Method for simultaneous determination of nine components in safflower medicinal composition

Abstract

The present invention provides a method for simultaneously determining nine components in a safflower pharmaceutical composition, belonging to the field of drug detection technology. The method comprises preparing a reference solution I by taking uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin, and p-hydroxysafflower yellow A; preparing a reference solution II by taking p-hydroxycinnamic acid and kaempferol 3-O-rutinoside; preparing a test solution by taking the safflower pharmaceutical composition; and performing high-performance liquid chromatography on the reference solution I, the reference solution II, and the test solution, respectively. The contents of the nine components in the safflower pharmaceutical composition are then calculated using an external standard method. The present invention utilizes a method for determining the contents of nine chemical components in a safflower pharmaceutical composition by high-performance liquid chromatography at one time, thereby achieving control of multiple index components in the safflower pharmaceutical composition.

Description

Technical Field

[0001] The present invention relates to drug detection technology, in particular to a method for simultaneously determining nine components in a safflower medicinal composition. Background Art

[0002] Safflower injection is a sterile aqueous solution extracted and refined from safflower medicinal materials. It has the effect of promoting blood circulation and removing blood stasis, and is used to treat occlusive cardiovascular and cerebrovascular diseases, coronary heart disease, and vasculitis.

[0003] The quality standard for safflower injection is the national drug standard WS3-B-3825-98-2012, and currently only the content of one chemical component, hydroxysafflower yellow A, is measured. Literature review reveals that currently, methods for determining the active ingredients in safflower injection only measure the content of kaempferol or quercetin compounds individually, or simultaneously measure the content of uridine, guanosine, and adenosine. There are no methods for simultaneously measuring the content of more than three components. Therefore, to achieve simple, rapid, and multi-index quality control of safflower injection, the present invention uses high-performance liquid chromatography (HPLC) with gradient elution to simultaneously determine the content of nine compounds in safflower injection, achieving excellent separation and accuracy. Summary of the Invention

[0004] In view of the above problems, the present invention provides a method for simultaneously determining nine components in a safflower medicinal composition.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A method for simultaneously determining nine components in a safflower medicinal composition, comprising the following steps:

[0007] Prepare a reference solution I by taking seven ingredients, namely, uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin and p-hydroxysafflor yellow A;

[0008] Prepare reference solution II by taking two components, p-hydroxycinnamic acid and kaempferol 3-O-rutinoside;

[0009] Prepare a test solution by taking the safflower medicinal composition;

[0010] The reference solution I, the reference solution II and the test solution were respectively subjected to high performance liquid chromatography detection, and then the external standard method was used to calculate the contents of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin and hydroxysafflower yellow in the safflower medicinal composition;

[0011] Among them, the detection wavelength for uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside and 5-hydroxymethylfurfural is 252-256nm;

[0012] The detection wavelength for p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, and syringin was 262–270 nm;

[0013] The detection wavelength of hydroxysafflor yellow A is 408-412 nm.

[0014] Furthermore, the elution mode of the high performance liquid chromatography detection is gradient elution;

[0015] The mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is a 0.1-0.2 wt% glacial acetic acid aqueous solution;

[0016] The gradient elution conditions are:

[0017] 0-15 min, 2% → 5% mobile phase A, 98% → 95% mobile phase B;

[0018] 15-30 min, 5%→25% mobile phase A, 95%→75% mobile phase B.

[0019] Furthermore, the column temperature of the high performance liquid chromatography detection is 25-40°C, the flow rate is 0.8-1.2 mL / min, and the chromatographic column is filled with octadecylsilane bonded silica gel, preferably phenomenex Gemini-C18, 4.6×250 mm 5 μm; the injection volumes of the reference solution I, the reference solution II, and the test solution are all 10-30 μL.

[0020] Furthermore, the reference solution I contains uridine 10-80 μg / mL, guanosine 10-60 μg / mL, adenosine 10-70 μg / mL, p-hydroxybenzoic acid 10-70 μg / mL, 5-hydroxymethylfurfural 10-70 μg / mL, syringin 1-70 μg / mL, and p-hydroxysafflor yellow A 1-65 μg / mL;

[0021] The reference solution II contains 8 to 50 μg / mL of p-hydroxycinnamic acid and 10 to 80 μg / mL of kaempferol 3-O-rutinoside.

[0022] Furthermore, the solvent of the reference solution I is water;

[0023] The solvent of reference solution II is 48-52 vol% acetonitrile in water;

[0024] The test solution is prepared by diluting the safflower medicinal composition with water by 2 to 10 times its volume;

[0025] The theoretical plate numbers of the nine components in the high performance liquid chromatography detection process are all above 10,000.

[0026] A method for simultaneously determining nine components in a safflower medicinal composition, comprising the following steps:

[0027] A total of seven components, namely, uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin, and p-hydroxysafflor yellow A, were used to prepare a series of reference solutions I with different concentrations.

[0028] Prepare reference solution series II with different concentrations of p-hydroxycinnamic acid and kaempferol 3-O-rutinoside.

[0029] Different concentrations of reference substance series solution I and reference substance series solution II were respectively taken for high performance liquid chromatography detection, and standard curves corresponding to different chemical components were drawn according to the concentrations of different chemical components and the peak areas measured corresponding to the concentrations of the corresponding chemical components;

[0030] Prepare a test solution by taking the safflower medicinal composition;

[0031] The test solution is subjected to high performance liquid chromatography detection, and the peak area of the corresponding chemical component obtained is substituted into the corresponding standard curve, and the content of the corresponding chemical component in the safflower medicinal composition is obtained by calculation;

[0032] Among them, the detection wavelength for uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside and 5-hydroxymethylfurfural is 252-256nm;

[0033] The detection wavelength for p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, and syringin was 262–270 nm;

[0034] The detection wavelength of hydroxysafflor yellow A is 408-412 nm.

