System and Method for Identifying Shenqi Fuzheng Injection

Abstract

A system for identifying Shenqi Fuzheng injection includes a mechanism for establishing a profile of a sample to be tested; a mechanism for establishing a characteristic fingerprint profile of Shenqi Fuzheng injection as a standard fingerprint profile; and a mechanism for comparing the profile of the sample to be tested with the standard fingerprint profile to distinguish between authentic Shenqi Fuzheng injection and counterfeit Shenqi Fuzheng injection.

A method for establishing a Shenqi Fuzheng injection fingerprint spectrum, including: employing an ultra-high voltage liquid chromatography mass spectrometer to test the Shenqi Fuzheng injection, the chromatography conditions including: chromatographic column: Agilent Zorbax Eclipse Plus C18, 2.1 mm100 mm, 1.8 m; mobile phase: mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is 0.1% formic acid acetonitrile solution; and employing gradient elution procedure.

Claims

1. A system for identifying Shenqi Fuzheng injection, the system comprising: means for establishing a profile of a sample to be tested; means for establishing a characteristic fingerprint profile of Shenqi Fuzheng injection as a standard fingerprint profile; and means for comparing the profile of the sample to be tested with the standard fingerprint profile to distinguish between authentic Shenqi Fuzheng injection and counterfeit Shenqi Fuzheng injection; wherein, the standard fingerprint profile comprises 18 characteristic peaks, and a retention time and a mass number of the 18 characteristic peaks are as follows: Peak 1:7.1 min (471.2083), Peak 2: 7.5 min (491.1195), Peak 3: 8.1 min (441.1919), Peak 4: 8.6 min (309.1555), Peak 5: 9.2 min (187.0976), Peak 6: 9.9 min (441.1766), Peak 7: 10.9 min (593.1876), Peak 8: 11.3 min (507.1508), Peak 9: 11.7 min (463.1610), Peak 10: 12.7 min (991.5119), Peak 11: 13.4 min (991.5119), Peak 12: 13.7 min (829.4591), Peak 13: 14.4 min (871.4697), Peak 14: 14.8 min (871.4697), Peak 15: 15.1 min (871.4697), Peak 16: 15.5 min (913.4650), Peak 17: 15.9 min (913.4650), and Peak 18: 16.3 min (913.4650); wherein Peak 2 and Peak 12 are calycosin glucoside and astragaloside IV, respectively; wherein the Shenqi Fuzheng injection characteristic fingerprint profile takes a control astragaloside IV as a reference peak, by which a relative retention time of each characteristic peak is calculated, as follows: Peak 1: 0.52, Peak 2: 0.54, Peak 3: 0.59, Peak 4: 0.62, Peak 5: 0.66, Peak 6: 0.72, Peak 7: 0.79, Peak 8: 0.82, Peak 9: 0.85, Peak 10: 0.92, Peak 11: 0.97, Peak 12: 1.00, Peak 13: 1.04, Peak 14: 1.07, Peak 15: 1.10, Peak 16: 1.13, Peak 17: 1.16, and Peak 18: 1.19; wherein, when comparing, the sample is identified as authentic Shenqi Fuzheng injection if the relative retention time fluctuates within 5% of the above specified values, and a ratio of an area of calycosin glucoside peak and the astragaloside IV peak to an area of a corresponding reference peak is 0.5-1.5.

2. The system of claim 1, wherein the means for establishing the profile of the samples to be tested and the means for establishing the characteristic fingerprint profile of Shenqi Fuzheng injection as the standard fingerprint profile each independently comprise ultra-high pressure liquid chromatography-mass spectrometer.

