provided, identify to have outstanding advantage for Chinese medicine analysis of complex ingredient and structure.
Given this, high resolving power, high-quality degree of accuracy, the high speed detection technology that the present invention uses LC/MS-IT-TOF set up a kind of method of express-analysis traditional Chinese medicine complicated ingredient, has great importance and general applicability for the quality control of traditional Chinese medicine.
Summary of the invention
The purpose of this invention is to provide a kind of new traditional Chinese medicine analysis of complex ingredient and structure authentication method, finish as follows based on LC/MS-IT-TOF high resolving power, high-quality degree of accuracy, high speed detection technology:
1, adopting certain method that traditional Chinese medicine is carried out full composition extracts:
Should extraction principal ingredient as much as possible, remove auxiliary material again as far as possible and disturb, select suitable extracting method according to the traditional Chinese medicine injection self-characteristic.Extracting method generally includes liquid-liquid extraction, Solid-Phase Extraction, filtration etc., wherein the liquid-liquid extraction solvent comprises normal butyl alcohol, ethyl acetate, ether, chloroform, benzene, normal hexane, cyclohexane etc., Solid-Phase Extraction can be selected the solid phase pillar of different fillers according to actual conditions, filters then and selects different model, the filter membrane in aperture, filter paper according to actual conditions.
2, use LC/MS-IT-TOF, under certain chromatogram mass spectrum condition, carry out the negative ions full scan and obtain total ions chromatogram:
The LC/MS-IT-TOF that uses among the present invention is the high-end mass spectrometer of the up-to-date release of Tianjin, island company, be the present unique connection ion trap and the tandem mass spectrometry technology of flight time mass spectrum, possessed advantages such as high resolving power, high-quality degree of accuracy, high speed detection simultaneously, thereby realize both can doing multi-stage ms, can reach the power of high-quality precision again, but the present invention is not limited to the LC/MS-IT-TOF mass spectrometer that Tianjin, island company produces.In the sample introduction process of a sample, can obtain the information of positive and negative ion pattern simultaneously, only need 0.1 second just can finish MS
1Automatic switchover between the positive and negative ion pattern and scanning and MS
2The switching each other and the scanning of positive and negative ion pattern.Select chromatogram (length, internal diameter, the filler that comprise chromatographic column according to the traditional Chinese medicine injection self-characteristic, column temperature, flow velocity, moving phase is formed, gradient etc.), mass spectrum condition (each setup parameter of instrument), full scan (SCAN) obtains its total ions chromatogram under the positive and negative ion pattern.
3, from total ions chromatogram, seek, determine to have the leading ion peak of suitable intensity, i.e. principal ingredient:
LC/MS-IT-TOF can utilize compressed ion injection mode (CCI technology) to trap whole chromatographic peaks through the LC wash-out as much as possible.According to actual conditions, determine from total ion current, to extract have suitable intensity and retention time ion as the main target composition, and carry out the multistage cracked of next step.
4,, obtain the multi-stage ms (MS of each principal ingredient by collision induced dissociation (CID)
n) data:
The molion of each principal ingredient that previous step obtains can be used as the cracked leading ion of secondary, and the high strength leading ion of secondary in cracked can also be as three grades of cracked leading ions, and is by that analogy, cracked step by step.In the present invention, three grades of cracked fragmention information just are enough to finish constituent analysis, although LC/MS-IT-TOF can finish ten grades cracked.By collision induced dissociation, can obtain the multi-stage ms information such as accurate molecular weight of fragmentation pathway, molion and the fragmention of principal ingredient ion, and every grade MS
nMass spectrogram can both be accomplished high resolving power, high-quality degree of accuracy and high sensitivity simultaneously.
5, based on accurate molecular weight, the chemical formula of predictive molecule ion and fragmention in certain error range:
LC/MS-IT-TOF is present unique mass spectrometer that can guarantee every grade of MS high-quality degree of accuracy, whole instrument has been equipped with internal temperature control function (comprising tof tube) completely, thereby guarantee the ion flight distance and the ion accelerating voltage of high stability, this just means that more multistage MS can obtain extraordinary quality stability in analyzing.Use the Formula Predictor software in the LC/MS solution workstation, can be based on accurate molecular weight (being accurate to 0.0001Da), the chemical formula of (0-10ppm) predictive molecule ion and fragmention in certain error range.