[0035] Furthermore, the elution mode of the high performance liquid chromatography detection is gradient elution;

[0036] The mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is a 0.1-0.2 wt% glacial acetic acid aqueous solution;

[0037] The gradient elution conditions are:

[0038] 0-15 min, 2% → 5% mobile phase A, 98% → 95% mobile phase B;

[0039] 15-30 min, 5%→25% mobile phase A, 95%→75% mobile phase B.

[0040] Furthermore, the column temperature of the high performance liquid chromatography detection is 25-40°C, the flow rate is 0.8-1.2 mL / min, the chromatographic column is filled with octadecylsilane bonded silica gel, preferably phenomenex Gemini-C18, 4.6×250 mm 5 μm; the injection volume of the reference solution I, the reference solution II and the test solution are all 10-30 μL; the detector is a diode array detector.

[0041] Furthermore, in the different reference substance series solutions I, the concentration of uridine was between 10.01 and 60.05 μg / mL, the concentration of guanosine was between 8.99 and 53.93 μg / mL, the concentration of adenosine was between 9.44 and 56.61 μg / mL, the concentration of p-hydroxybenzoic acid glycoside was between 9.82 and 58.91 μg / mL, the concentration of 5-hydroxymethylfurfural was between 9.60 and 57.62 μg / mL, the concentration of syringin was between 11.80 and 70.82 μg / mL, and the concentration of p-hydroxysafflor yellow A was between 1 and 65 μg / mL;

[0042] The concentrations of p-hydroxycinnamic acid in the different reference substance series solutions II ranged from 9.23 to 55.38 μg / mL, and the concentrations of kaempferol 3-O-rutinoside ranged from 12.22 to 73.33 μg / mL;

[0043] The solvent of reference solution series I is water;

[0044] The solvent of reference solution series II is 48-52 vol% acetonitrile in water;

[0045] The test solution is prepared by diluting the safflower medicinal composition with water by 2 to 10 times its volume;

[0046] The theoretical plate numbers of the nine components in the high performance liquid chromatography detection process are all above 10,000.

[0047] Furthermore, the standard curves corresponding to the different chemical components are as follows:

[0048] The standard curve corresponding to uridine is y=46.13562x+1.73333, R 2 =0.99992;

[0049] The standard curve corresponding to guanosine is y=42.86719x-0.26667, R 2 =0.99995;

[0050] The standard curve corresponding to adenosine is y=50.50161x+4.46667, R 2 =0.99997;

[0051] The standard curve corresponding to p-hydroxybenzoic acid glycosides is 43.57603x-2.06667, R 2 =0.99994;

[0052] The standard curve corresponding to 5-hydroxymethylfurfural is y=43.04129x-3.60000, R 2 =0.99994;

[0053] The standard curve corresponding to p-hydroxycinnamic acid is y=105.70592x+19.66667, R 2 =0.99993;

[0054] The standard curve corresponding to kaempferol 3-O-rutinoside is y=15.47304x-0.06667, R 2 =0.99992;

[0055] The standard curve corresponding to syringin is y=40.00366x+1.46667, R 2 =0.99992;

[0056] The standard curve corresponding to hydroxysafflor yellow A is y=40.31518x-18.93333, R 2 =0.99946.

[0057] The beneficial effects of the method for simultaneously determining nine components in the safflower medicinal composition of the present invention are:

[0058] The present invention utilizes a method for determining the contents of nine chemical components, including uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin, and hydroxysafflower yellow A, in a safflower medicinal composition by high performance liquid chromatography at one time, thereby achieving control of multiple index components in the safflower medicinal composition.

[0059] The present invention determines suitable detection wavelengths for different components by ultraviolet scanning of the ultraviolet absorption of the above-mentioned nine components. Among them, the detection wavelengths of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside and 5-hydroxymethylfurfural are 252-256 nm; the detection wavelengths of p-hydroxycinnamic acid, kaempferol 3-O-rutinoside and syringin are 262-270 nm; and the detection wavelength of hydroxysafflor yellow A is 408-412 nm.

[0060] The present invention can simultaneously determine the contents of nine components in a safflower medicinal composition by adjusting process parameters during the elution process, thereby achieving a good separation effect without mutual interference, and providing accurate measurement results, thereby achieving the purpose of multi-index quality control of the safflower medicinal composition. The operation is simple, quick, and easy to master.

