CN104777260A - Brucea javanica oil emulsion injection liquid quality control method - Google Patents

Abstract

The invention provides a brucea javanica oil emulsion injection liquid quality control method including the following steps: a methanol/dichloromethane solution containing 1,2-dilinoleic acid-3-glyceryl oleate, 1,2-dilinoleic acid-3-glyceryl linoleate and glycerol trioleate is used as a reference substance solution I; a methanol/dichloromethane solution containing brucea javanica oil is used as a reference substance solution II; a methanol/dichloromethane solution containing brucea javanica oil emulsion injection liquid is used as a sample solution; and liquid chromatography graphs of the reference substance solution I, the reference substance solution II and the sample solution are respectively measured. According to the method, both the content and fingerprint can be measured by the same method, the method is quantitative and qualitative, and a rapid, simple and accurate and multi-angle brucea javanica oil emulsion injection liquid quality control method is provided.

Description

Java brucea fruit oil emulsion injection method of quality control

Technical field

The present invention relates to a kind of Java brucea fruit oil emulsion injection method of quality control.

Background technology

Java brucea fruit oil emulsion injection quality standard is recorded in the Sanitation Ministry medicine standard Traditional Chinese medicine historical preparation the 14, standard number: WS3-B-2739-97, this standard content measures and adopts titrimetry to survey total acid content, and method falls behind, and can not the composition of actual response brucea fruit oil.

A lot of bibliographical information is also had to adopt vapor-phase chromatography to survey oleic acid or linoleic content, but the method first specificity is bad, as in brucea fruit oil, 85% is triglyceride, oleic acid is for being transformed after hydrolysis in its body, and free fatty acid content is lower, be mainly the fatty glyceride of combining form; Secondly, in gas chromatographic analysis process, because column temperature is very high, easily make detection composition oleic acid, linoleic double bond rupture or produce the heterogeneous phenomenon of double bond, causing detecting and forbidden.

The content assaying method of current paper report is the content adopting HPLC-ELSD method to measure triglyceride, as Kong Lingfeng etc. adopts HPLC-ELSD method to measure the content of olein in Java brucea fruit oil emulsion injection, but the method only measures a kind of composition in brucea fruit oil 8 kinds of compositions, and the pre-treatment of test sample is comparatively loaded down with trivial details, the steps such as mobile phase dissolves, ultrasonic, cooling, dilution, filtration.Tian Jinmiao etc. adopt HPLC-ELSD method to measure the content of four kinds of triglyceride in brucea fruit oil, but four kinds of compositions that in four kinds of non-brucea fruit oils of triglyceride, 8 kinds of compositions that the method measures, content is the highest, the representative non-optimal of assay, and the pre-treatment of test sample is equally comparatively loaded down with trivial details, the steps such as mobile phase dissolves, ultrasonic, cooling, dilution, filtration.

In finger-print context of detection, Guo Hui etc. establish the HPLC-ELSD finger-print of brucea fruit oil, but the HPLC-ELSD finger-print of Java brucea fruit oil emulsion injection there is no report; Preparation is because emulsifying agent, osmotic pressure regulator etc. in Fat Emulsion are to the interference detected, and its pre-treating method directly can not apply mechanically the pre-treating method of brucea fruit oil, need study voluntarily and grope; And mobile phase is three solvent compositions, operation inconvenience, mixing easily produces the problems such as bubble; Simultaneously, No. 3 peaks 1 in figure, 2-dilinoleic acid-3-tripalmitin, No. 8 peaks 1,2-bis-oleic acid-3-tristerin peak area ratio is too small, according to " technical requirement (provisional) of traditional Chinese medicine finger-print research " peak area size to each total peak and the regulation of corresponding ratio thereof, as too small, can there is larger error during similarity-rough set in peak area ratio.

Summary of the invention

Inaccurate to the result existed in Java brucea fruit oil emulsion injection quality control in order to solve prior art, the problems such as complex steps, the embodiment of the present invention provides a kind of Java brucea fruit oil emulsion injection method of quality control.

One aspect of the present invention, relate to a kind of Java brucea fruit oil emulsion injection method of quality control, described method comprises:

With the methanol/dichloromethane solution containing 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein as object of reference solution I;

Using the methanol/dichloromethane solution containing brucea fruit oil as object of reference solution II;

Using the methanol/dichloromethane solution containing Java brucea fruit oil emulsion injection as need testing solution;

Measure the chromatograms of object of reference solution I, object of reference solution II and need testing solution respectively.

Specifically, described method comprises:

Get every 1ml containing 1,2-dilinoleic acid-3-olein 0.20mg, 1,2-bis-oleic acid-3-glyceryl linoleate 0.50mg and olein 0.66mg, methyl alcohol: methylene chloride is the solution of 1: 1, as object of reference solution I;

Get every 1ml containing 1mg brucea fruit oil, methyl alcohol: methylene chloride is that the solution of 1: 1 is as object of reference solution II;

Get containing Java brucea fruit oil emulsion injection, methyl alcohol: methylene chloride is that the solution of 1: 1 is as need testing solution;

Accurate absorption object of reference solution I, object of reference solution II and each 5 μ l of need testing solution respectively, injection liquid chromatography, measures its collection of illustrative plates.

More specifically, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 60-70: 40-30 is mobile phase; Flow velocity is 0.3-0.8ml per minute; Column temperature 30-40 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute.Being preferably, take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 65: 35 is mobile phase; Flow velocity is 0.5ml per minute; Column temperature 35 DEG C; Evaporative light-scattering detector; Analysis time is preferably 65 minutes.

Further, above-mentioned Java brucea fruit oil emulsion injection method of quality control, also comprises the step with standard diagram comparison.Described standard diagram measures by the following method:

Choose some batches of Java brucea fruit oil emulsion injections, measure its liquid chromatography according to method described in any one of claim 1-4, by chromatographic fingerprinting similarity evaluation system, generate Java brucea fruit oil emulsion injection standard finger-print.

Again further, above-mentioned Java brucea fruit oil emulsion injection method of quality control, also comprises the step that component content measures.Described component content measures and refers to,

With the methanol/dichloromethane solution containing 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein as object of reference solution I;

Using the methanol/dichloromethane solution containing Java brucea fruit oil emulsion injection as need testing solution;

Measure the chromatograms of V1 volume object of reference solution I, V2 volume object of reference solution I and V3 volume need testing solution respectively, wherein, V1 < V3 < V2;

With external standard two-point method logarithmic equation calculating composition content, formula is as follows:

In formula:

A is peak area

N is sampling volume, μ l;

C is concentration, mg/ml;

V is sample volume, ml;

A is the slope of equation;

B is intercept.

Particularly, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 60-70: 40-30 is mobile phase; Flow velocity is 0.3-0.8ml per minute; Column temperature 30-40 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute.Preferably, take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 65: 35 is mobile phase; Flow velocity is 0.5ml per minute; Column temperature 35 DEG C; Evaporative light-scattering detector; Analysis time is preferably 65 minutes.

In standard chromatograms provided by the present invention, the relative retention time RRT of Trilinoleyl glyceride (No. 1 peak) is 0.49, 1, the relative retention time RRT of 2-dilinoleic acid-3-olein (No. 2 peaks) is 0.62, 1, the relative retention time RRT of 2-dilinoleic acid-3-tripalmitin (No. 3 peaks) is 0.66, 1, the relative retention time RRT of 2-bis-oleic acid-3-glyceryl linoleate (No. 4 peaks) is 0.78, the relative retention time RRT of 1-palmitic acid-2-oleic acid-3-glyceryl linoleate (No. 5 peaks) is 0.83, the relative retention time RRT of olein (No. 6 peaks) is 1.The relative retention time RRT of 1-stearic acid-2-oleic acid-3-glyceryl linoleate (No. 7 peaks) is 1.03,1, the relative retention time RRT of 2-bis-oleic acid-3-tripalmitin (No. 8 peaks) is 1.07, the relative retention time RRT of 1,2-bis-oleic acid-3-tristerin (No. 9 peaks) is 1.33.