6,, obtain the possible structure of institute of each predicted chemical formula, structure promptly to be selected by database search:
Applying Internet (for example: Google (http://www.google.com), ChemExper (http://www.chemexper.com)) or CA data of optical disk storehouses such as (american chemical index), with each predicted chemical formula is that keyword is searched for, just can obtain the possible structure of its institute, structure promptly to be selected.
7, from structure to be selected, pick out the structure that can produce all corresponding fragment ions, promptly prove conclusively structure:
According to the multi-stage ms information that the 4th step obtained, in all structures to be selected, seek the structure that can produce all corresponding fragment ions one by one, the structure to be selected that can not produce is excluded, and satisfactory structure to be selected is the structure that will look for.
The present invention has the following advantages:
1, the Chinese medicine compound injection is carried out full constituent analysis, the gained result meets the mass action characteristics of Chinese medicine;
2, do not rely on standard items, use cracked information of multi-stage ms and accurate mass measurement and can carry out complicated ingredient (comprise known and not principal component) and analyze and the structure evaluation;
3, LC/MS-IT-TOF used in the present invention is the present unique connection ion trap and the tandem mass spectrometry technology of flight time mass spectrum, possessed characteristics such as high resolving power, high-quality degree of accuracy, high speed detection simultaneously, thereby realize both can doing multi-stage ms, can reach the power of high-quality precision again.It has the following advantages: the MS that (1) is every grade
nCan both guarantee the high-quality degree of accuracy; (2) every grade MS
nMass spectrogram can both be accomplished high resolving power, high-quality degree of accuracy and high sensitivity simultaneously; (3) fabulous quality stability; (4) switch between the positive and negative ion pattern at a high speed, and guarantee high mass accuracy; (5) MS/MS analytic function automatically can automatically switch from MS to MS in the mensuration process
nMensuration; (6) carry out the correction of MS working order automatically, and carry out the optimization correction of MS axle.
4, the present invention is feasible fast, the chemical constitution structural information that single injected sampling analysis (can finish in 1h usually) can obtain to enrich, and the present invention simultaneously has the characteristics of general applicability, is not limited to the analysis of specific Chinese medicine or particular category traditional Chinese medicine ingredients.
Description of drawings
Fig. 1: the total ions chromatogram of MAILUONING ZHUSHEYE under negative ion mode.
Fig. 2: the multistage cracked (MS of the representative composition of MAILUONING ZHUSHEYE (No. 41 peak)
n) figure.(a) one-level mass spectrogram; (b) mass-to-charge ratio is the second order ms figure of 389.1061 ion; (c) mass-to-charge ratio is three grades of mass spectrograms of 345.1119 ion in the second order ms; (d) mass-to-charge ratio is three grades of mass spectrograms of 209.0443 ion in the second order ms.
Fig. 3: the fracture approach of No. 41 peak theveside of MAILUONING ZHUSHEYE (theveside).
Embodiment
Embodiment 1: the MAILUONING ZHUSHEYE analysis method for complex composition
A: MAILUONING ZHUSHEYE is carried out full composition extract:
With commercially available MAILUONING ZHUSHEYE (No.200611032, Nanjing Jinting Pharmaceutical Co., Ltd. produce) with 10 times of ultrapure water dilutions after, acidifying, 5 times of saturated normal butyl alcohols of water gaging extract, and get organic layer and volatilize, the moving phase dissolving is to be analyzed.
B: use LC/MS-IT-TOF, full scan obtains the total ions chromatogram of MAILUONING ZHUSHEYE:
Instrument: LC/MS-IT-TOF (Tianjin, island, Japan).