[0061] The method of the present invention has strong specificity, high accuracy, high precision, and good linearity;

[0062] The present invention completes the content determination of 9 components through only one determination, and can quickly and conveniently obtain the quality of the safflower medicinal composition. It solves the problem that the detection method in the prior art can only determine the content of 3 or less components and cannot comprehensively control the quality of the safflower medicinal composition, thereby achieving the purpose of controlling the quality of the safflower medicinal composition by determining the content of 9 chemical components. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 is a chromatogram of the reference solution I in Example 1 of the present invention at a wavelength of 254 nm;

[0064] Figure 2 is a chromatogram of the reference solution II in Example 1 of the present invention at a wavelength of 266 nm;

[0065] Figure 3 is a chromatogram of the reference solution I in Example 1 of the present invention at a wavelength of 410 nm;

[0066] Figure 4 is a chromatogram of the reference solution I in Example 1 of the present invention at a wavelength of 266 nm;

[0067] Figure 5 is a chromatogram of the test solution at a wavelength of 254 nm in Example 1 of the present invention;

[0068] Figure 6 is a chromatogram of the test solution at a wavelength of 266 nm in Example 1 of the present invention;

[0069] Figure 7 is a chromatogram of the test solution at a wavelength of 410 nm in Example 1 of the present invention;

[0070] Figure 8 is a standard curve of uridine at a wavelength of 254 nm in Example 6 of the present invention;

[0071] Figure 9 This is a standard curve of guanosine at a wavelength of 254 nm in Example 6 of the present invention;

[0072] Figure 10This is a standard curve of adenine nucleoside at a wavelength of 254 nm in Example 6 of the present invention;

[0073] Figure 11 This is a standard curve of p-hydroxybenzoic acid glycosides at a wavelength of 254 nm in Example 6 of the present invention;

[0074] Figure 12 This is a standard curve of 5-hydroxymethylfurfural at a wavelength of 254 nm in Example 6 of the present invention;

[0075] Figure 13 This is a standard curve of p-hydroxycinnamic acid at a wavelength of 266 nm in Example 6 of the present invention;

[0076] Figure 14 This is a standard curve of kaempferol 3-O-rutinoside at a wavelength of 266 nm in Example 6 of the present invention;

[0077] Figure 15 This is a standard curve of syringin at a wavelength of 266 nm in Example 6 of the present invention;

[0078] Figure 16 This is a standard curve of hydroxysafflor yellow A at a wavelength of 410 nm in Example 6 of the present invention. DETAILED DESCRIPTION

[0079] The technical solutions in the embodiments of the present invention are described clearly and completely below. Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0080] Example 1 A method for simultaneously determining nine components in a safflower medicinal composition

[0081] This example is a method for simultaneously determining nine components in a safflower medicinal composition, using safflower injection produced by Shenwei Pharmaceutical Group Co., Ltd. for determination, specifically comprising the following steps:

[0082] 1) Preparation of test solution and reference solution

[0083] 11) Preparation of test solution

[0084] Accurately measure safflower injection (batch number: HH001), add 5 times the volume of water to dilute (i.e. N = 5), shake well to obtain the test solution.

[0085] 12) Preparation of reference solution

[0086] Weigh uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin and p-hydroxysafflower yellow A respectively to prepare reference solution I with a concentration of C. 对照品 Among them, the content of uridine is 40μg / mL, the content of guanosine is 30μg / mL, the content of adenine is 35μg / mL, the content of p-hydroxybenzoic acid glycoside is 35μg / mL, the content of 5-hydroxymethylfurfural is 30μg / mL, the content of syringin is 35μg / mL, the content of p-hydroxysafflor yellow A is 30μg / mL (i.e., C 对照品-尿嘧啶核苷 =40μg / mL, C 对照品-鸟嘌呤 =30μg / mL, C 对照品-腺嘌呤核苷 =35μg / mL, C 对照品-对羟基苯甲酸苷 =35μg / mL, C 对照品-5-羟甲基糖醛 =30μg / mL, C 对照品-紫丁香苷 =35μg / mL, C 对照品-对羟基红花黄色素A =30 μg / mL);

[0087] Prepare reference solution II with a concentration of C using p-hydroxycinnamic acid and kaempferol 3-O-rutinoside. 对照品 , wherein the content of p-hydroxycinnamic acid is 25 μg / mL and the content of kaempferol 3-O-rutinoside is 40 μg / mL (i.e. C 对照品-对羟基肉桂酸 =25μg / mL, C 对照品-山萘酚3-O-芸香糖苷 =40 μg / mL).

[0088] 2) High performance liquid chromatography detection

[0089] 21) Take reference solution I and reference solution II and perform high performance liquid chromatography to obtain the peak area A corresponding to the concentration of different components. 对照品 , that is, A 对照品-尿嘧啶核苷 、A 对照品-鸟嘌呤 、A 对照品-腺嘌呤核苷 、A 对照品-对羟基苯甲酸苷 、A 对照品-5-羟甲基糖醛 、A 对照品-紫丁香苷 、A 对照品-对羟基红花黄色素A 、A 对照品-对羟基肉桂酸 、A 对照品-山萘酚3-O-芸香糖苷 .

[0090] The HPLC conditions for detecting reference solution I and reference solution II are as follows:

[0091] Chromatographic column: phenomenex Gemini-C18, 4.6 × 250 mm, 5 μm;

[0092] Mobile phase: acetonitrile (A)-0.2 wt% glacial acetic acid aqueous solution (B);

[0093] Elution program: 0-15 min, 2%→5% mobile phase A, 98%→95% mobile phase B;

[0094] 15-30 min, 5% → 25% mobile phase A, 95% → 75% mobile phase B;

[0095] Flow rate: 0.8 mL / min;

[0096] Column temperature: 30°C;

[0097] Detection wavelength: 254nm, 266nm, 410nm;

[0098] In the case of a single injection, different detection wavelengths were used for detection, specifically: 254 nm (detection of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural), 266 nm (detection of p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin), 410 nm (detection of hydroxysafflor yellow A);

[0099] Injection volume: The injection volume of reference solution I and reference solution II is 10 μL, that is, I 对照品 =10 μL;

[0100] Instrument: Agilent high performance liquid chromatograph, diode array detector, quaternary gradient pump.