3. The system of claim 1, further comprising means for detecting saponins in Shenqi Fuzheng injection.

4. A method for identifying Shenqi Fuzheng injection, comprising the following steps: step (1): establishing a profile of the samples to be tested; step (2): establishing a characteristic fingerprint profile of Shenqi Fuzheng injection as the standard fingerprint profile; step (3): comparing the profile of the samples to be tested in step (1) with the standard fingerprint profile in step (2) to distinguish between true and false; wherein, the standard fingerprint profile in step (2) comprises 18 characteristic peaks, and the retention time and the mass number of the 18 characteristic peaks are as follows: Peak 1:7.1 min (471.2083), Peak 2: 7.5 min (491.1195), Peak 3: 8.1 min (441.1919), Peak 4: 8.6 min (309.1555), Peak 5: 9.2 min (187.0976), Peak 6: 9.9 min (441.1766), Peak 7: 10.9 min (593.1876), Peak 8: 11.3 min (507.1508), Peak 9: 11.7 min (463.1610), Peak 10: 12.7 min (991.5119), Peak 11: 13.4 min (991.5119), Peak 12: 13.7 min (829.4591), Peak 13: 14.4 min (871.4697), Peak 14: 14.8 min (871.4697), Peak 15: 15.1 min (871.4697), Peak 16: 15.5 min (913.4650), Peak 17: 15.9 min (913.4650), Peak 18: 16.3 min (913.4650); wherein Peak 2 and Peak 12 are calycosin glucoside and astragaloside IV, respectively; wherein the Shenqi Fuzheng injection characteristic fingerprint profile takes the control astragaloside IV as a reference peak, by which the relative retention time of each characteristic peak is calculated, as follows: Peak 1: 0.52, Peak 2: 0.54, Peak 3: 0.59, Peak 4: 0.62, Peak 5: 0.66, Peak 6: 0.72, Peak 7: 0.79, Peak 8: 0.82, Peak 9: 0.85, Peak 10: 0.92, Peak 11: 0.97, Peak 12: 1.00, Peak 13: 1.04, Peak 14: 1.07, Peak 15: 1.10, Peak 16: 1.13, Peak 17: 1.16, Peak 18: 1.19; wherein, when comparing, the relative retention time fluctuates within 5% of the above specified values, and the ratio of the area of the calycosin glucoside peak and the astragaloside IV peak to the area of the corresponding reference peak is 0.5-1.5.

5. The method according to claim 4, wherein, the profile of the samples to be tested in step (1) was established by the following method, which comprising testing the samples to be tested by ultra-high pressure liquid chromatography-mass spectrometer, wherein the method comprises the following steps: (1) preparation of control solution: accurately weighing an appropriate amount of calycosin glucoside or astragaloside IV, and then adding methanol to prepare a solution containing 0.004 mg of calycosin glucoside per ml or 0.006 mg of astragaloside IV per ml, respectively; (2) preparation of test sample solution: filtering the samples to be tested through a 0.22 m microporous filter membrane; (3) determination: accurately aspirating 5 l of the control solution or the test sample solution, respectively, and then injecting the solutions into a ultra-high pressure liquid chromatography-mass spectrometer, conducting determination according to the following conditions to obtain the fingerprint profile; wherein the chromatographic conditions include the followings: chromatographic column: Agilent Zorbax Eclipse Plus C18, 2.1 mm100 mm, 1.8 m; mobile phase: mobile phase A is 0.1% (v/v) formic acid aqueous solution, mobile phase B is 0.1% (v/v) formic acid acetonitrile solution; using gradient elution according to the following elution program, wherein the proportions of the mobile phases are all volume percentages: 0-0.5 min, mobile phase A is 95%, mobile phase B is 5%; 0.5-10 min, mobile phase A is 95%-75%, mobile phase B is 5%-25%; 10-15 min, mobile phase A is 75%-45%, mobile phase B is 25%-55%; 15-18 min, mobile phase A is 45%-0%, mobile phase B is 55%-100%; 18-20 min, mobile phase A is 0%, mobile phase B is 100%. flow rate: 0.35 ml/min; column temperature: 40 C.; the ion source is an ESI source, and detection is operated in negative ion mode; atomized gas pressure: 35 psig; dry gas temperature: 350 C.; dry gas flow rate: 10 L/min; capillary voltage: 3,500 V; voltage at capillary exit: 135 V.