Chromatographic parameter is as follows: column type, Synergi C
18Hydro-RP 80A, 250mm * 4.6mm i.d., 4 μ m (Phenomenex, USA); Column temperature, 35 ℃; Sample size, 5 μ l; Flow velocity, 0.8ml/min; Moving phase is formed, and water (contains 0.025% formic acid, A) and methyl alcohol (B); Gradient, 0-15min:8-12% (B), 15-40min:12-60% (B), 40-50min:60% (B), 50-55min:60-8% (B), 55-65min:8% (B).
The mass spectrum parameter is as follows: ionization mode (Ionization mode), ESI (-); Spray gas flow velocity (Nebulizinggas flow rate), 1.5L/min; Dry gas pressure (Drying gas pressure), 0.1Mpa; Applied voltage (Appliedvoltage) ,-3.5KV; CDL voltage (CDL voltage), Constant mode; CDL temperature (CDL temperature), 200 ℃; Analytic type (Analysis mode), MS measurement; Measurement range (Measurement range), AutoMS/MS mode (MS:m/z 100-1500; MS
n: m/z 50-1500); The ion accumulation time (Ion accumulation time), 30msec.
In the above conditions, full scan obtains the total ions chromatogram of MAILUONING ZHUSHEYE under negative ion mode, sees Fig. 1.
C: from total ions chromatogram, seek, determine to have the leading ion peak of suitable intensity, i.e. principal ingredient:
In Fig. 1, find the principal ingredient of 66 quasi-molecular ions (Intensity＞800,000) altogether, its accurate molecular weight ([M-H] as MAILUONING ZHUSHEYE
-), retention time sees Table 1.
Table 1. MAILUONING ZHUSHEYE analysis of complex ingredient
| Sequence number | Retention time (min) | [M-H] - | Chemical formula | Constituent analysis |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 a 29 b 30 31 32 33 34 35 a 35 b 36 37 | 5.5 31.1 25.0 20.0 35.9 27.3 31.1 42.0 3.5 36.9 15.8 46.2 51.2 30.8 23.8/15.0 42.3 31.4 7.0 43.2 44.7 34.8 29.8 33.5 42.5 43.3 41.4 25/36.2 14.2 28.1 28.1 32.5 26.8/32.1/32.8 28.3 41.8 29.0/22.9 24.8 24.8 26.2/31.1 32.8 | 128.0376 135.0459 137.0269 153.0202 163.0414 175.0620 179.0382 187.0996 191.0605 193.0519 201.0769 201.1124 209.1190 210.0759 215.0562 243.1215 253.0702 257.0279 263.1286 271.0982 281.0667 281.1222 281.1390 287.0568 301.1303 312.1218 313.0723 315.0731 325.0937 325.0937 335.0767 337.0930 339.1080 342.1303 353.0903 357.0600 357.0600 359.0792 365.2050 | C 5H 7NO 3 C 8H 8O 2 C 7H 6O 3 C 7H 6O 4 C 9H 8O 3 C 7H 12O 5 C 9H 8O 4 C 9H 16O 4 C 7H 12O 6 C 10H 10O 4 C 9H 14O 5 C 10H 18O 4 C 12H 18O 3 C 10H 13NO 4 C 9H 12O 6 C 12H 20O 5 C 12H 14O 6 C 10H 10O 8 C 15H 20O 4 C 16H 16O 4 C 13H 14O 7 C 11H 22O 8 C 15H 22O 5 C 15H 12O 6 C 14H 22O 7 C 18H 19NO 4 C 17H 14O 6 C 13H 16O 9 C 15H 18O 8 C 15H 18O 8 C 16H 16O 8 C 16H 18O 8 C 16H 20O 8 C 19H 21NO 5 C 16H 18O 9 C 18H 14O 8 C 18H 14O 8 C 18H 16O 8 C 24H 30O 3 | 2-Oxo-3-pyrrolidinecarboxylic acid 3，4-Dihydroxy-cinnamene 4-Hydroxybenzoic acid 3，4-Dihydroxybenzoic acid ρ-coumaric acid 2-Hydroxy-2，3，3-trimethylbutanedioic acid Caffeic acid Ovatic acid Quinic acid Ferulic acid 1- |