[0101] The theoretical plate number calculated based on the chromatographic peaks of each component should not be less than 10,000.

[0102] Wherein, the chromatogram of reference substance solution I at 254nm wavelength is shown in Figure 1 In the figure, a is uridine, b is guanosine, c is adenosine, d is p-hydroxybenzoic acid glycoside, and e is 5-hydroxymethylfurfural;

[0103] The chromatogram of reference solution II at 266 nm is shown in Figure 2 , in the figure, f is p-hydroxycinnamic acid, g is kaempferol-3-O-rutinoside;

[0104] The chromatogram of reference solution I at 410 nm wavelength is shown in Figure 3 , h in the figure is hydroxysafflor yellow A;

[0105] The chromatogram of reference solution I at 266 nm wavelength is shown in Figure 4 , i in the figure is syringin.

[0106] 22) Take the test solution and perform HPLC under the above chromatographic conditions, except that the injection volume of the test solution is 30 μL (i.e., 1 供试品 =30 μL), and the peak area A of the corresponding chemical components contained in the test solution was obtained.供试品 ; i.e. A 供试品-尿嘧啶核苷 、A 供试品-鸟嘌呤 、A 供试品-腺嘌呤核苷 、A 供试品-对羟基苯甲酸苷 、A 供试品-5-羟甲基糖醛 、A 供试品-紫丁香苷 、A 供试品-对羟基红花黄色素A 、A 对照品-对羟基肉桂酸 、A 对照品-山萘酚3-O-芸香糖苷 .

[0107] Among them, the chromatogram of the test solution at a wavelength of 254nm is shown in Figure 5 In the figure, a is uridine, b is guanosine, c is adenosine, d is p-hydroxybenzoic acid glycoside, and e is 5-hydroxymethylfurfural;

[0108] The chromatogram of the test solution at a wavelength of 266 nm is shown in Figure 7 In the figure, f is p-hydroxycinnamic acid, g is kaempferol-3-O-rutinoside, and i is syringin;

[0109] The chromatogram of the test solution at a wavelength of 410 nm is shown in Figure 8 , h in the figure is hydroxysafflor yellow A.

[0110] Then according to the formula, the content of the corresponding component in safflower injection C = (A 供试品 ×I 对照品 ×C 对照品 ×N) / (A 对照品 ×I 供试品 ), calculate the content of the corresponding components in safflower injection.

[0111] A 供试品 ——Peak area of the corresponding component in the test solution;

[0112] A 对照品 ——Peak area of the corresponding component in reference solution I or reference solution II;

[0113] I 供试品 ——Injection volume of test solution, μL;

[0114] I 对照品 ——Injection volume of reference solution I or reference solution II, μL;

[0115] C 对照品 ——Concentration of reference solution, μg / mL;

[0116] N——Dilution multiple of safflower injection when preparing the test solution.

[0117] Take uridine as an example:

[0118] The content of uridine in safflower injection 尿嘧啶核苷 =(A供试品-尿嘧啶核苷 ×I 对照品 ×C 对照品-尿嘧啶核苷 ×N) / (A 对照品-尿嘧啶核苷 ×I 供试品 ).

[0119] The same applies to other chemical components.

[0120] In this embodiment, the uridine content of the safflower injection is 206.2 μg / mL, the guanosine content is 56.28 μg / mL, the adenine content is 124.0 μg / mL, the p-hydroxybenzoic acid content is 97.1 μg / mL, the 5-hydroxymethylfurfural content is 26.8 μg / mL, the p-hydroxycinnamic acid content is 80.0 μg / mL, the kaempferol 3-O-rutinoside content is 104.9 μg / mL, the syringin content is 89.2 μg / mL, and the p-hydroxysafflower yellow A content is 429.8 μg / mL.

[0121] Examples 2-5 Method for Simultaneously Determining Nine Components in a Carthamus Tinctorius Medicinal Composition

[0122] Examples 2 to 5 are methods for simultaneously determining nine components in a safflower medicinal composition. The steps are substantially the same as those in Example 1, with the only difference being the process parameters. See Table 1 for details.

[0123] Table 1 List of process parameters in Examples 2 to 5

[0124]

[0125]

[0126] In the case of a single injection, different detection wavelengths were used for detection, specifically: 252-256 nm (detection of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural), 264-268 nm (detection of p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin), 408-412 nm (detection of hydroxysafflor yellow A);

[0127] The process parameters and steps of other parts of Examples 2 to 5 are the same as those of Example 1.

[0128] Example 6 A method for simultaneously determining nine components in a safflower medicinal composition

[0129] b1) Preparation of test solution and reference solution

[0130] b11) Preparation of test solution

[0131] The test solution was prepared according to the method of step 11) in Example 1, wherein the steps and amounts were the same as in Example 1.

[0132] b12) Preparation of reference solution series with different concentrations

[0133] Weigh approximately 5 mg of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, and 5-hydroxymethylfurfural reference substances accurately, place in a 10 mL volumetric flask, dissolve in water and dilute to the mark, shake well, and prepare the mixed reference substance stock solution I.

[0134] Weigh approximately 4 mg of p-hydroxycinnamic acid and 6 mg of kaempferol-3-0-rutinoside accurately, place in a 10 mL volumetric flask, dissolve in water and dilute to the mark, shake well, and use as mixed reference substance stock solution II.