6. The method according to claim 4, wherein, the Shenqi Fuzheng injection characteristic fingerprint profile in step (2) was established by the following method, which comprises testing Shenqi Fuzheng injection by ultra-high pressure liquid chromatography-mass spectrometer, wherein the method comprises the following steps: (1) preparation of control solution: accurately weighing an appropriate amount of calycosin glucoside or astragaloside IV, and then adding methanol to prepare a solution containing 0.004 mg of calycosin glucoside per ml or 0.006 mg of astragaloside IV per ml, respectively; (2) preparation of test sample solutions: filtering Shenqi Fuzheng injection through a 0.22 m microporous filter membrane; (3) determination: accurately aspirating 5 l of the control solution or the test sample solution, respectively, and then injecting the solutions into a ultra-high pressure liquid chromatography-mass spectrometer, conducting determination according to the following conditions to obtain the Shenqi Fuzheng injection fingerprint profile; wherein the chromatographic conditions include the followings: chromatographic column: Agilent Zorbax Eclipse Plus C18, 2.1 mm100 mm, 1.8 m; mobile phase: mobile phase A is 0.1% (v/v) formic acid aqueous solution, mobile phase B is 0.1% (v/v) formic acid acetonitrile solution; using gradient elution according to the following elution program, wherein the proportions of the mobile phases are all volume percentages: 0-0.5 min, mobile phase A is 95%, mobile phase B is 5%; 0.5-10 min, mobile phase A is 95%-75%, mobile phase B is 5%-25%; 10-15 min, mobile phase A is 75%-45%, mobile phase B is 25%-55%; 15-18 min, mobile phase A is 45%-0%, mobile phase B is 55%-100%; 18-20 min, mobile phase A is 0%, mobile phase B is 100%. flow rate: 0.35 ml/min; column temperature: 40 C.; the ion source is an ESI source, and detection is operated in negative ion mode; atomized gas pressure: 35 psig; dry gas temperature: 350 C.; dry gas flow rate: 10 L/min; capillary voltage: 3,500 V; voltage at capillary exit: 135 V; (4) determination of common characteristic peaks to obtain the standard fingerprint profile: comparing multiple Shenqi Fuzheng injection fingerprint profiles obtained from the above method, picking out 18 common characteristic peaks as the standard fingerprint profile.

7. A method for detecting saponins in Shenqi Fuzheng injection, the method comprising: preparing chromatographic conditions for HPLC-ELSD as followings: chromatographic column: Discovery C-18, 5 m, 2504.6 mm; mobile phase: mobile phase A is acetonitrile, mobile phase B is water; and using gradient elution according to the following elution program, wherein the proportions of the mobile phases are all volume percentages: 0-30 min, mobile phase A is 12-25%, mobile phase B is 88-75%; 30-40 min, mobile phase A is 25%-32%, mobile phase B is 75%-68%; 40-55 min, mobile phase A is 32%-55%, mobile phase B is 68%-45%; 55-68 min, mobile phase A is 55%-90%, mobile phase B is 45%-10%; 68-72 min, mobile phase A is 90%, mobile phase B is 10%.

8. The method according to claim 7, wherein, the chromatographic conditions also include the followings: flow rate: 1.0 ml/min; column temperature: 30 C.; injection volume: 20 l.

9. The method according to claim 7, wherein the conditions of the ELSD detector include the followings: the temperature of drift tube was 55 C.; atomizing power was 60%; gain value was 20; gas pressure was 25 psig.

10. The method according to claim 7, further comprising preparation of control solutions by the following steps: accurately weighing an appropriate amount of astragaloside IV, and adding methanol to prepare a solution containing 0.10 mg of astragaloside IV per ml.

11. The method according to claim 7, further comprising preparation of a test sample solution by the following step: fractioning the Shenqi Fuzheng Injection on a macroporous resin, eluting the Shenqi Fuzheng Injection with water and, firstly 30 v/v % ethanol solution and then 70 v/v % ethanol solution to remove polysaccharides, protein and salts, concentrating the Shenqi Fuzheng Injection, setting the volume, and filtering the Shenqi Fuzheng Injection.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0105] FIG. 1 illustrates a total ion chromatogram (TIC) of the Shenqi Fuzheng injection provided by the present invention, wherein the arrows from left to right indicate the characteristic peaks 1 to 18, respectively;

[0106] FIG. 2 illustrates an extracted ion chromatogram (EIC) of the Shenqi Fuzheng injection, wherein the arrows from left to right indicate the characteristic peaks 1 to 18, respectively;

[0107] FIG. 3 illustrates an extracted ion chromatogram (EIC) of the control mixture, wherein Peaks 2 and 12 represent calycosin glucoside and astragaloside IV, sequentially;

[0108] FIG. 4 is a graph illustrating the comparison of the Shenqi Fuzheng injection fingerprint profile of the present invention with a counterfeit, wherein 1 showing the certified Shenqi Fuzheng injection, 2 showing the counterfeit (presumed as Danshen Injection), and 3 showing a Danshen infusion solution;

[0109] FIG. 5 is a chromatogram obtained by using the mobile phase shown in table 8;

[0110] FIG. 6 is a chromatogram obtained by using the mobile phase shown in table 9;

[0111] FIG. 7 is a chromatogram obtained by using the mobile phase shown in table 10;

[0112] FIG. 8 is a chromatogram obtained by using the mobile phase shown in table 11;

[0113] FIG. 9 is a chromatogram obtained on Kromasil 100-5 C18 column;

[0114] FIG. 10 is a chromatogram obtained on Diamonsil C18 column;

[0115] FIG. 11 is a chromatogram obtained on Discovery C18 column;