Java brucea fruit oil emulsion injection method of quality control provided by the invention at least achieves following beneficial effect:

The present invention adopts HPLC-ELSD method to measure 3 kinds of compositions that in brucea fruit oil, content is the highest: 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein, and test sample pretreatment process easily, create a kind of advanced, easy, accurately, more representative content assaying method.

The present invention adopts HPLC-ELSD method to set up standard finger-print and the fingerprint detection method of Java brucea fruit oil emulsion injection, pre-treating method is simple and efficient, extraction ratio is high, mobile phase component is few, more convenient operation, improve the peak area ratio at the minimum total peak of two peak areas, fingerprint atlas detection method is more accurate, adopt two kinds of structural identification methods mutually to confirm, improve the accuracy of structural identification.

The present invention's same method both surveyed content, also detected finger-print, quantitatively existing, also difinite quality, provided the Java brucea fruit oil emulsion injection method of quality control of quick, easy, accurate a, multi-angle.

The HPLC-ELSD finger-print of the brucea fruit oil that Guo Hui etc. set up is when structural confirmation, HPLC-MS method is only adopted to infer structure, No. 1 peak (oleic acid leukotrienes glyceryl linoleate), No. 4 peaks (1, the sub-acid glyceride of 3 two oleic acid-2-leukotrienes), No. 5 peaks (Oleic Linoleic tripalmitin), there is mistake in the structural identification of No. 8 peaks (palmitic acid linoleic acid tristerin), the present invention adopts HPLC-MS and reference substance Comparison Method two kinds of methods to infer structure, structural identification accuracy is higher, correct for the mistake in document.

Summary of the invention

Java brucea fruit oil emulsion injection standard finger-print prepared by the present invention, can see Fig. 1.

Java brucea fruit oil emulsion injection chemical constitution study

HPLC-MS method is adopted to carry out structure analysis to the chemical composition of refining brucea fruit oil, compound structure is inferred according to analysis result, buy reference substance, then reference substance Comparison Method is adopted, according to the comparison of retention time between HPLC-ELSD working sample and reference substance, and HPLC-MS method is to the reference substance bought and the analyses and comparison of sample ions figure, the principal ingredient triglyceride chemical composition structure of final confirmation Java brucea fruit oil emulsion injection, i.e. Trilinoleyl glyceride, 1, 2-dilinoleic acid-3-olein, 1, 2-dilinoleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-glyceryl linoleate, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1, 2-bis-oleic acid-3-tripalmitin and 1, 2-bis-oleic acid-3-tristerin.

Finger-print is studied

By the selection (optimization of the selection of detecting device, the selection of chromatographic column, mobile phase, the selection of column temperature, the selection of flow velocity, the determination of analysis time) of chromatographic condition, the preparation (selection of extracting method, the selection of diluting solvent) of need testing solution, the determination of each chromatographic peak and the selection of object of reference, Method validation (specificity test, precision test, replica test, stability test), with the detection method determining finger-print comparing (olive oil, soybean oil, sesame oil, edible wet goods) etc. of similar products.

The generation of standard finger-print

According to preparation method and the chromatographic condition determined of above-mentioned need testing solution, determine the finger-print of 19 batches of Java brucea fruit oil emulsion injections altogether, according to different product batch numbers, have chosen 10 batch samples wherein, generate Java brucea fruit oil emulsion injection standard finger-print

Java brucea fruit oil emulsion injection finger-print sample determination

Compare with standard finger-print, the similarity evaluation 2.0 editions adopting state-promulgated pharmacopoeia to provide, has carried out the mensuration of fingerprint similarity to 19 batch samples.The sample similarity of 95% is greater than 0.998 as a result, has the sample similarity of 100% to be greater than 0.99.

Olein class assay

Adopt the content of HPLC-ELSD method Simultaneously test 9 principal component, and through mobile phase, chromatographic column, flow velocity, the selection of column temperature, test sample preparation method selects, Method validation (test by specificity, linear relationship is investigated, precision test, replica test, recovery test, stability test) etc. research, set up olein class content assaying method, according to said method determine 18 batches of Java brucea fruit oil emulsion injections, 9 component contents, because part reference substance is expensive, in order to take into account cost, select 3 compositions that content is higher: 1, 2-dilinoleic acid-3-olein, 1, 2-bis-oleic acid-3-glyceryl linoleate and olein are index components.

Assay pre-treating method of the present invention is convenient, simple, can reach simultaneously and extract completely.Assay of the present invention selects 3 kinds of compositions that in injecting Java brucea fruit oil emulsion injection agent, content is the highest: 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein, assay method is more representative, more accurately.During finger-print research of the present invention, two kinds of methods are adopted mutually to confirm, improve the accuracy of structural identification, after deliberation: No. 1 peak should be Trilinoleyl glyceride but not oleic acid leukotrienes glyceryl linoleate, No. 4 peaks should be 1,2-bis-oleic acid-3-glyceryl linoleate but not 1,3 two oleic acid-2-glyceryl linolenates, No. 5 peaks should be 1-palmitic acid-2-oleic acid-3-glyceryl linoleate but not Oleic Linoleic tripalmitin, No. 8 peaks should be 1,2-oleic acid-3-tripalmitin but not palmitic acid linoleic acid tristerin, correct for the mistake of bibliographical information.Fingerprint atlas detection method mobile phase of the present invention is grouped into by two kinds of one-tenth, easy and simple to handle, quick, and remains the chemical composition (peak area ratio of two smallest peaks improves) in test sample to greatest extent.Fingerprint atlas detection method of the present invention can get rid of the interference of auxiliary material etc., and improves the peak area ratio at the minimum total peak (No. 3 peaks, No. 8 peaks) of two peak areas, and fingerprint atlas detection method is more accurate.Same Way both can survey content, also can survey finger-print, existing quantitatively difinite quality again, provided the Java brucea fruit oil emulsion injection method of quality control of quick, easy, accurate a, multi-angle, guaranteed the steady quality of injection, controlled.

Experimental example 1: finger-print, measures with reference to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).

Chromatographic condition and system suitability

Take octadecylsilane chemically bonded silica as filling agent; Be mobile phase with the mixed solution of acetonitrile/methylene chloride (acetonitrile: methylene chloride for 60-70: 40-30, preferably 65: 35); Flow velocity is 0.3-0.8ml per minute, preferred 0.5ml; Column temperature 30-40 DEG C, preferably 35 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute, preferably 65 minutes.Number of theoretical plate calculates should be not less than 5000 by olein peak, and No. 2 peaks corresponding with standard finger-print in test sample and the degree of separation at No. 3 peaks should be more than 1.5.

The preparation of object of reference solution

Get 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein reference substance appropriate, accurately weighed, add ethanol/methylene mixed solution (methyl alcohol: methylene chloride is 1: 1) and make every 1ml containing 1, the solution of 2-dilinoleic acid-3-olein 0.20mg, 1,2-bis-oleic acid-3-glyceryl linoleate 0.50mg and olein 0.66mg, as object of reference solution I.Get brucea fruit oil reference extract more appropriate, accurately weighed, add ethanol/methylene mixed solution (methyl alcohol: methylene chloride is 1: 1) and make the solution of every 1ml containing 1mg, as object of reference solution II.

The preparation of need testing solution

Get this product 0.3g, put in 10ml measuring bottle, add ethanol/methylene mixed solution (methyl alcohol: methylene chloride is 1: 1) and, to scale, shake up, to obtain final product.

Assay method

Accurate absorption object of reference solution I, II and each 5 μ l of need testing solution respectively, injection liquid chromatography, measure, after deduction solvent peak, the similarity evaluation that can provide by state-promulgated pharmacopoeia calculates, and the similarity of test sample finger-print and standard finger-print must not lower than 0.95.