[0135] Weigh approximately 6 mg of syringin and 5 mg of 4-hydroxysafflor yellow A accurately, place in a 10 mL volumetric flask, add 50% acetonitrile water to dissolve and dilute to the scale, shake well, and use as the mixed reference substance stock solution III.

[0136] Accurately measure 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the mixed reference substance stock solution I, respectively, and add them to a 10 mL volumetric flask. Then, dilute them to volume with water to prepare 6 reference substance series solutions I with different concentration gradients.

[0137] Accurately pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the mixed reference substance stock solution II into a 10 mL volumetric flask, and dilute to volume with water to prepare 6 reference substance series solutions II with different concentration gradients.

[0138] Accurately pipette 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the mixed reference substance stock solution III into a 10 mL volumetric flask, and dilute to volume with 50% acetonitrile water to prepare 6 reference substance series solutions III with different concentration gradients.

[0139] b2) High performance liquid chromatography detection

[0140] b21) Taking different concentrations of reference substance series solution I, different concentrations of reference substance series solution II, and different concentrations of reference substance series solution III, high performance liquid chromatography was performed under the chromatographic conditions of Example 1 to obtain the peak area of the corresponding chemical component corresponding to the concentration determination. According to the concentration of the different chemical components and the peak area of the corresponding chemical component corresponding to the concentration determination, a standard curve corresponding to the different chemical components was drawn, and the concentration of the different chemical components was used as the abscissa (i.e., x, in μg / mL), and the peak area of the corresponding chemical component corresponding to the concentration determination was used as the ordinate (i.e., y).

[0141] Taking uridine as an example, when the concentration of uridine is 10.00838 μg / mL, x=10.00838 μg / mL, and the peak area corresponding to this concentration is the vertical coordinate y, and so on for other chemical components.

[0142] The standard curves drawn using reference solution series I with different concentrations, reference solution series II with different concentrations, and reference solution series III with different concentrations are as follows:

[0143] The standard curve corresponding to uridine is y=46.13562x+1.73333, R 2 =0.99992, see Figure 8 ;

[0144] The standard curve corresponding to guanosine is y=42.86719x-0.26667, R 2 =0.99995, see Figure 9 ;

[0145] The standard curve corresponding to adenosine is y=50.50161x+4.46667, R 2 =0.99997, see Figure 10 ;

[0146] The standard curve corresponding to p-hydroxybenzoic acid glycosides is 43.57603x-2.06667, R 2 =0.99994, see Figure 11 ;

[0147] The standard curve corresponding to 5-hydroxymethylfurfural is y=43.04129x-3.60000, R 2 =0.99994, see Figure 12 ;

[0148] The standard curve corresponding to p-hydroxycinnamic acid is y=105.70592x+19.66667, R 2 =0.99993, see Figure 13 ;

[0149] The standard curve corresponding to kaempferol 3-O-rutinoside is y=15.47304x-0.06667, R 2 =0.99992, see Figure 14 ;

[0150] The standard curve corresponding to syringin is y=40.00366x+1.46667, R 2 =0.99992, see Figure 15 ;

[0151] The standard curve corresponding to hydroxysafflor yellow A is y=40.31518x-18.93333, R 2 =0.99946, see Figure 16 .

[0152] b22) The test solution was subjected to HPLC under the chromatographic conditions of Example 1 to obtain the peak areas corresponding to the nine components contained in the test solution, and the peak areas of the corresponding chemical components were substituted into the corresponding standard curves, and the obtained C 类比-相应成分 Substitute the value into the following formula to calculate the concentration of each of the 9 components in the test solution.

[0153] The formula is the content of the corresponding component in safflower injection C = (C 类比-相应成分 ×I 对照品 ×N) / I 供试品 , calculate the content of the corresponding components in safflower injection.

[0154] Taking uridine as an example, the peak area (A) of uridine measured under the chromatographic conditions of Example 1 is 对照品-尿嘧啶核苷 ) was substituted into the standard curve corresponding to uridine to calculate the concentration of uridine in the test solution (C 类比-尿嘧啶核苷 );

[0155] The content of uridine in safflower injection is C=(C 类比-相应成分 ×I 对照品 ×N) / I 供试品 , and so on for the calculation methods of other chemical components.

[0156] In this embodiment, the uridine content of the safflower injection is 206.2 μg / mL, the guanosine content is 56.28 μg / mL, the adenine content is 124.0 μg / mL, the p-hydroxybenzoic acid content is 97.1 μg / mL, the 5-hydroxymethylfurfural content is 26.8 μg / mL, the p-hydroxycinnamic acid content is 80.0 μg / mL, the kaempferol 3-O-rutinoside content is 104.9 μg / mL, the syringin content is 89.2 μg / mL, and the p-hydroxysafflower yellow A content is 429.8 μg / mL.

[0157] In addition, in actual application, when the standard curve is known, the test solution can be directly prepared for HPLC detection and then directly substituted into the standard curve for calculation. There is no need to repeatedly prepare different concentrations of reference solution I, different concentrations of reference solution II, and different concentrations of reference solution I for measurement and draw the standard curve.

[0158] Examples 7-10 Method for Simultaneously Determining Nine Components in Carthamus Tinctorius Medicinal Composition

[0159] Examples 7 to 10 are methods for simultaneously determining nine components in a safflower medicinal composition. The steps are substantially the same as those in Example 6, with the only difference being the process parameters. See Table 2 for details.