| 38 39 a 39 b 39 c 40 a 40 b 41 42 43 44 45 46 47 48 49 50 51 52 a 52 b 53 a 53 b 53 c 54 55 a 55 b 56 57 58 59 60 61 62 63 64 65 66 | 28.8/33.1/33.6 29.9 29.9 29.9 29.2/28 29.2/28 30.2 36.1/36.4 35.2/32.1 41.6/43.4/43.7 36.5 40.8 38.4 24.1 46.4 45.4 35.2/32.1 44/47.7 30.8 44.3 44.3 44.3 39.8 44.6 44.6 30.1/36.7/37.1/39.3 44.3 40.1 27.0 40.1 46.7/53.0 40.1 38.1 41.0 43.5 39.4 | 367.0992 373.1146 373.1146 373.1146 375.1309 375.1309 389.1061 389.1228 403.1267 409.1515 429.1398 431.0931 447.0947 461.1671 461.2103 467.2141 471.1111 475.1831 475.1831 475.2627 475.2627 475.2627 479.2135 507.2980 507.2980 515.1172 521.2673 525.3014 531.1200 541.2957 571.1789 593.2869 623.1986 637.2056 651.2232 783.2645 | C 17H 20O 9 C 16H 22O 10 C 16H 22O 10 C 16H 22O 10 C 16H 24O 10 C 16H 24O 10 C 16H 22O 11 C 20H 22O 8 C 17H 24O 11 C 20H 26O 9 C 19H 26O 11 C 21H 20O 10 C 21H 20O 11 C 20H 30O 12 C 22H 22O 11 C 20H 36O 12 C 27H 20O 8 C 21H 32O 12 C 21H 32O 12 C 27H 40O 7 C 27H 40O 7 C 27H 40O 7 C 21H 36O 12 C 28H 44O 8 C 28H 44O 8 C 25H 24O 12 C 28H 42O 9 C 28H 46O 9 C 25H 24O 13 C 31H 42O 8 C 29H 32O 12 C 27H 46O 14 C 29H 36O 15 C 30H 38O 15 C 31H 40O 15 C 36H 48O 19 | (3or 4or 5)-O-Feruloylquinic acid Ketologanic acid Gardoside Geniposidic acid Loganic acid Mussaenosidic acid Theveside 3，3′，4′，5，5′，7-Hexahydroxyflavanone； (2R，3R)-form，Hexa-Me ether Theviridoside (3or 4or 5)-O-Caffeoylquinic acid；(E)-form， Butyl ester Regaloside G Cosmosiin Luteolin 7-g1ucoside Decaffeoylacteoside Thermopsoside Ebracteatoside C Unknow Kanokoside A Cistanoside E Lucidenic acid G Lucidenic acid H Lucidenic acid C Secolonitoside 25-Hydroxyatrotosterone B Atrotosterone C (1，3or 1，4or 1，5or 3，4or 3， |
Glc: glucose.
D:, obtain the multi-stage ms (MS at each leading ion peak by collision induced dissociation (CID)
n) data:
In Fig. 1, the dehydrogenation ion ([M-H] of each composition
-) can be used as the cracked leading ion of secondary, and the high strength leading ion of secondary in cracked can also be as three grades of cracked leading ions, by that analogy, cracked step by step.In the present invention, three grades of cracked fragmention information just are enough to finish constituent analysis, although LC/MS-IT-TOF can finish ten grades cracked.
Therefore the data magnanimity of these 66 kinds of compositions only specify with No. 41 composition as an example.Shown in Fig. 2 (a), No. 41 principal ingredient dehydrogenation ion ([M-H]
-) accurate molecular weight be 389.1061, can be used as its 2 grades of cracked leading ions.In Fig. 2 (b), the accurate molecular weight of leading ion is 345.1119 and 209.0443, can be used as its 3 grades of cracked leading ions.Just the multistage cracked data of each principal ingredient have been obtained thus.