In Java brucea fruit oil emulsion injection standard finger-print, peak 1 is Trilinoleyl glyceride, and peak 2 is 1,2-dilinoleic acid-3-olein, peak 3 is 1,2-dilinoleic acid-3-tripalmitin, peak 4 is 1,2-bis-oleic acid-3-glyceryl linoleate, and peak 5 is 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, peak 6 is olein, peak 7 is 1-stearic acid-2-oleic acid-3-glyceryl linoleate, and peak 8 is 1,2-bis-oleic acid-3-tripalmitin, peak 9 is 1,2-bis-oleic acid-3-tristerin.

Reference color spectral condition:

Agilent 1100 series of high efficiency liquid chromatograph (Agilent Chemstation workstation)

Chromatographic column: Agilent Zorbax Extend C18 (250mm × 4.6mm, 5 μm)

Detecting device: Alltech 2000 type evaporative light-scattering detector

Detector parameters: drift tube temperature: 95 DEG C; Flow rate of carrier gas: 2.5L per minute

Specific experiment process is as follows:

(1) Java brucea fruit oil emulsion injection chemical constitution study

Java brucea fruit oil emulsion injection is Java brucea medicinal material ligroin extraction, therefore water soluble ingredient is not retained, be mostly liposoluble constituent, present stage generally believes that the anticancer active constituent of brucea fruit oil is the unsaturated fatty acid of certain proportioning, because unsaturated fatty acid mainly exists with the form of triglyceride in brucea fruit oil, therefore adopt the constituent structure of HPLC-MS to refining brucea fruit oil to infer, then the retention time of HPLC-ELSD method comparative sample and reference substance is adopted, the mass spectrogram of HPLC-MS method analysis of control product and sample, thus putative structure is confirmed, and carry out GC-MS analysis to the volatile ingredient beyond its main chemical compositions with through the composition of silicagel column enrichment.

1HPLC-MS analyzes the composition of refining brucea fruit oil

1.1 instruments, reagent and reagent

Instrument: liquid chromatography mass instrument Agilent 6430 Triple Quad LC/MS (Agilent company of the U.S.).

Reagent: methylene chloride and acetonitrile are chromatographically pure, German Merck company.

Reagent: refining brucea fruit oil (lot number: 20100906, Zhejiang Province Jiuxu Pharmaceutical Co., Ltd).

1.2 experimental technique

Mass Spectrometry Conditions: atmosphere pressure chemical ion source (APCI), positive ion mode.Spray voltage 4500V, capillary temperature 350 DEG C, APCI evaporating temperature 350 DEG C, corona electric current 4 μ A.Sheath gas (high-purity liquid nitrogen) flow velocity 5L/min.Mass spectrum mass scan range m/z300 ~ 900.

Liquid phase chromatogram condition: according to Java brucea fruit oil emulsion injection finger-print chromatographic condition, chromatographic column is Agilent Extend C18 (4.6mm × 250mm, 5 μm); Mobile phase acetonitrile-isopropyl alcohol-normal hexane (66: 18: 16), flow velocity 0.3ml/min, column temperature 40 DEG C.

The preparation of need testing solution: get refining brucea fruit oil 10g, put in 25ml measuring bottle, add isopropanol to scale, shake up, to obtain final product.Sample size: 2 μ l.

1.3 compounds are inferred

In APCI mass spectrum, it is a lot of that triglyceride is cracked into diglyceride correlative study, a large amount of documents shows that fatty acid is connected to positions different in glycerine, there is some difference for cracking diglyceride relative abundance out, in APCI mass spectrum, generally there is the diglyceride peak of molecular ion peak and some cracking fatty acid.From mass spectrogram, can analyze and obtain 9 kinds of compounds.

By MS chromatogram see compound 1 molecular ion peak be m/z879.6 [M+H]+, after sloughing m/z280.4 ion, obtain the ion of m/z599.4,280.4 be linoleic molecular weight, the molecular weight of binding compounds 1 and only have a fragmention m/z599.4, infer that compound 1 is containing identical fatty acid, i.e. 3 linoleic acid, infer that compound 1 may be Trilinoleyl glyceride thus.

By the mass spectrum of compound 2 see molecular ion peak be m/z881.6 [M+H]+, obtain 2 characteristic fragmentions 599.3 and 601.2, be the fragment that compound 2 sloughs m/z280.4 and m/z282.5 gained simultaneously respectively.280.4 is linoleic molecular weight, and 282.5 is the molecular weight of oleic acid, and to sum up, therefore, we infer that compound 2 may be dilinoleic acid olein.

By the mass spectrum of compound 3 see molecular ion peak be m/z855.6 [M+H]+, obtain 2 characteristic fragmentions 599.3 and 575.8, be the fragment that compound 3 sloughs m/z256.4 and m/z280.4 gained simultaneously respectively.This shows there are two kinds of fatty acid in structure, again because the molecular weight of palmitic acid is 256.42, linoleic molecular weight is 280.4, and the abundance of m/z599.3 is apparently higher than m/z575.8, therefore infers that compound 3 may be dilinoleic acid tripalmitin.

By the mass spectrum of compound 4 see molecular ion peak be m/z883.6 [M+H]+, obtain 2 characteristic fragmentions 601.4 and 603.16, be the fragment that compound 4 sloughs m/z282.2 and m/z280.4 gained simultaneously respectively.And linoleic molecular weight is 280.4, the molecular weight of oleic acid is 282.4, therefore infer that two kinds of fatty acid are respectively linoleic plus oleic acid, but because the abundance of the m/z601.4 after de-oiling acid is apparently higher than m/z603.16, this shows in compound 4 structure containing two oleic acid, a linoleic acid, therefore infers that this compound 4 may be two Oleic Linoleic glyceride.

By the mass spectrum of compound 5 see molecular ion peak be m/z857.6 [M+H]+, obtain 3 characteristic fragmentions m/z601.4, m/z577.2 and m/z575.2, be the fragment that compound 5 sloughs m/z256.2 and m/z280.4 and m/z282.4 gained simultaneously respectively.Therefore three kinds of fatty acid may be had in this compound structure of deducibility, and the molecular weight of oleic acid is 282.4, linoleic molecular weight is 280.4, and the molecular weight of palmitic acid is 256.4, infers that compound 5 may be Oleic Linoleic tripalmitin according to fatty acid molecule amount.

By the mass spectrum of compound 6 see molecular ion peak be m/z885.6 [M+H]+, obtain may only having a fatty acid in 1 characteristic fragmention m/z603.2, therefore this compound structure of deducibility, m/z603.2 is the fragmention that m/z885.6 sloughs m/z282.4 and obtains, and the molecular weight of oleic acid is 282.4, infer this compound only containing oleic acid, m/z339.0 may be that m/z603.2 continues to slough a fatty acid and obtains, and therefore compound 6 may be olein.

By the mass spectrum of compound 7 see molecular ion peak be m/z885.6 [M+H]+, obtain 3 characteristic fragmentions m/z601.4, m/z605.2 and m/z603.2, be the fragmention that m/z885.6 sloughs m/z284.4 and m/z280.4 and m/z282.4 and obtains simultaneously respectively.Stearic acid molecule amount is 284.4, and the molecular weight of oleic acid is 282.4, and linoleic molecular weight is 280.4, infers that compound 7 may be stearic acid Oleic Linoleic glyceride according to stearic acid, oleic acid, linoleic structure and molecular weight.

By the mass spectrum of compound 8 see molecular ion peak be m/z859.7 [M+H]+, obtaining 2 characteristic fragmentions m/z577.3, m/z603.3, is the fragmention that m/z859.7 sloughs m/z282.4, m/z256.4 and obtains respectively.The molecular weight of oleic acid is 282.4, and the molecular weight of palmitic acid is 256.4, and deducibility two kinds of fatty acid are respectively oleic acid and palm oil.But the abundance of m/z577.3 is apparently higher than m/z603.3, two oleic acid in description architecture, may be had, a palmitic acid, therefore infer that compound 8 may be two oleic acid tripalmitins according to the structure and molecular weight of sloughing fatty acid.