[0160] Table 2 List of process parameters in Examples 7 to 10

[0161]

[0162]

[0163] In the case of a single injection, different detection wavelengths were used for detection, specifically: 252-256 nm (detection of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural), 264-268 nm (detection of p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin), 408-412 nm (detection of hydroxysafflor yellow A);

[0164] The process parameters and steps of other parts of Examples 7 to 10 are the same as those of Example 6.

[0165] Experimental Example 1 Methodological Investigation

[0166] This experimental example investigates the method for simultaneously determining 9 components in a safflower medicinal composition, mainly examining the precision, intra-day stability, repeatability, and sample recovery of the content determination method.

[0167] 1. Precision

[0168] Three different concentrations of mixed reference solution I of low, medium and high were prepared with uridine, guanosine, adenosine, p-hydroxybenzoic acid nucleoside, 5-hydroxymethylfurfural, syringin and hydroxysafflor yellow A. The three different concentrations of mixed reference solution I were measured 6 times using the chromatographic conditions and injection volume in Example 1, and the results were analyzed. The specific results are shown in the table below.

[0169] Table 3 Concentration list of three different concentrations of mixed reference solution I

[0170]

[0171]

[0172] Table 4 Precision test results list 1 (peak area)

[0173]

[0174]

[0175] The data in Table 4 are the peak areas obtained when 6 replicates were performed on the mixed reference solution I with three different concentrations: low, medium, and high. As can be seen from Table 4, the RSD (%) of uridine is ≤0.70, the RSD (%) of guanosine is ≤0.73, the RSD (%) of adenosine is ≤0.85, the RSD (%) of p-hydroxybenzoic acid glycoside is ≤0.98, the RSD (%) of 5-hydroxymethylfurfural is ≤1.45, the RSD (%) of syringin is ≤1.57, and the RSD (%) of hydroxysafflor yellow A is ≤1.58, indicating that the precision of this method is good.

[0176] Three mixed reference solutions II with low, medium, and high concentrations of p-hydroxycinnamic acid and kaempferol-3-O-rutinoside were prepared. The three mixed reference solutions II with different concentrations were measured six times using the chromatographic conditions and injection volumes in Example 1, and the results were analyzed. The specific results are shown in the table below.

[0177] Table 5 Concentration list of three different concentrations of mixed reference solution II

[0178]

[0179] Table 6 Precision test results list 2 (peak area)

[0180]

[0181] The data in Table 6 show the peak areas obtained from six replicate measurements of mixed reference solution II at low, medium, and high concentrations. As can be seen from Table 6, the RSD (%) for p-hydroxycinnamic acid is ≤1.15, and the RSD (%) for kaempferol-3-O-rutinoside is ≤1.57, indicating that this method has good precision.

[0182] 2. Intraday Stability

[0183] Take safflower injection (batch number: HH001) and prepare a test solution W according to the method in Example 1. The obtained test solution W is placed at room temperature in the dark, and at 0, 1, 2, 6, 12, and 18 h, 10 μL of the test solution W is accurately measured and injected into the liquid chromatograph respectively. The chromatographic conditions in Example 1 are used for determination, and the relative standard deviation of the peak area is calculated 6 times. The specific results are shown in the table below.

[0184] Table 7 Summary of intra-day stability test results (peak area)

[0185] name 0 1h 2h 6h 12h 18h RSD% Uridine 1911 1921 1906 1930 1872 1942 1.26 Guanosine 485 493 492 492 476 479 1.51 Adenosine 1252 1261 1261 1239 1240 1264 0.89 Parabens 842 851 853 825 828 856 1.58 5-Hydroxymethylfurfural 231 235 227 225 235 235 1.93 p-Hydroxycinnamic acid 1697 1702 1685 1673 1706 1704 0.76 Kaempferol-3-O-rutinoside 322 325 316 317 328 326 1.53 Syringin 718 731 701 710 728 726 1.62 Hydroxysafflor Yellow A 3448 3472 3420 3427 3471 3467 0.67

[0186] As can be seen from Table 7, the RSD (%) of uridine in the test solution W is ≤1.26, the RSD (%) of guanosine is ≤1.51, the RSD (%) of adenosine is ≤0.89, the RSD (%) of p-hydroxybenzoic acid glycoside is ≤1.58, the RSD (%) of 5-hydroxymethylfurfural is ≤1.93, the RSD (%) of p-hydroxycinnamic acid is ≤0.76, the RSD (%) of kaempferol-3-O-rutinoside is ≤1.53, the RSD (%) of syringin is ≤1.62, and the RSD (%) of hydroxysafflor yellow A is ≤0.67, indicating that the method has good intraday stability.

[0187] 3. Repeatability Investigation

[0188] The same batch of safflower injection (batch number: HH001, 6 parts) was taken and 6 parts of test solution C were prepared according to the method in Example 1. The test solution C was measured using the chromatographic conditions and injection volume in Example 1, and the results were analyzed. The specific results are shown in the table below.