[0116] FIG. 12 is a chromatogram obtained at a flow rate of 0.8 ml/min;

[0117] FIG. 13 is a chromatogram obtained at a flow rate of 1.0 ml/min;

[0118] FIG. 14 is a chromatogram obtained at a flow rate of 1.2 ml/min;

[0119] FIG. 15 is a chromatogram with an injection volume of 10 l;

[0120] FIG. 16 is a chromatogram with an injection volume of 20 l;

[0121] FIG. 17 is a chromatogram obtained by extracting with n-butanol;

[0122] FIG. 18 is a chromatogram obtained by purifying on D.sub.101 macroporous resin directly;

[0123] FIG. 19 is a chromatogram obtained by purifying on D.sub.101 macroporous resin after concentration;

[0124] FIG. 20 is a chromatogram obtained after 120 min;

[0125] FIG. 21 is a chromatogram of astragaloside IV;

[0126] FIG. 22 is a chromatogram of calycosin glucoside;

[0127] FIG. 23 is a chromatogram of negative control solution without radix astragali;

[0128] FIG. 24 is a chromatogram of negative control solution without radix codonopsis;

[0129] FIG. 25 is a chromatogram of negative control solution without radix astragali and radix codonopsis;

[0130] FIG. 26 is control fingerprint of saponins of Shenqi Fuzheng Injection (Peak S: astragaloside IV);

[0131] FIG. 27 is a spectrum of Shenqi Fuzheng Injection by HPLC-ELSD method;

[0132] FIG. 28 is a total ionic spectrum of Shenqi Fuzheng Injection by HPLC-MS, wherein Peak 1: Astragaloside V, Peak 2: Astragaloside VI, Peak 3: Astragaloside IV, Peak 4: Isoastragaloside II, Peak 5: Astragaloside II, Peak 6: Cyclocephaloside II,

[0133] FIG. 29 is a spectrum of control solution of Astragaloside V by HPLC-ELSD method;

[0134] FIG. 30 is a spectrum of control solution of Astragaloside VI by HPLC-ELSD method;

[0135] FIG. 31 is a spectrum of control solution of Astragaloside IV by HPLC-ELSD method;

[0136] FIG. 32 is a spectrum of control solution of Isoastragaloside II by HPLC-ELSD method;

[0137] FIG. 33 is a spectrum of control solution of Astragaloside II by HPLC-ELSD method; and

[0138] FIG. 34 is a spectrum of control solution of Cyclocephaloside II by HPLC-ELSD method.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0139] The technical solutions of the present invention will be further described in details in combination with specific examples.

Example 1

[0140] 1. Instrument and Test Drug

[0141] 1.1 Instruments:

[0142] Agilent LC/MSD (1290UHPLC dual-gradient pump, built-in vacuum degasser, 100-bit automatic sampler, intelligent column oven, high-precision quadrupole tandem time-of-flight mass spectrometer system); Chromatographic column: Agilent Zorbax Eclipse Plus C18 (2.1 mm100 mm, 1.8 m).

[0143] 1.2 Test Drug:

[0144] Shenqi Fuzheng injection, provided by Livzon Group Limin Pharmaceutical Factory. Reagents acetonitrile and formic acid used in the experiments were both chromatographically pure, and the water was ultrapure water.

[0145] 2. Method and Result

[0146] 2.1 Preparation of Test Sample Solution:

[0147] Shenqi Fuzheng injection was filtered through a 0.22 m microporous filter membrane.

[0148] 2.2 Preparation of Mixed Control Solution:

[0149] An appropriate amount of calycosin glucoside and astragaloside IV was accurately weighed, and methanol was then added to prepare a solution containing 0.004 mg of calycosin glucoside and 0.006 mg of astragaloside IV per ml, respectively.

[0150] 2.3 Chromatographic Conditions:

[0151] Chromatographic column was Agilent Zorbax Eclipse Plus C18, 2.1 mm100 mm, 1.8 m; Mobile phases were 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B); Column temperature was 40 C.; Injection volume was 5 l; A gradient elution program as shown in Table 2 was used:

TABLE-US-00002 TABLE 2 Gradient Elution Program Time (min) Mobile Phase A (%) Mobile Phase B (%) 0-0.5 95 5 0.5-10 95 .fwdarw. 75 5 .fwdarw. 25 10-15 75 .fwdarw. 45 25 .fwdarw. 55 15-18 45 .fwdarw. 0 55 .fwdarw. 100 18-20 0 100

[0152] 2.4 Mass Spectrometry Conditions:

[0153] The ion source was an ESI source, detection was operated in negative ion mode; Atomized gas pressure: 35 psig; Dry gas temperature: 350 C.; Dry gas flow rate: 10 L/min; Vcap Capillary voltage: 3,500 V; Voltage at capillary exit: 135 V.