E: based on accurate molecular weight, the chemical formula of predictive molecule ion and fragmention in certain error range:
Use LC/MS-IT-TOF (Tianjin, island, Japan) the Formula Predictor software in the LC/MS solution workstation, can be based on accurate molecular weight (being accurate to 0.0001Da), the chemical formula of (0-10ppm) predictive molecule ion and fragmention in certain error range.The chemical formula prediction of No. 41 composition sees Table 2.
Table 2. is based on No. 41 composition chemical formula prediction of accurate molecular weight
| The source | Actual measurement (m/z) | Chemical formula a | Prediction (m/z) | Error (mDa) | Error (ppm) | DBE b |
| MS 1 MS 2 MS 2 MS 2 MS 2 MS 2 MS 2 MS 2 MS 2 MS 2 MS 3 (345.1119) MS 3 (345.1119) MS 3 (209.0443) MS 3 (209.0443) MS 3 (209.0443) | 389.1061 371.0936 345.1119 209.0443 183.0664 179.0557 165.0565 143.0364 139.0043 121.0333 179.0593 165.0591 191.0350 165.0572 139.0043 | C 16H 22O 11 C 23H 18O 6 C 30H 14O C 23H 16O 5 C 16H 20O 10 C 22H 18O 4 C 15H 22O 9 C 11H 22O 12 C 10H 10O 5 C 9H 12O 4 C 6H 12O 6 C 9H 10O 3 C 6H 8O 4 C 6H 4O 4 C 7H 6O 2 C 6H 12O 6 C 9H 10O 3 C 10H 8O 4 C 9H 10O 3 C 6H 4O 4 | 389.1084 389.1025 389.0966 371.0919 371.0978 345.1127 345.1186 345.1033 209.0450 183.0657 179.0556 165.0552 143.0344 139.0031 121.0290 179.0556 165.0552 191.0344 165.0552 139.0031 | 2.3 -3.6 -9.5 -1.7 4.2 0.8 6.7 -8.6 0.7 -0.7 -0.1 -1.3 -2.0 -1.2 -4.3 -3.7 -3.9 -0.6 -2.0 -1.2 | 5.91 -9.25 -24.41 -4.58 11.32 2.32 19.41 -24.92 3.35 -3.82 -0.56 -7.88 -13.98 -8.63 -35.53 -20.66 -23.63 -3.14 -12.12 -8.63 | 6.0 15.0 24.0 16.0 7.0 14.0 5.0 1.0 6.0 4.0 1.0 5.0 3.0 5.0 5.0 1.0 5.0 7.0 5.0 5.0 |
aChemical formula prediction (atom span): C[0-100]; H[0-200]; 0[0-50].
bTwo keys and ring etc. value.
F:, obtain the possible structure of institute of each predicted chemical formula, structure promptly to be selected by database search:
Applying Internet (for example: Google (http://www.google.com), ChemExper (http://www.chemexper.com)) or CA (american chemical index), with each predicted chemical formula is that keyword is searched for, and just can obtain its institute might structure.As shown in table 3, find 19 kinds on the possible structure of No. 41 composition altogether.
The possible structure (m/z:389.1061) of No. 41 composition of table 3.
| Sequence number | Chemical formula a | Possible structure |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 | C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 16H 22O 11 C 23H 18O 6 C 23H 18O 6 C 23H 18O 6 C 23H 18O 6 | Allose；β-D-Pyranose-form，Penta-Ac Altrose；α-D-Pyranose-form，Penta-Ac Eustomoside Forsythide Fructose；D-form，1，3，4，5，6-Penta-Ac Kingiside；Parent acid Monotropein Oleoside 1，2，3，4，6-Penta-O- |
aPredicted chemical formula C
30H
14The corresponding construction of O does not exist.