[0189] Table 8 Repeatability test results (peak area)

[0190] name 1 2 3 4 5 6 RSD% Uridine 1911 1934 1893 1927 1936 1941 0.95 Guanosine 485 490 481 490 491 491 0.84 Adenosine 1252 1266 1226 1259 1266 1261 1.21 Parabens 842 853 822 849 849 849 1.34 5-Hydroxymethylfurfural 231 233 229 234 229 228 1.05 p-Hydroxycinnamic acid 1697 1709 1683 1708 1682 1720 0.90 Kaempferol-3-O-rutinoside 322 320 315 324 316 326 1.36 Syringin 718 715 725 721 713 723 0.65 Hydroxysafflor Yellow A 3448 3402 3487 3494 3420 3487 1.13

[0191] As can be seen from Table 8, uridine RSD (%) ≤ 0.95, guanosine RSD (%) ≤ 0.84, adenosine RSD (%) ≤ 1.21, p-hydroxybenzoic acid glycoside RSD (%) ≤ 1.34, 5-hydroxymethylfurfural RSD (%) ≤ 1.05, p-hydroxycinnamic acid RSD (%) ≤ 0.90, kaempferol-3-O-rutinoside RSD (%) ≤ 1.36, syringin RSD (%) ≤ 0.65, p-hydroxysafflower yellow RSD (%) ≤ 1.13. The results show that the method has good reproducibility.

[0192] 4. Sample recovery test

[0193] Take the same batch number of safflower injection (batch number: HH001), accurately measure 1.3 mL and place it in a 10 ml volumetric flask, add 1.7 ml, 3.5 ml, and 5.5 ml of the mixed reference substance stock solution I prepared in Example 6, respectively, mix well, and adjust the volume to the scale value to obtain different sample amounts of the sample recovery test solution I. For each group of sample amounts of the sample recovery test solution I, 3 portions were prepared in parallel, for a total of 9 portions. The chromatographic conditions in Example 1 were used to measure the different sample amounts of the sample recovery test solution I in each group. The specific results are shown in the table below.

[0194] Table 9 Sample recovery test results list 1

[0195]

[0196]

[0197] As can be seen from Table 9, the RSD (%) of uridine is ≤1.15, the RSD (%) of guanosine is ≤0.95, the RSD (%) of adenosine is ≤1.12, the RSD (%) of p-hydroxybenzoic acid glycoside is ≤1.45, the RSD (%) of 5-hydroxymethylfurfural is ≤1.02, the RSD (%) of syringin is <1.13, and the RSD (%) of hydroxysafflor yellow A is <0.87%. It can be seen that the recovery rate of this method is good.

[0198] Take the same batch number of safflower injection (batch number: HH001), accurately measure 2 mL and place it in a 10 ml volumetric flask, add 1.7 ml, 3.5 ml, and 5.5 ml of the mixed reference substance stock solution I prepared in Example 6, respectively, and mix well. Add water to the scale value to obtain different sample amounts of the sample recovery test solution II. For each group of sample amounts of the sample recovery test solution II, 3 portions were prepared in parallel, for a total of 9 portions. The chromatographic conditions in Example 1 were used to measure the different sample amounts of the sample recovery test solution II in each group. The specific results are shown in the table below.

[0199] Table 10 Sample recovery test results list 1

[0200] name p-Hydroxycinnamic acid Kaempferol-3-O-rutinoside Low concentration 1 101.3 101.8 Low concentration 2 101.7 100.2 Low concentration 3 101.9 99.4 Medium concentration 1 101.2 99.1 Medium concentration 2 99.8 98.4 Medium concentration 3 98.7 100.5 High concentration 1 101.1 101.5 High concentration 2 100.4 98.5 High concentration 3 99.2 101.0

[0201] As can be seen from Table 10, the RSD (%) of p-hydroxycinnamic acid is ≤1.11, and the RSD (%) of kaempferol-3-O-rutose is ≤1.26, which shows that the recovery rate of this method is good.

[0202] Experimental Example 2 Determination of different batches of test samples

[0203] Five batches of safflower injection with different batch numbers were used to prepare test solution B according to the method in Example 1. The test solution B was subjected to 9 component content determination according to the chromatographic conditions and calculation method in Example 1. Each test solution B was measured in parallel twice, and the average value was calculated. The results are shown in the table below.

[0204] Table 11 Summary of the determination results of 9 components in different batches of test samples

[0205]

[0206] As can be seen from Table 11, the contents of active ingredients in different batches of safflower injection are different, which shows that the current preparation method of safflower injection needs to be improved. The present invention provides a good technical means for the quality control of the subsequent production of safflower pharmaceutical compositions.

[0207] Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

Claims

1. A method for simultaneously determining nine components in a safflower medicinal composition, characterized in that: The safflower medicinal composition is safflower injection; The method comprises the following steps: Prepare a reference solution I by taking seven ingredients, namely, uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin and p-hydroxysafflor yellow A; Prepare reference solution II by taking two components, p-hydroxycinnamic acid and kaempferol 3-O-rutinoside; Prepare a test solution by taking the safflower medicinal composition; The reference solution I, the reference solution II and the test solution were respectively subjected to high performance liquid chromatography detection, and then the external standard method was used to calculate the contents of uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, syringin and hydroxysafflower yellow in the safflower medicinal composition; Among them, the detection wavelength for uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside and 5-hydroxymethylfurfural is 252~256nm; The detection wavelength for p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, and syringin was 262–270 nm; The detection wavelength for hydroxysafflor yellow A is 408-412 nm; The elution mode of the high performance liquid chromatography detection is gradient elution; The mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is a 0.1-0.2 wt% glacial acetic acid aqueous solution; The gradient elution conditions are: 0-15 min, 2%→5% mobile phase A, 98%→95% mobile phase B; 15-30 min, 5%→25% mobile phase A, 95%→75% mobile phase B; The chromatographic column for high performance liquid chromatography detection uses octadecylsilane bonded silica gel as a filler.

2. The method for simultaneously determining 9 components in a safflower medicinal composition according to claim 1, characterized in that: The column temperature of the high performance liquid chromatography detection is 25-40° C. and the flow rate is 0.8-1.2 mL / min.