By the mass spectrum of compound 9 see molecular ion peak be m/z887.7 [M+H]+, obtaining 2 characteristic fragmentions m/z605.4, m/z603.3, is the fragmention that m/z887.7 sloughs m/z282.4, m/z284.4 and obtains respectively.And the molecular weight of oleic acid is 282.4, stearic acid molecule amount is 284.4, and deducibility two kinds of fatty acid are respectively oleic acid and stearic acid.The abundance of m/z605.4 is apparently higher than m/z603.3 simultaneously, may have two oleic acid, a stearic acid in description architecture, therefore infers that compound 9 may be two oleic acid tristerins according to the structure and molecular weight of sloughing fatty acid.

By the analysis of LC-MS to ingredient in refining brucea fruit oil, result infers the structure of 9 triglyceride constituents.Be respectively linolein, dilinoleic acid olein, dilinoleic acid tripalmitin, two Oleic Linoleic glyceride, palmitic acid oleic acid glyceryl linoleate, olein, stearic acid Oleic Linoleic glyceride, two oleic acid tripalmitins, two oleic acid tristerins.

2. reference substance Comparison Method

Use 9 reference substances, contrast each composition retention time through HPLC-ELSD chromatogram, and HPLC-APCI-MS detects the chromatogram ion figure of reference substance, confirms the structure of 9 chemical compositions in refining brucea fruit oil.

2.1HPLC-ELSD method

2.1.1 instrument

Agilent 1200 series of high efficiency liquid chromatograph (Agilent company of the U.S. is equipped with the online degasser of G1322A, G1311A quaternary pump, G1329A automatic sampling instrument, G1316A column oven, Chemstation chromatographic work station); CP-225D electronic balance (Beijing Sai Duolisi company); Evaporative light-scattering detector (Alltech2000ES).

2.1.2 reagent

Methyl alcohol (land, Shanghai all chemical reagent factories, analyze pure); Methylene chloride (Chemical Reagent Co., Ltd., Sinopharm Group analyzes pure); Acetonitrile (MERCK company, chromatographically pure); Methylene chloride (TEDIA company of the U.S., chromatographically pure).

2.1.3 reagent

Reference substance Trilinoleyl glyceride, 1,2-dilinoleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tristerin, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate is purchased from LarodanAB company, and olein, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate are purchased from SIGMA company.

Java brucea fruit oil emulsion injection 20100504 (Zhejiang Province Jiuxu Pharmaceutical Co., Ltd provides).

2.1.4 experimental technique

Chromatographic condition: according to Java brucea fruit oil emulsion injection finger-print chromatographic condition take octadecylsilane chemically bonded silica as filling agent; Acetonitrile-dichloromethane (65: 35) is mobile phase isocratic elution, record 65min chromatographic data; Flow velocity: 0.5ml/min; Column temperature: 35 DEG C; Evaporative light-scattering detector condition: drift tube temperature 90 DEG C; Gas flow rate 2.5L/min.

The preparation of reference substance solution: get each reference substance appropriate, dilute with ethanol/methylene mixed solution (methyl alcohol: methylene chloride is 1: 1).

The preparation of need testing solution: prepare by Java brucea fruit oil emulsion injection determining fingerprint pattern need testing solution preparation method.Precision takes Java brucea fruit oil emulsion injection 0.3g, puts in 10ml measuring bottle, adds ethanol/methylene mixed solution (methyl alcohol: methylene chloride is 1: 1) and is diluted to scale, shake up, to obtain final product.

2.1.5 measurement result

In Java brucea fruit oil emulsion injection finger-print, employing reference substance contrasts, confirm the structure of chromatographic fingerprinting 9 main chromatographic peaks altogether, be respectively Trilinoleyl glyceride, 1,2-dilinoleic acid-3-olein, 1,2-dilinoleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-glyceryl linoleate, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1,2-bis-oleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tristerin.

2.2 LC-APCI-MS verify reference substance structure

Adopt LC-APCI-MS method to carry out structural identification to the reference substance bought, contrast with the deduction compound structure in refining brucea fruit oil sample, thus confirm the structure of triglyceride constituents in refining brucea fruit oil.

By the analysis to HPLC-MS chemical composition chromatography of ions figure, we infer 9 kinds of possible triglyceride compounds, through HPLC-ELSD chromatogram, the reference substance bought and Java brucea fruit oil emulsion injection sample retention time are contrasted again, found that the chromatographic peak retention time of reference substance and sample is basically identical, we adopt LC-APCI-MS to carry out structural confirmation to the reference substance bought simultaneously, find that parent ion and the fragmention of reference substance and chemical composition in sample are consistent, therefore in Java brucea fruit oil emulsion injection, ingredient is Trilinoleyl glyceride respectively, 1, 2-dilinoleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-tristerin, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1, 2-dilinoleic acid-3-olein, 1, 2-bis-oleic acid-3-glyceryl linoleate, olein, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate.

(2) finger-print research

1 instrument, reagent and reagent

1.1 instrument

Agilent 1200 series of high efficiency liquid chromatograph (Agilent company of the U.S. is equipped with the online degasser of G1322A, G1311A quaternary pump, G1329A automatic sampling instrument, G1316A column oven, Chemstation chromatographic work station); CP-225D electronic balance (Beijing Sai Duolisi company); Evaporative light-scattering detector (Alltech2000ES).

1.2 reagent

Methyl alcohol (land, Shanghai all chemical reagent factories); Methylene chloride (Chemical Reagent Co., Ltd., Sinopharm Group); Acetonitrile (MERCK company); Methylene chloride (TEDIA company of the U.S.).

1.3 reagent

Reference substance Trilinoleyl glyceride, 1,2-dilinoleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tristerin, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate is purchased from LarodanAB company, and olein, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate are purchased from SIGMA company.

Java brucea fruit oil emulsion injection 19 batches (Zhejiang Province Jiuxu Pharmaceutical Co., Ltd provides), sees the following form:

The selection of 2 chromatographic conditions

The selection of 2.1 detecting devices

Because of this product in ultraviolet region substantially without absorption, once adopted UV-detector (with 270nm as determined wavelength) to connect with evaporative light-scattering detector to detect, signal measured by result UV-detector because of response too little, baseline noise is large and evaporative light-scattering detector gained signal is more satisfactory, therefore adopts common detector evaporative light-scattering detector to detect.

The selection of 2.2 chromatographic columns

Investigated four kinds of different chromatographic columns listed in Tab.2-1-2 in test, result Agilent Extend-C18 chromatographic column can be separated the composition in Java brucea fruit oil emulsion injection preferably, and peak shape is better, therefore selects this chromatographic column.

The chromatographic column that different manufacturers selected by the present invention is produced:

The optimization of 2.3 mobile phases

2.3.1 type of elution is investigated

With acetonitrile/methylene chloride mixed solution for mobile phase, Agilent Extend-C18 (250 × 4.6mm, 5 μm) be chromatographic column, investigated respectively different proportion isocratic elution mode and program gradient elution (mode one: 0 to 30 minute, acetonitrile ratio becomes 40% from 60%; Mode two: 0 to 15 minutes, acetonitrile ratio is 55%, and within 15 to 40 minutes, acetonitrile ratio is 0%) separating effect.

Compare acetonitrile and the methylene chloride of different proportion, compare gradient elution program simultaneously, result is better with the chromatographic peak peak shape of acetonitrile/methylene chloride constant ratio wash-out, and the separation of each chromatographic peak is ideal.Consider that analyzed each composition capacity factor measure is close, therefore adopt constant ratio wash-out.In addition, from the result of gradient elution, when adopting the mobile phase wash-out of strong eluting power (methylene chloride 100%), other chromatographic peak is there is not in chromatogram, illustrate that when adopting constant ratio (acetonitrile: methylene chloride is 65: 35) wash-out, measured 9 main chromatographic peaks are outward substantially without the chromatographic peak of other compositions, most composition can be embodied in this chromatogram.