G: from structure to be selected, pick out the structure that can produce all corresponding fragment ions, promptly prove conclusively structure:
No. 41 composition is accredited as theveside (theveside), its accurate molecular weight ([M-H]
-) be 389.1061.Fragmention (m/z) 371.0936,345.1119,209.0443,183.0664,165.0565 loses H by parent ion respectively
2O, CO
2, Glc ﹠amp; H
2O, Glc ﹠amp; CO
2, Glc ﹠amp; CO
2﹠amp; H
2O forms.Fragmention (m/z) 179.0557 and 143.0364 is accredited as glucose and anhydroglucose.The fracture approach of No. 41 composition theveside (theveside) is seen Fig. 3.
As shown in table 1, be divided into and separate out the wherein structure of 63 kinds of compositions, illustrate that the present invention has high feasibility.Because do not find the structure to be selected of suitable (can produce the fragmention of actual measurement), the structure of 3 kinds of compositions does not have analyzedly to go out, but obtains its accurate molecular weight and multistage fragmentation of ions information.Footmark below the sequence number (a or b) expression does not have identical parent nucleus but isomerism body structure with identical accurate molecular weight.
But because the limitation of mass spectrophotometry self, under the prerequisite that lacks the respective standard product, the present invention can not differentiate for isomers, may be 1,3 or 1,4 or 1,5 or 3,4 or 3,5 or 4 as No. 56 composition ,-dioxy caffeoyl guinic acid.
Claims (4)
1. new traditional Chinese medicine analysis method for complex composition based on LC/MS-IT-TOF is characterized in that being made up of following several steps:
A: adopt certain method that traditional Chinese medicine is carried out full composition and extract;
B: use high-resolution, highly sensitive LC/MS-IT-TOF, under certain chromatogram mass spectrum condition, carry out the total ions chromatogram (TIC) that negative ions full scan (scan) obtains traditional Chinese medicine;
C: from total ions chromatogram, seek, determine the to have suitable intensity leading ion peak of (determining strength criterion as required), i.e. principal ingredient;
D:, obtain the multi-stage ms (MS of each principal ingredient by collision induced dissociation (CID)
n) data, comprise the information such as accurate molecular weight of fragmentation pathway, molion and the fragmention of principal ingredient ion;
E: based on accurate molecular weight (being accurate to 0.0001Da), the chemical formula of (0-100ppm or 0-50mDa) predictive molecule ion and fragmention in certain error range;
F:, obtain meeting the possible structure of institute of each predicted chemical formula, structure promptly to be selected by database search;
G: from structure to be selected, pick out the structure that can produce all corresponding fragment ions, promptly prove conclusively structure.
2. traditional Chinese medicine as claimed in claim 1, it is characterized in that Chinese crude drug through extract, make behind the purifying for the solution that injects in the body (acupuncture point, subcutaneous, muscle, intravenous injection and drip-feed etc.), emulsion fluid and for facing with before being mixed with the powder of solution or the sterile preparation of strong solution.The present invention is that example is specifically described with the MAILUONING ZHUSHEYE, and MAILUONING ZHUSHEYE is on the basis of famous hospital " Simiao Yongan Tang ", the Chinese medicine compound prescription injection that is prepared from by honeysuckle, radix scrophulariae, the root of bidentate achyranthes, stem of noble dendrobium four traditional Chinese medicine material.
3. as the described extracting method of claim 1a, it is characterized in that comprising liquid-liquid extraction, Solid-Phase Extraction, filtration etc., wherein the liquid-liquid extraction solvent comprises normal butyl alcohol, ethyl acetate, ether, chloroform, benzene, normal hexane, cyclohexane etc., Solid-Phase Extraction comprises the solid phase pillar of various models, filters the filter membrane, the filter paper that comprise various models.
4. as the described LC/MS-IT-TOF of claim 1b, it is characterized in that by day island proper Tianjin company produce or the IT-MS that produces by other company
nForm with the TOF-MS applied in any combination.Its chromatographic condition is characterised in that chromatographic column, column length 100-300mm, internal diameter 2-16mm, filler granularity 2-7 μ m; Column temperature, 10-70 ℃; Sample size, 1-50 μ l; Flow velocity, 0.01-1.50ml/min; Moving phase is formed, water (containing acid or alkali) and organic solvent (methyl alcohol, acetonitrile etc.); Carry out gradient elution or isocratic elution.Its mass spectrum condition is characterised in that the setting range that institute's each parameter of use instrument is allowed.