[0154] 2.5 Determination Method

[0155] 5 l of the control solution or the test sample solution was accurately aspirated, respectively, then injected into the liquid chromatography-mass spectrometer, respectively, determination was conducted, recording the spectra for 20 minutes.

[0156] 2.6 Determination of Common Characteristic Peaks

[0157] The common characteristic peaks were sorted out by comparing the total ion chromatograms (TIC) of 100 batches of Shenqi Fuzheng injection, see details in FIG. 1; the extracted ion chromatogram (EIC) was obtained by using the ion mass number of these common characteristic peaks, see details in FIG. 2 (specifically, a series of target ions were extracted from FIG. 1 by using the qualitative analysis software in the data analysis software Masshunter and utilizing the function of ion extraction, and thereby obtaining FIG. 2); then the retention time (Rt) of each common characteristic peak was marked, so as to obtain the Shenqi Fuzheng injection characteristic fingerprint profile. There were 18 common characteristic peaks, with a retention time (Rt) and a mess number as follows: 7.1 min (471.2083), 7.5 min (491.1195), 8.1 min (441.1919), 8.6 min (309.1555), 9.2 min (187.0976), 9.9 min (441.1766), 10.9 min (593.1876), 11.3 min (507.1508), 11.7 min (463.1610), 12.7 min (991.5119), 13.4 min (991.5119), 13.7 min (829.4591), 14.4 min (871.4697), 14.8 min (871.4697), 15.1 min (871.4697), 15.5 min (913.4650), 15.9 min (913.4650), 16.3 min (913.4650), wherein the chromatographic peaks with a Rt of 7.5 min and 13.7 min were verified as calycosin glucoside and astragaloside IV respectively, see details in FIG. 3; Peak S was a peak corresponding to an astragaloside IV reference peak, the retention time of each characteristic peaks was calculated, wherein the retention time should fluctuate within 5% of the specified values, which were listed in an order of 0.52, 0.54, 0.59, 0.62, 0.66, 0.72, 0.79, 0.82, 0.85, 0.92, 0.97, 1.00, 1.04, 1.07, 1.10, 1.13, 1.16, 1.19; the ratio of the peak area of calycosin glucoside and astragaloside IV to the peak area of their corresponding reference should be within 0.5-1.5.

[0158] 2.7 Precision Assay:

[0159] The test sample solution from the same Shenqi Fuzheng injection was injected for continuously 6 times, followed by extracting the characteristic peaks and marking the retention time. Peak 12 of astragaloside IV was taken as Peak S, and then the relative retention time of each of other characteristic peaks were calculated. The results showed that the RSD values of the relative retention time of each characteristic peak was all less than 1%, and the precision of the instrument was excellent. The results of the precision assay are shown in Table 3:

TABLE-US-00003 TABLE 3 Results of Precision Assay 1 2 3 4 5 6 RSD (%) 1 0.517 0.517 0.517 0.516 0.516 0.517 0.14 2 0.542 0.542 0.542 0.541 0.541 0.542 0.09 3 0.586 0.586 0.586 0.585 0.585 0.586 0.08 4 0.623 0.624 0.624 0.623 0.623 0.624 0.08 5 0.661 0.661 0.661 0.661 0.661 0.661 0.04 6 0.716 0.715 0.716 0.715 0.716 0.716 0.06 7 0.786 0.786 0.787 0.787 0.788 0.787 0.08 8 0.820 0.819 0.820 0.820 0.820 0.821 0.08 9 0.847 0.847 0.848 0.848 0.849 0.848 0.06 10 0.923 0.922 0.923 0.922 0.923 0.923 0.01 11 0.972 0.972 0.972 0.972 0.972 0.972 0.02 S 1.000 1.000 1.000 1.000 1.000 1.000 0.00 13 1.044 1.044 1.044 1.045 1.044 1.044 0.02 14 1.074 1.074 1.074 1.074 1.074 1.074 0.02 15 1.096 1.096 1.096 1.096 1.096 1.096 0.01 16 1.129 1.129 1.128 1.129 1.129 1.129 0.03 17 1.155 1.155 1.155 1.156 1.156 1.155 0.02 18 1.189 1.189 1.189 1.189 1.189 1.189 0.02

[0160] 2.8 Stability Assay:

[0161] The test sample solution from the same Shenqi Fuzheng injection was injected at 0 hour, at 1 hour, at 2 hours, at 4 hours, at 8 hours, and at 12 hours, respectively, followed by extracting the characteristic peaks and marking the retention time. Peak 12 of astragaloside IV was taken as Peak S, and then the relative retention time of each of other characteristic peaks was calculated. The results showed that the RSD values of the relative retention time of each characteristic peak was all less than 1%), and the test sample solution remained stable within 12 hours during standing. The results of the stability assay are shown in Table 4:

TABLE-US-00004 TABLE 4 Results of Stability Assay 0 1 2 4 8 12 RSD (%) 1 0.517 0.516 0.516 0.517 0.517 0.517 0.15 2 0.542 0.541 0.541 0.542 0.541 0.543 0.11 3 0.586 0.586 0.585 0.586 0.586 0.587 0.09 4 0.624 0.623 0.623 0.624 0.623 0.624 0.09 5 0.662 0.662 0.661 0.662 0.661 0.662 0.05 6 0.716 0.716 0.715 0.716 0.715 0.716 0.06 7 0.787 0.786 0.786 0.788 0.787 0.786 0.07 8 0.820 0.820 0.819 0.820 0.820 0.821 0.04 9 0.848 0.848 0.847 0.848 0.848 0.847 0.04 10 0.922 0.922 0.923 0.923 0.922 0.923 0.02 11 0.972 0.972 0.972 0.972 0.972 0.972 0.01 S 1.000 1.000 1.000 1.000 1.000 1.000 0.00 13 1.044 1.044 1.044 1.044 1.044 1.045 0.01 14 1.074 1.074 1.074 1.074 1.074 1.074 0.01 15 1.096 1.096 1.096 1.096 1.096 1.096 0.02 16 1.129 1.129 1.129 1.129 1.129 1.129 0.02 17 1.155 1.156 1.156 1.155 1.156 1.156 0.02 18 1.189 1.190 1.189 1.189 1.189 1.190 0.04

[0162] 2.9 Repeatability Assay:

[0163] Six Shenqi Fuzheng injection of the same batch were taken, prepared according to the method of preparing the test sample solution, and then injected, respectively, followed by extracting the characteristic peaks and marking the retention time. Peak 12 of astragaloside IV was taken as peak S, and then the relative retention time of each of other characteristic peaks was calculated, The results showed that the RSD values of the relative retention time of each characteristic peak was all less than 1%, and the method has good repeatability. The results of the repeatability assay are shown in Table 5:

TABLE-US-00005 TABLE 5 Results of Repeatability Test 1 2 3 4 5 6 RSD (%) 1 0.512 0.511 0.511 0.512 0.511 0.517 0.45 2 0.537 0.536 0.536 0.536 0.536 0.542 0.45 3 0.581 0.581 0.580 0.580 0.581 0.586 0.40 4 0.619 0.619 0.618 0.619 0.618 0.624 0.38 5 0.655 0.655 0.655 0.655 0.655 0.661 0.37 6 0.711 0.710 0.710 0.710 0.709 0.716 0.36 7 0.782 0.782 0.781 0.782 0.781 0.786 0.25 8 0.815 0.815 0.815 0.815 0.815 0.820 0.25 9 0.843 0.844 0.844 0.844 0.844 0.848 0.18 10 0.922 0.922 0.922 0.922 0.922 0.923 0.03 11 0.972 0.972 0.972 0.972 0.972 0.972 0.01 S 1.000 1.000 1.000 1.000 1.000 1.000 0.00 13 1.045 1.044 1.045 1.044 1.044 1.044 0.02 14 1.074 1.075 1.075 1.075 1.074 1.074 0.03 15 1.096 1.097 1.097 1.096 1.096 1.096 0.04 16 1.129 1.129 1.130 1.129 1.128 1.129 0.05 17 1.156 1.156 1.156 1.156 1.155 1.155 0.04 18 1.190 1.190 1.190 1.190 1.190 1.189 0.04

[0164] 2.10 Intermediate Precision:

[0165] Shenqi Fuzheng injections taken from the same batch were assayed according to the method described above, respectively, except for under the variable factors such as on different dates and by different analysts.