3. The method for simultaneously determining 9 components in a safflower medicinal composition according to claim 1, characterized in that: Reference solution I contains uridine 10-80 μg / mL, guanosine 10-60 μg / mL, adenosine 10-70 μg / mL, p-hydroxybenzoic acid 10-70 μg / mL, 5-hydroxymethylfurfural 10-70 μg / mL, syringin 1-70 μg / mL, and p-hydroxysafflor yellow A 1-65 μg / mL; Reference solution II contained 8-50 μg / mL of p-hydroxycinnamic acid and 10-80 μg / mL of kaempferol 3-O-rutinoside.

4. The method for simultaneously determining 9 components in a safflower medicinal composition according to claim 1, characterized in that: The solvent of reference solution I is water; The solvent of reference solution II is 48-52 vol% acetonitrile in water; The test solution is prepared by diluting the safflower medicinal composition with water to 2 to 10 times its volume.

5. A method for simultaneously determining nine components in a safflower medicinal composition, characterized in that: The safflower medicinal composition is safflower injection; The method comprises the following steps: A total of seven components, namely, uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside, 5-hydroxymethylfurfural, syringin, and p-hydroxysafflor yellow A, were used to prepare a series of reference solutions I with different concentrations. Prepare reference solution series II with different concentrations of p-hydroxycinnamic acid and kaempferol 3-O-rutinoside. Different concentrations of reference substance series solution I and reference substance series solution II were respectively taken for high performance liquid chromatography detection, and standard curves corresponding to different chemical components were drawn according to the concentrations of different chemical components and the peak areas of the corresponding chemical components; Prepare a test solution by taking the safflower medicinal composition; The test solution is subjected to high performance liquid chromatography detection, and the peak area of the corresponding chemical component obtained is substituted into the corresponding standard curve, and the content of the corresponding chemical component in the safflower medicinal composition is obtained by calculation; Among them, the detection wavelength for uridine, guanosine, adenosine, p-hydroxybenzoic acid glycoside and 5-hydroxymethylfurfural is 252~256nm; The detection wavelength for p-hydroxycinnamic acid, kaempferol 3-O-rutinoside, and syringin was 262–270 nm; The detection wavelength for hydroxysafflor yellow A is 408-412 nm; The elution mode of the high performance liquid chromatography detection is gradient elution; The mobile phase A of the gradient elution is acetonitrile, and the mobile phase B is a 0.1-0.2 wt% glacial acetic acid aqueous solution; The gradient elution conditions are: 0-15 min, 2%→5% mobile phase A, 98%→95% mobile phase B; 15-30 min, 5%→25% mobile phase A, 95%→75% mobile phase B; The chromatographic column for high performance liquid chromatography detection uses octadecylsilane bonded silica gel as a filler.

6. The method for simultaneously determining 9 components in a safflower medicinal composition according to claim 5, characterized in that: The column temperature of the high performance liquid chromatography detection is 25-40° C. and the flow rate is 0.8-1.2 mL / min.

7. The method for simultaneously determining 9 components in a safflower medicinal composition according to claim 5, characterized in that: In the different reference substance series solutions I, the concentrations of uridine ranged from 10.01 to 60.05 μg / mL, the concentrations of guanosine ranged from 8.99 to 53.93 μg / mL, the concentrations of adenosine ranged from 9.44 to 56.61 μg / mL, the concentrations of p-hydroxybenzoic acid glycoside ranged from 9.82 to 58.91 μg / mL, the concentrations of 5-hydroxymethylfurfural ranged from 9.60 to 57.62 μg / mL, the concentrations of syringin ranged from 11.80 to 70.82 μg / mL, and the concentrations of p-hydroxysafflor yellow A ranged from 1 to 65 μg / mL; The concentrations of p-hydroxycinnamic acid in the different reference substance series solutions II ranged from 9.23 to 55.38 μg / mL, and the concentrations of kaempferol 3-O-rutinoside ranged from 12.22 to 73.33 μg / mL; The solvent of reference solution series I is water; The solvent of reference solution series II is 48-52 vol% acetonitrile in water; The test solution was prepared by diluting the safflower medicinal composition with water by 2 to 10 times its volume; The theoretical plate numbers of the nine components in the high performance liquid chromatography detection process are all above 10,000.

8. The method for simultaneously determining nine components in a safflower medicinal composition according to claim 5, wherein: The standard curves corresponding to the different chemical components are as follows: The standard curve corresponding to uridine is y=46.13562x+1.73333, R 2 =0.99992; The standard curve corresponding to guanosine is y=42.86719x-0.26667, R 2 =0.99995; The standard curve corresponding to adenine nucleoside is y=50.50161x+4.46667, R 2 =0.99997; The standard curve corresponding to p-hydroxybenzoic acid glycosides is 43.57603x-2.06667, R 2 =0.99994; The standard curve corresponding to 5-hydroxymethylfurfural is y=43.04129x-3.60000, R 2 =0.99994; The standard curve corresponding to p-hydroxycinnamic acid is y=105.70592x+19.66667, R 2 =0.99993; The standard curve corresponding to kaempferol 3-O-rutinoside is y=15.47304x-0.06667, R 2 =0.99992; The standard curve corresponding to syringin is y=40.00366x+1.46667, R 2 =0.99992; The standard curve corresponding to hydroxysafflor yellow A is y=40.31518x-18.93333, R 2 =0.99946.

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