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| CNA2007101333914A CN101153866A (en) | 2007-10-19 | 2007-10-19 | Novel analysis method for complex composition of traditional Chinese medicine injection based on LC/MS-IT-TOF |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102507786A (en) * | 2011-11-11 | 2012-06-20 | 中国科学院成都生物研究所 | Structure identification method of components of traditional Chinese medicine |
| US9359275B2 (en) | 2012-02-23 | 2016-06-07 | Children's Medical Center Corporation | Natural product antibiotics and analogs thereof |
| CN104359968B (en) * | 2014-11-14 | 2016-10-05 | 通化华夏药业有限责任公司 | Realize to the Fast Classification of chemical composition in Ixeris Sonchifolia Hance injection and qualification based on UPLC-Q-TOF-MS technology |
| CN106588664A (en) * | 2016-10-26 | 2017-04-26 | 华南理工大学 | Water-soluble caffeoyl structural grease, and preparation method and application thereof |
| CN109270187A (en) * | 2018-11-02 | 2019-01-25 | 江苏省中医院 | A kind of Chinese materia medica preparation quality evaluating method based on metabolism group Yu full ingredient semi-quantitative analysis |
| CN109632997A (en) * | 2018-12-21 | 2019-04-16 | 广东方制药有限公司 | The extraction and measuring method of regallity glycosides B in a kind of lily medicinal material |
| CN111239315A (en) * | 2020-01-20 | 2020-06-05 | 金陵药业股份有限公司 | UPLC-DAD-MS-based analysis method for researching mailuoning injection fingerprint |
| CN111991525A (en) * | 2020-09-02 | 2020-11-27 | 中国药科大学 | Effective component group of Simiao pills and preparation method and application thereof |
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- 2007-10-19 CN CNA2007101333914A patent/CN101153866A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507786A (en) * | 2011-11-11 | 2012-06-20 | 中国科学院成都生物研究所 | Structure identification method of components of traditional Chinese medicine |
| US9359275B2 (en) | 2012-02-23 | 2016-06-07 | Children's Medical Center Corporation | Natural product antibiotics and analogs thereof |
| CN104359968B (en) * | 2014-11-14 | 2016-10-05 | 通化华夏药业有限责任公司 | Realize to the Fast Classification of chemical composition in Ixeris Sonchifolia Hance injection and qualification based on UPLC-Q-TOF-MS technology |
| CN106588664A (en) * | 2016-10-26 | 2017-04-26 | 华南理工大学 | Water-soluble caffeoyl structural grease, and preparation method and application thereof |
| CN109270187A (en) * | 2018-11-02 | 2019-01-25 | 江苏省中医院 | A kind of Chinese materia medica preparation quality evaluating method based on metabolism group Yu full ingredient semi-quantitative analysis |
| CN109270187B (en) * | 2018-11-02 | 2022-05-10 | 江苏省中医院 | Chinese medicine preparation quality evaluation method based on metabonomics and full-ingredient semi-quantitative analysis |
| CN109632997A (en) * | 2018-12-21 | 2019-04-16 | 广东方制药有限公司 | The extraction and measuring method of regallity glycosides B in a kind of lily medicinal material |
| CN109632997B (en) * | 2018-12-21 | 2021-11-30 | 广东一方制药有限公司 | Extraction and determination method of liliflorin B in lily medicinal material |
| CN111239315A (en) * | 2020-01-20 | 2020-06-05 | 金陵药业股份有限公司 | UPLC-DAD-MS-based analysis method for researching mailuoning injection fingerprint |
| CN111991525A (en) * | 2020-09-02 | 2020-11-27 | 中国药科大学 | Effective component group of Simiao pills and preparation method and application thereof |
| CN111991525B (en) * | 2020-09-02 | 2022-05-03 | 中国药科大学 | Effective component group of Simiao pills and preparation method and application thereof |
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