2.3.2 the investigation of mobile phase solvent system

Be chromatographic column with Agilent Extend-C18 (250 × 4.6mm, 5 μm), the good flow phase system of more several separating effects according to the literature, as acetonitrile/tetrahydrofuran, acetonitrile/acetone, acetonitrile/methylene chloride etc., result is better with acetonitrile/methylene chloride systematic position effect.

The selection of 2.4 column temperatures

Be chromatographic column with Agilent Extend-C18 (250 × 4.6mm, 5 μm) in test, compare different chromatographic column column temperatures: the separating effect of 30 DEG C, 35 DEG C, 40 DEG C.When other peak separating effect no significant differences, during column temperature 35 DEG C, the chromatographic peak separating effect that retention time is about 65min is better, and retention time is moderate, so select column temperature to be 35 DEG C.

The selection of 2.5 flow velocitys

Be chromatographic column with Agilent Extend-C18 (250 × 4.6mm, 5 μm) in test, compare the separating effect of different flow velocity 0.3ml/min, 0.5ml/min, 0.8ml/min.As can be seen from collection of illustrative plates: the separating effect impact of different in flow rate on the larger main chromatographic peak of peak area is little, but when flow velocity is less, the separating effect of some small peak is better, in order to take into account appearance time and separating effect, finally flow velocity is selected to be 0.5ml/min.

The determination of 2.6 analysis times

With acetonitrile: methylene chloride be 65: 35 mixed solution be mobile phase, measure the need testing solution chromatogram of 130 minutes, as can be seen from collection of illustrative plates: the composition that evaporative light detecting device can detect within 65 minutes completely by wash-out, therefore select detection time be 65 minutes.

In sum, determine that the chromatographic condition of Java brucea fruit oil emulsion injection fingerprint spectrum method is: take octadecylsilane chemically bonded silica as filling agent; Acetonitrile: methylene chloride be 65: 35 mixed solution be mobile phase isocratic elution, record 65 minutes chromatographic datas; Flow velocity 0.5ml/min; Column temperature: 35 DEG C; Evaporative light detecting device condition: drift tube temperature 90 DEG C, gas flow rate 2.5L/min.

The preparation of 3 need testing solutions

The selection of 3.1 extracting method

Because this product is the folk prescription parenteral solution that refining brucea fruit oil adds auxiliary material and makes, and refining brucea fruit oil is directly obtained by Java brucea medicinal material extract, by test, adopt direct dilution method just can well detect the principal ingredient of Java brucea fruit oil emulsion injection, therefore adopt direct dilution method to prepare need testing solution.

The selection of 3.2 dilute solutions

This product is oil emulsion injection, and common solvent is difficult to sample is dissolved completely, through comparing, adopts methyl alcohol _:methylene chloride is the mixed solution of 1: 1, comparatively acetonitrile: methylene chloride be 65: 35 the dissolubility of mixed solution to sample relatively good, therefore adopt methyl alcohol: methylene chloride is that the mixed solution of 1: 1 is as diluting solvent.

The determination of 4 each chromatographic peaks and the selection of object of reference:

In Java brucea fruit oil emulsion injection composition Study part, we infer compound structure by HPLC-MS, the retention time of HPLC-ELSD chromatogram comparison reference substance and sample, and LC-APCI-MS verification sample and reference substance molion flow graph, 9 chromatographic peaks of having confirmed in sample are followed successively by Trilinoleyl glyceride, 1, 2-dilinoleic acid-3-olein, 1, 2-dilinoleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-glyceryl linoleate, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1, 2-bis-oleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-tristerin.Because the content of 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein is higher, be assay index components, simultaneously also as finger-print object of reference.

Because Java brucea fruit oil emulsion injection is the parenteral solution that refining brucea fruit oil is made through emulsification, consider the difference of instrument, personnel etc. between different experiments room, judge for the ease of result, make method reappearance better, adding refining brucea fruit oil is object of reference.

5 Method validation

Determine, on chromatographic condition, need testing solution preparation method basis, to have investigated the specificity of method, precision, reappearance and stability further above-mentioned.

5.1 specificity tests

Solvent blank: get this product blank solvent, carries out determining fingerprint pattern, and result shows that solvent blank does not affect determining fingerprint pattern.

5.2 precision test

Precision takes Java brucea fruit oil emulsion injection, need testing solution 1 part is prepared by method described in () 3, press (one) 2 chromatographic condition continuous sample introduction 6 times, record chromatogram, and by the similarity evaluation that state-promulgated pharmacopoeia can provide, its similarity is evaluated, the mean value measured with 6 times calculates for contrasting, and result shows that this law precision is better.

Java brucea fruit oil emulsion injection finger-print precision test similarity-rough set result is as shown in the table:

5.3 replica test

Precision takes Java brucea fruit oil emulsion injection 6 parts, need testing solution is prepared by method described in () 3, measure by (one) 2 chromatographic condition, record chromatogram, and by the similarity evaluation that state-promulgated pharmacopoeia can provide, its similarity is evaluated, the mean value measured with 6 times calculates for contrasting, and result shows that this law has good repeatability.

Java brucea fruit oil emulsion injection finger-print replica test similarity-rough set result is as shown in the table:

5.4 stability test

Precision measures 1 part of need testing solution of replica test, by chromatographic condition described in () 2, measure at regular intervals, record chromatogram, and by the similarity evaluation that state-promulgated pharmacopoeia can provide, its similarity is evaluated, compare with the contrast color spectrogram generated, result shows that need testing solution is stable in 48 lab scales.

Java brucea fruit oil emulsion injection finger-print stability test similarity-rough set result is as shown in the table:

The generation of 6 Java brucea fruit oil emulsion injection standard finger-prints

According to preparation method and the chromatographic condition determined of above-mentioned need testing solution, determine the finger-print of 19 batches of Java brucea fruit oil emulsion injections altogether, according to different product batch numbers, have chosen 10 batch samples wherein, generate Java brucea fruit oil emulsion injection standard finger-print.

As shown in the table for 10 batch samples generating Java brucea fruit oil emulsion injection standard finger-print:

7 Java brucea fruit oil emulsion injection finger-print sample determinations

Use the Java brucea fruit oil emulsion injection standard finger-print of above-mentioned generation to contrast, the similarity evaluation 2.0 editions adopting state-promulgated pharmacopoeia to provide, has carried out the mensuration of fingerprint similarity to 19 batch samples.

The Similarity Measure result of Java brucea fruit oil emulsion injection 19 batch sample is as shown in the table:

According to 19 batches of Java brucea fruit oil emulsion injection similarity measurement results, the sample similarity of 95% is greater than 0.998, has the sample similarity of 100% to be greater than 0.99, comprehensive and similar products comparative result, limit is fixed tentatively as must not lower than 0.95.

8.3.2.4.2 with the comparing of similar products

In order to investigate the difference between this product and similar products, we have collected common different classes of grease on market, adopt text condition to determine brucea fruit oil, olive oil, soybean oil, sesame oil, coix seed oil, eat the different classes of grease of wet goods.

Brucea fruit oil sample has nine peaks, and wherein the 2nd, 4, No. 6 peak content is comparatively large, and namely 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein account for more than 60% of total peak area; Olive oil sample collection of illustrative plates has 6 peaks, and wherein the 2nd, 4, No. 5 peak content is comparatively large, and account for more than 80% of total peak area, these three peaks may be respectively two Oleic Linoleic glyceride, olein, two oleic acid tripalmitins; Soybean oil sample collection of illustrative plates has 4 larger peaks, accounts for more than 60% of total peak area, and these five peaks may be respectively linolein, dilinoleic acid olein, dilinoleic acid tripalmitin, two Oleic Linoleic glyceride; Sesame oil sample collection of illustrative plates has 9 peaks, and wherein the 1st, 2, No. 4 peak content is comparatively large, and account for more than 60% of total peak area, these three peaks may be respectively linolein, dilinoleic acid olein, two Oleic Linoleic glyceride; The collection of illustrative plates of coix seed oil and edible oil and brucea fruit oil is also different.Result shows that different types of grease difference is comparatively large, namely the kind of triglyceride or ratio different, the specificity of this law is strong.