[0166] 2.10.1 Different Analysis Dates:

[0167] Shenqi Fuzheng injections taken from the same batch were prepared according to the method of preparing test sample solution on different dates, respectively, and then three of the test sample solutions were injected in parallel, followed by extracting the characteristic peaks and marking the retention time. Peak 12 of astragaloside IV was taken as Peak S, and then the relative retention time of each of other characteristic peaks. The results showed that the RSD values of the relative retention time of each characteristic peak was all less than 1%, as shown in Table 6:

TABLE-US-00006 TABLE 6 Results of analysis on different dates Date 1 Date 2 RSD (%) 1 0.517 0.518 0.517 0.516 0.516 0.517 0.19 2 0.541 0.541 0.542 0.542 0.541 0.541 0.08 3 0.585 0.586 0.585 0.585 0.585 0.586 0.03 4 0.623 0.624 0.623 0.623 0.623 0.623 0.07 5 0.661 0.661 0.661 0.661 0.661 0.661 0.05 6 0.715 0.715 0.715 0.715 0.715 0.715 0.01 7 0.786 0.786 0.787 0.786 0.786 0.786 0.03 8 0.819 0.820 0.820 0.820 0.820 0.819 0.02 9 0.847 0.847 0.848 0.848 0.848 0.847 0.03 10 0.923 0.923 0.923 0.923 0.922 0.922 0.01 11 0.972 0.972 0.973 0.972 0.972 0.972 0.02 S 1.000 1.000 1.000 1.000 1.000 1.000 0.00 13 1.044 1.044 1.044 1.045 1.044 1.044 0.02 14 1.074 1.074 1.074 1.074 1.074 1.074 0.01 15 1.096 1.097 1.096 1.096 1.096 1.096 0.03 16 1.128 1.129 1.129 1.129 1.128 1.128 0.03 17 1.155 1.156 1.155 1.156 1.156 1.155 0.03 18 1.189 1.189 1.190 1.189 1.189 1.188 0.04

[0168] 2.10.2 Different Analysts:

[0169] Shenqi Fuzheng injections taken from the same batch were prepared according to the method of preparing test sample solution by different analysts, respectively, and three of the test sample solution were injected in parallel, followed by extracting the characteristic peaks and marking the retention time. Peak 12 of astragaloside IV was taken as Peak S, and then the relative retention time of each of other characteristic peaks was calculated. The results showed that the RSD values of the relative retention time of each characteristic peak was all less than 1%, as shown in Table 7:

TABLE-US-00007 TABLE 7 Results of analysis by different analysts Analyst 1 Analyst 2 RSD (%) 1 0.516 0.518 0.519 0.519 0.519 0.519 0.18 2 0.542 0.544 0.544 0.544 0.543 0.544 0.16 3 0.586 0.588 0.588 0.589 0.586 0.587 0.18 4 0.623 0.625 0.625 0.626 0.624 0.625 0.16 5 0.661 0.663 0.663 0.663 0.661 0.663 0.17 6 0.714 0.716 0.717 0.717 0.714 0.716 0.19 7 0.786 0.788 0.789 0.789 0.785 0.788 0.19 8 0.819 0.821 0.822 0.822 0.819 0.821 0.15 9 0.848 0.848 0.849 0.849 0.847 0.848 0.07 10 0.922 0.922 0.923 0.923 0.922 0.923 0.03 11 0.972 0.972 0.972 0.972 0.972 0.972 0.03 S 1.000 1.000 1.000 1.000 1.000 1.000 0.00 13 1.044 1.044 1.044 1.044 1.044 1.044 0.02 14 1.074 1.074 1.074 1.074 1.074 1.074 0.01 15 1.096 1.095 1.095 1.095 1.096 1.096 0.02 16 1.128 1.129 1.129 1.128 1.128 1.128 0.02 17 1.155 1.155 1.155 1.155 1.155 1.156 0.03 18 1.189 1.188 1.188 1.188 1.189 1.189 0.04

Example 2 Identification of Shenqi Fuzheng Injection

[0170] In recent years, as the use of Shenqi Fuzheng injection is increasing in clinical, some criminals are motivated by economic interest and counterfeit Shenqi Fuzheng injection with other varieties for sale to make huge profits, which results in a great negative impact on the brands for Shenqi Fuzheng injection, and has caused significant economic loss to these enterprises which produce and sell Shenqi Fuzheng injections legally. These counterfeit Shenqi Fuzheng injections have almost the same appearance as the real ones, so it is hard to distinguish the real from the fake.

[0171] In this example, the method described in Example 1 was adopted to test the certified Shenqi Fuzheng injection (provide by Livzon Group Limin Pharmaceutical Factory), a suspected sample and a Danshen injection, so as to establish the corresponding fingerprint profiles. The results were shown in FIG. 4. It can be seen that the fingerprint profile of the suspected sample is completely different from that of the certified Shenqi Fuzheng injection, the components of the counterfeit were inferred by using the precise molecular weight provided by mass spectrometry. It was substantially confirmed that the components of the counterfeit were derived from Danshen.