Accompanying drawing explanation

Fig. 1: Java brucea fruit oil emulsion injection standard finger-print prepared by the embodiment of the present invention.

Wherein, peak 1 is Trilinoleyl glyceride, and peak 2 is 1,2-dilinoleic acid-3-olein, peak 3 is 1,2-dilinoleic acid-3-tripalmitin, peak 4 is 1,2-bis-oleic acid-3-glyceryl linoleate, and peak 5 is 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, peak 6 is olein, peak 7 is 1-stearic acid-2-oleic acid-3-glyceryl linoleate, and peak 8 is 1,2-bis-oleic acid-3-tripalmitin, peak 9 is 1,2-bis-oleic acid-3-tristerin.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.

Embodiment 2: assay (triglyceride is example)

Measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).

Chromatographic condition and system suitability are with under [finger-print] item.

The preparation of reference substance solution is with object of reference solution I under [finger-print] item.

The preparation of need testing solution is with under [finger-print] item.

Determination method

Accurate absorption reference substance solution 5 μ l, 10 μ l, need testing solution 5 ~ 10 μ l respectively, injection liquid chromatography, measures its collection of illustrative plates, calculates, to obtain final product with external standard two-point method logarithmic equation.

LgA＝aLg(NC)+b

In formula:

A is peak area

N is sampling volume, μ l;

C is concentration, mg/ml;

V is sample volume, ml;

A is the slope of linear equation LgA=aLg (NC)+b;

B is the intercept of linear equation LgA=aLg (NC)+b.

Specific experiment process is as follows:

In Java brucea fruit oil emulsion injection primary standard, assay item mainly comprises two parts, Part I surveys total acid content by titrimetry, Part II uses gas chromatography determination oleic acid content, find by testing us, poor by the method collimation of gas chromatography determination oleic acid content, and oleic acid is not the characteristic chemical constituent of Java brucea fruit oil emulsion injection, therefore the method does not have specificity.By to the constituent analysis of refining brucea fruit oil, we recognize, triglyceride constituents is refining Java brucea main body of oil, therefore this experimental study sets up HPLC-ELSD method Simultaneously test Java brucea fruit oil emulsion injection, refine the method for triglyceride component content in brucea fruit oil and Java brucea medicinal material, through Method validation, show that method is feasible, and respectively to many batches of injections, intermediate and medicinal material carry out sample determination, and result shows that the content of medicinal material, intermediate and parenteral solution index components is basicly stable.

Total acid content: same to primary standard, does not make an amendment.

Olein assay: the content adopting HPLC-ELSD method Simultaneously test 9 principal component, and all carry out Method validation, because part reference substance is expensive, in order to take into account cost, only select reference substance to be easy to get, 3 compositions that content is higher list standard in, are respectively 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein.

1 instrument, reagent and reagent

1.1 instrument

Agilent 1200 series of high efficiency liquid chromatograph (Agilent company of the U.S. is equipped with the online degasser of G1322A, G1311A quaternary pump, G1329A automatic sampling instrument, G1316A column oven, Chemstation chromatographic work station); CP-225D electronic balance (Beijing Sai Duolisi company); Evaporative light dispersion dispersion (Alltech2000ES).

1.2 reagent

Methyl alcohol (land, Shanghai all chemical reagent factories, analyze pure); Methylene chloride (chemical drug reagent company limited of traditional Chinese medicines group, analyzes pure); Acetonitrile (MERCK company, chromatographically pure): methylene chloride (TEDIA company of the U.S., chromatographically pure).

1.3 reagent

Reference substance Trilinoleyl glyceride, 1,2-dilinoleic acid-3 tripalmitin, 1,2-bis-oleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-tristerin, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3 glyceryl linoleate is purchased from LarodanAB company, and olein, 1-palmitic acid-2 oleic acid-3-glyceryl linoleate are purchased from SIGMA company.

Java brucea fruit oil emulsion injection 18 batches (Zhejiang Province Jiuxu Pharmaceutical Co., Ltd provides), lot number refers to following table.

The determination of 2 experimental programs

The selection of 2.1 mobile phases, chromatographic column, flow velocity, column temperature

With reference to Java brucea fruit oil emulsion injection fingerprint spectrum method, under this condition, each composition all can be better separated.

2.2 test sample preparation methods select

Select using methyl alcohol: methylene chloride be the mixed solution of 1: 1 as diluting solvent, direct dilute sample, with this understanding, the need testing solution comparison of ingredients of gained is stablized.

In sum, determine that Java brucea fruit oil emulsion injection content assaying method is:

Chromatographic condition

Stationary liquid: Agilent ExtendC 18 chromatographic column (250 × 4.6mm, 5um)

Mobile phase: with acetonitrile: methylene chloride be 63: 35 mixed solution be mobile phase wash-out

Flow rate of mobile phase: 0.5ml/min

Column temperature: 35 DEG C

Sampling volume: 5ul, 10ul, 15ul

Evaporative light dispersion dispersion: drift tube temperature 95 DEG C

Nitrogen flow rate 2.5L/min

The preparation of reference substance solution

It is appropriate that precision takes reference substance, adds methyl alcohol: methylene chloride be 1: 1 mixed solution make every 1ml respectively containing the solution of 0.06mg, 0.20mg, 0.05mg, 0.05mg, 0.07mg, 0.66mg, 0.08mg, 0.23mg, 0.22mg.

The preparation of need testing solution

Precision takes Java brucea fruit oil emulsion injection 0.3g, puts in 10ml volumetric flask, adds methyl alcohol: methylene chloride be 1: 1 mixed solution be diluted to scale, shake up, to obtain final product.

3 Method validation

3.1 specificity tests

The interference of solvent and auxiliary material; This product is the aseptic injection emulsion that refining brucea fruit oil is made, and specificity tests paper examines reagent blank.Get methyl alcohol: methylene chloride be the mixed solution of 1: 1 as blank solvent, measure by fixed chromatographic condition; Show that solvent measures mutually noiseless to sample size.

3.2 linear relationships are investigated

Precision measures the mixed solution 2 of nine reference substances, 5, 10, 15, 20ul injection liquid chromatography, measures peak area, with the logarithm of sample size (ug) (X) for horizontal ordinate, the logarithm (Y) of peak area is ordinate, does typical curve, and with the regression equation that least square method calculates, result shows, Trilinoleyl glyceride, 1.2-dilinoleic acid-3-olein, 1,2-dilinoleic acid-3-tripalmitin, 1,2-bis-oleic acid-3 glyceryl linoleate, 1-palmitic acid-2 oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1,2-bis-oleic acid-3-tripalmitin, 1,2-bis-oleic acid--3-tristerin is 0.1118 ~ 1.1178ug at sample size respectively, 0.4056 ~ 4.0560ug, 0.1078 ~ 1.0784ug, 1.0001 ~ 10.0008ug, 0.1367 ~ 1.3665ug, 1.3276 ~ 13.2762ug, 0.1650 ~ 1.6503ug, 0.4664 ~ 4.6640ug, in good linear relationship within the scope of 0.4394 ~ 4.3936ug.

9 component linear relations are investigated and be the results are shown in following table

3.3 precision test

Get Java brucea fruit oil emulsion injection sample, need testing solution portion is prepared according to the preparation method of above-mentioned need testing solution, repeat sample introduction 6 times, measure peak area, calculate content, result Trilinoleyl glyceride RSD is 0.8% (n=6), 1, 2-linoleic acid-3-olein RSD is 0.5% (n=6), 1, 2-linoleic acid-3-tripalmitin RSD is 2.5% (n=6), 1, 2-oleic acid-3-glyceryl linoleate RSD is 0.3% (n=6), 1-palmitic acid-2-oleic acid-3 glyceryl linoleate RSD is 0.8% (n=6), olein RSD is 0.2% (n=6), 1-stearic acid-2-oleic acid-3-glyceryl linoleate RSD is 1.0% (n=6), 1, 2-oleic acid-3-tripalmitin RSD is 0.7% (n=6), 1, 2-oleic acid-3 tristerin RSD is 0.4% (n=6), see the following form:

3.4 replica test

Precision takes 6 parts, Java brucea fruit oil emulsion injection sample, and above-mentioned experimental program prepares need testing solution, measures the content that peak area also calculates 9 reference substances respectively.Result olein average content is 1.7482mg/ml, RSD is 1.9% (n=6), 1, 2-dilinoleic acid-3-olein average content is 6.6965mg/ml, RSD is 1.1% (n=6), 1, 2-dilinoleic acid-3-tripalmitin average content is 1.6894mg/ml, RSD is 3.5% (n=6), 1, 2-bis-oleic acid-3-glyceryl linoleate average content is 23.3456mg/ml, RSD is 0.6% (n=6), 1-palmitic acid-2-oleic acid-3 glyceryl linoleate average content is 2.6846mg/ml, RSD is 1.2% (n=6), olein average content is 21.9433mg/ml, RSD is 0.7% (n=6), 1-stearic acid-2-oleic acid-3-glyceryl linoleate average content is 2.5540mg/ml, RSD is 0.8% (n=6), 1, 2-bis-oleic acid-3-tripalmitin average content is 7.2624mg/ml, RSD is 0.8% (n=6), 1, 2-bis-oleic acid-3 tristerin average content is 5.3703mg/ml, RSD is 0.7% (n=6), the results are shown in following table.：

3.5 accuracy testings (recovery test)

Adopt average recovery determination method, precision measures 6 parts, the Java brucea fruit oil emulsion injection sample of known content, and precision adds the mixed solution of 9 reference substances in right amount respectively, and according to test sample, preparation method prepares need testing solution, measure peak area, calculate average recovery rate and RSD%.Result display Trilinoleyl glyceride average recovery rate is 101.7%, RSD is 1.6%; 1,2-linoleic acid-3-olein average recovery rate is 101.3%, RSD is 1.8%; 1,2-linoleic acid-3-tripalmitin average recovery rate is 101.7%, RSD is 2.1%; 1,2-oleic acid-3-glyceryl linoleate average recovery rate is 101.2%, RSD is 2.5%; 1-palmitic acid-2-oleic acid-3 glyceryl linoleate average recovery rate is 102.0%, RSD is 1.5%; Olein average recovery rate is 100.8%, RSD is 1.9%; 1-stearic acid-2-oleic acid-3-glyceryl linoleate is 98.1%, RSD is 1.1%; 1,2-oleic acid-3-tripalmitin average recovery rate is 99.2%, RSD be 1.4%, 1,2-oleic acid-3-tristerin average recovery rate be 100.5%, RSD is 1.2%, the results are shown in following table:

3.6 stability test

Get Java brucea fruit oil emulsion injection need testing solution, respectively the injection liquid chromatography when 0,1,6,12,24,48h, record peak area, calculates content.Result shows, in need testing solution, each tested composition is good at 48h internal stability, Trilinoleyl glyceride, 1, 2-dilinoleic acid-3-olein, 1, 2-dilinoleic acid-3-palmitic acid grease, 1, 2-bis-oleic acid-3-glyceryl linoleate, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1, 2-bis-oleic acid-3-tripalmitin, 1, 2-bis-oleic acid-3-tristerin RSD is respectively 2.3%, 1.6%, 3.5%, 1.2%, 1.3%, 1.0%, 1.8%, 0.9%, 1.2%.

The stability test of Java brucea fruit oil emulsion injection need testing solution the results are shown in following table:

3.7 sample determination

As stated above, 18 batches of Java brucea fruit oil emulsion injections, 9 component contents are determined altogether.Trilinoleyl glyceride, 1 in each batch sample, 2-dilinoleic acid-3-olein, 1,2-dilinoleic acid-3-tripalmitin, 1,2-bis-oleic acid-3-glyceryl linoleate, 1-palmitic acid-2-oleic acid-3-glyceryl linoleate, olein, 1-stearic acid-2-oleic acid-3-glyceryl linoleate, 1, the content of 2-bis-oleic acid-3-tripalmitin and 1,2-, bis-oleic acid-tristerin is followed successively by:

20110801 batches, 3.3752mg, 8.5696mg, 2.5020mg, 22.4312mg, 3.2758mg, 18.9933mg, 2.9271mg, 7.9161mg, 5.5264mg;

20110802 batches, 3.2873mg, 8.3813mg, 2.4504mg, 22.0879mg, 3.1955mg, 18.6328mg, 2.8691mg, 7.7766mg, 5.4251mg.

20110803 batches, 3.3708mg, 8.5898mg, 2.5138mg, 22.3992mg, 3.2794mg, 18.9769mg, 2.9347mg, 7.9291mg, 5.5705mg.

20110306 batches, 2.3147mg, 8.7503mg, 2.1430mg, 30.2930mg, 3.7163mg, 28.2713mg, 3.7060mg, 11.1162mg, 9.1349mg.

20101203 batches, 2.0949mg, 7.2754mg, 1.6829mg, 25.0583mg, 2.8818mg, 23.5274mg, 2.4831mg, 8.7055mg, 5.7097mg.

20101202 batches, 2.0127mg, 7.0032mg, 1.6315mg, 24.2829mg, 2.7893mg, 22.7570mg, 2.3942mg, 8.4020mg, 5.5407mg.

20100912 batches, 2.9238mg, 8.2716mg, 2.0384mg, 23.7986mg, 2.9531mg, 21.1827mg, 2.7264mg, 7.9912mg, 5.6462mg.

20100913 batches, 2.9399mg, 8.3134mg, 2.0574mg, 23.8408mg, 2.9469mg, 21.2417mg, 2.7259mg, 8.0128mg, 5.6631mg.

20100918 batches, 2.9139mg, 8.1606mg, 2.0817mg, 23.4005mg, 2.9761mg, 20.8481mg, 2.6170mg, 8.0574mg, 5.4410mg.

20100920 batches, 2.8660mg, 7.8690mg, 1.9841mg, 23.2648mg, 2.8873mg, 20.5537mg, 2.5263mg, 7.9150mg, 5.2518mg.

20101004 batches, 1.7900mg, 7.0017mg, 1.6094mg, 25.7780mg, 2.9935mg, 22.9486mg, 2.1288mg, 9.1424mg, 5.0405mg.

20101010 batches, 1.7421mg, 6.7386mg, 1.6118mg, 24.9701mg, 2.9577mg, 22.5666mg, 2.0289mg, 9.1039mg, 4.8675mg.

20101012 batches, 1.7444mg, 6.7121mg, 1.5846mg, 24.9750mg, 2.9569mg, 22.6150mg, 2.0127mg, 9.0953mg, 4.8846mg.

20101204 batches, 1.9260mg, 6.7777mg, 1.5434mg, 23.6461mg, 2.6777mg, 22.1436mg, 2.2996mg, 8.1749mg, 5.2695mg.

20101206 batches, 1.9404mg, 6.6952mg, 1.5646mg, 22.9802mg, 2.6801mg, 21.0120mg, 2.2881mg, 7.9211mg, 5.2405mg.

20101210 batches, 2.0001mg, 6.8798mg, 1.6012mg, 3.4438mg, 2.7406mg, 21.3591mg, 2.3482mg, 8.1071mg, 5.3723mg.

20110901 batches, 2.6437mg, 7.3353mg, 2.0978mg, 21.6334mg, 3.0870mg, 20.4844mg, 2.5877mg, 8.5867mg, 5.5189mg.

20110902 batches, 2.6528mg, 7.3483mg, 2.1133mg, 21.6554mg, 3.1016mg, 20.5104mg, 2.5993mg, 8.6063mg, 5.5300mg.

Consider inspection cost, can consider only to list content in higher by 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein are index components, limit according to 0.6 conversion of average result is: " the every 1g of this product is containing refining brucea fruit oil with 1; 2-dilinoleic acid-3-olein meter; must not be less than 4.6mg; with 1; 2-bis-oleic acid-3-glyceryl linoleate meter; must not be less than 14.3mg, in olein (C57H104O6), must not be less than 13.0mg ".

Embodiment 3: the Java brucea fruit oil emulsion injection method of quality control of recommendation

The information of integrated embodiment 1 and embodiment 2, those skilled in the art can draw Java brucea fruit oil emulsion injection method of quality control, can be such as the methods that the present embodiment is recommended.

[finger-print]

Measure with reference to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).

Chromatographic condition and system suitability

Take octadecylsilane chemically bonded silica as filling agent; Be mobile phase with the mixed solution of acetonitrile/methylene chloride (acetonitrile: methylene chloride for 60-70: 40-30, preferably 65: 35); Flow velocity is 0.3-0.8ml per minute, preferred 0.5ml; Column temperature 30-40 DEG C, preferably 35 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute, preferably 65 minutes.Number of theoretical plate calculates should be not less than 5000 by olein peak, and No. 2 peaks corresponding with standard finger-print in test sample and the degree of separation at No. 3 peaks should be more than 1.5.

The preparation of object of reference solution

Get 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein reference substance appropriate, accurately weighed, add methyl alcohol: methylene chloride be 1: 1 solution make every 1ml containing 1, the solution of 2-dilinoleic acid-3-olein 0.20mg, 1,2-bis-oleic acid-3-glyceryl linoleate 0.50mg and olein 0.66mg, as object of reference solution I.Get brucea fruit oil reference extract more appropriate, accurately weighed, add methanol dichloromethane (1: 1) solution and make the solution of every 1ml containing 1mg, as object of reference solution II.

The preparation of need testing solution

Get this product and be about 0.3g, accurately weighed, put in 10ml measuring bottle, add methyl alcohol: methylene chloride be the solution of 1: 1 to scale, shake up, to obtain final product.

Assay method

Accurate absorption object of reference solution I, II and each 5 μ l of need testing solution respectively, injection liquid chromatography, measure its collection of illustrative plates, after deduction solvent peak, the similarity evaluation that can provide by state-promulgated pharmacopoeia calculates, and the similarity of test sample finger-print and standard finger-print must not lower than 0.95.

Reference color spectral condition:

Agilent 1100 series of high efficiency liquid chromatograph (Agilent Chemstation workstation)

Chromatographic column: Agilent Zorbax Extend C18 (250mm × 4.6mm, 5 μm)

Detecting device: Alltech 2000 type evaporative light-scattering detector

Detector parameters: drift tube temperature: 95 DEG C; Flow rate of carrier gas: 2.5L per minute

[assay] triglyceride

Measure with reference to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).

Chromatographic condition and system suitability are with under [finger-print] item.

The preparation of reference substance solution is with object of reference solution I under [finger-print] item.

The preparation of need testing solution is with under [finger-print] item.

Determination method

Accurate absorption reference substance solution 5 μ l, 10 μ l, need testing solution 5 ~ 10 μ l respectively, injection liquid chromatography, measures its collection of illustrative plates, calculates, to obtain final product with external standard two-point method logarithmic equation.

LgA＝aLg(NC)+b

In formula:

A is peak area

N is sampling volume, μ l;

C is concentration, mg/ml;

V is sample volume, ml;

A is the slope of linear equation LgA=aLg (NC)+b;

B is the intercept of linear equation LgA=aLg (NC)+b.

The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.

Claims (10)

1. Java brucea fruit oil emulsion injection method of quality control, is characterized in that, described method comprises:

With the methanol/dichloromethane solution containing 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein as object of reference solution I;

Using the methanol/dichloromethane solution containing brucea fruit oil as object of reference solution II;

Using the methanol/dichloromethane solution containing Java brucea fruit oil emulsion injection as need testing solution;

Measure the chromatograms of object of reference solution I, object of reference solution II and need testing solution respectively.

2. Java brucea fruit oil emulsion injection method of quality control, is characterized in that, described method comprises:

Get every 1ml containing 1,2-dilinoleic acid-3-olein 0.20mg, 1,2-bis-oleic acid-3-glyceryl linoleate 0.50mg and olein 0.66mg, methyl alcohol: methylene chloride is the solution of 1: 1, as object of reference solution I;

Get every 1ml containing 1mg brucea fruit oil, methyl alcohol: methylene chloride is that the solution of 1: 1 is as object of reference solution II;

Get containing Java brucea fruit oil emulsion injection, methyl alcohol: methylene chloride is that the solution of 1: 1 is as need testing solution;

Accurate absorption object of reference solution I, object of reference solution II and each 5 μ l of need testing solution respectively, injection liquid chromatography, measures its collection of illustrative plates.

3. Java brucea fruit oil emulsion injection method of quality control described in claim 2, is characterized in that, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 60-70: 40-30 is mobile phase; Flow velocity is 0.3-0.8ml per minute; Column temperature 30-40 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute.

4. Java brucea fruit oil emulsion injection method of quality control described in claim 3, is characterized in that, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 65: 35 is mobile phase; Flow velocity is 0.5ml per minute; Column temperature 35 DEG C; Evaporative light-scattering detector; Analysis time is preferably 65 minutes.

5. Java brucea fruit oil emulsion injection method of quality control described in any one of claim 1-4, is characterized in that, described method also comprises the step with standard diagram comparison.

6. Java brucea fruit oil emulsion injection method of quality control described in claim 5, is characterized in that, described standard diagram measures by the following method:

Choose some batches of Java brucea fruit oil emulsion injections, measure its liquid chromatography according to method described in any one of claim 1-4, by chromatographic fingerprinting similarity evaluation system, generate Java brucea fruit oil emulsion injection standard finger-print.

7. Java brucea fruit oil emulsion injection method of quality control described in any one of claim 1-4, is characterized in that, described method also comprises the step that component content measures.

8. Java brucea fruit oil emulsion injection method of quality control described in claim 7, is characterized in that, described component content measures and refers to,

With the methanol/dichloromethane solution containing 1,2-dilinoleic acid-3-olein, 1,2-bis-oleic acid-3-glyceryl linoleate and olein as object of reference solution I;

Using the methanol/dichloromethane solution containing Java brucea fruit oil emulsion injection as need testing solution;

Measure the chromatograms of V1 volume object of reference solution I, V2 volume object of reference solution I and V3 volume need testing solution respectively, wherein, V1 < V3 < V2;

With following formulae discovery component content:

In formula:

A is peak area;

N is sampling volume, unit μ l;

C is concentration, unit mg/ml;

V is sample volume, units/ml;

A is the slope of linear equation LgA=aLg (NC)+b;

B is the intercept of linear equation LgA=aLg (NC)+b.

9. Java brucea fruit oil emulsion injection method of quality control described in claim 8, is characterized in that, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 60-70: 40-30 is mobile phase; Flow velocity is 0.3-0.8ml per minute; Column temperature 30-40 DEG C; Evaporative light-scattering detector; Analysis time is 40-130 minute.

10. Java brucea fruit oil emulsion injection method of quality control described in claim 9, is characterized in that, chromatographic condition is:

Take octadecylsilane chemically bonded silica as filling agent; With acetonitrile: methylene chloride is the mixed solution of the acetonitrile/methylene chloride of 65: 35 is mobile phase; Flow velocity is 0.5ml per minute; Column temperature 35 DEG C; Evaporative light-scattering detector; Analysis time is preferably 65 minutes.