[0172] Thus, the Shenqi Fuzheng injection fingerprint profile established by ultra high pressure liquid chromatography-mass spectrometer (UHPLC-MS) can be used to identify the authenticity of a Shenqi Fuzheng injection in a fast and accurate manner, and can also be used to analyze the counterfeit qualitatively and substantially confirm the source. If there is any counterfeit Shenqi Fuzheng injection with DanShen injection by criminals, it can be identified according to the method described above, i.e. comparing the fingerprint profile of the injection with that of the certified Shenqi Fuzheng injection, inferring the components of the counterfeit by using the precise molecular weight provided by mass spectrometry, substantially confirming the component source of the counterfeit.

[0173] Therefore, application of the Shenqi Fuzheng injection UHPLC-MS fingerprint profile can avoid counterfeiting, and ensure normal production and good circulation of the Shenqi Fuzheng injection so as to protect legitimate rights and interests of manufactures.

Example 3 Detection of Saponins in the Shenqi Fuzheng Injection by HPLC-ELSD Method

[0174] 1. Preparation of Test Sample

[0175] In order to detect saponins in the test sample, the test sample was pretreated and concentrated. Therefore, Shenqi Fuzheng Injection (obtained from Livzon Group Limin Pharmaceutical Factory) was fractionated on a macroporous resin, eluted with water and different concentration of ethanol solution (firstly 30 v/v % ethanol solution and then 70 v/v % ethanol solution is used) to remove polysaccharides, protein and salts, and then concentrated, made up to the mark, filtered, 20 l filtrate was injected into a liquid chromatography for analysis.

[0176] 2. Preparation of Control Sample

[0177] (1) Selection of Control Sample

[0178] The main component of saponins in Shenqi Fuzheng Injection is astragaloside IV, therefore, in this study, astragaloside IV was selected as the control sample. Astragaloside IV was obtained from National Institutes for Food and Drug Control.

[0179] (2) Preparation of Control Solution Containing Astragaloside IV

[0180] An appropriate amount of astragaloside IV was accurately weighed, and then methanol was added to prepare a solution containing 0.10 mg/mL astragaloside IV as the control solution.

[0181] 3. Detection

[0182] Detection was performed by high performance liquid chromatography (Chinese Pharmacopoeia, Part I, Appendix VI D, 2010 edition).

[0183] (1) Instruments and Reagents

[0184] Waters 2695 high performance liquid chromatograph, Waters 2420 ELSD detector, Empower2 workstation; Precisa 925M-202A electronic balance; chromatographically pure acetonitrile (TEDIA, USA); redistilled water.

[0185] (2) Chromatographic Conditions and Detection

[0186] {circle around (1)} Chromatographic Conditions

[0187] The chromatographic column used was Discovery C-18 (5 m, 2504.6 mm) column. Acetonitrile (A) and water (B) was used as mobile phases. Bivariate linear gradient elution was performed according to the table 16 below. Column temperature was 30 C. Flow rate was 1.0 ml/min; The temperature of drift tube was 55 C. Atomizing power was 60%. Gain value was 20. Gas pressure was 25 psig. The theoretical plate number should not be less than 3000 base on astragaloside IV.

TABLE-US-00008 TABLE 16 Time (minute)) Mobile phase A (%) Mobile phase B (%) Note 0~30 12.fwdarw.25 88.fwdarw.75 Equilibrating 30~40 25.fwdarw.32 75.fwdarw.68 the column 40~55 32.fwdarw.55 68.fwdarw.45 55~68 55.fwdarw.90 45.fwdarw.10 68~72 90 10 72~75 90.fwdarw.12 10.fwdarw.88 75~83 12 88

[0188] Detection Method

[0189] 20 l of the solution of the control sample and the solution of the test sample were accurately taken respectively, and then injected into the high performance liquid chromatography to detect, the chromatogram for 70 minutes was recorded.

[0190] a. Determination of Mobile Phase

[0191] Acetonitrile and water were used as the mobile phase, the effect of different gradient elution programs (shown in Table 8-11) was evaluated and the results were shown in FIG. 5-8.

TABLE-US-00009 TABLE 8 A % Time (min) (acetonitrile) B % (water) 0 2 98 80 100 0

TABLE-US-00010 TABLE 9 A % Time (min) (acetonitrile) B % (water) 0 2 98 8 5 95 18 12 88 40 25 75 50 32 68 60 48 52 65 55 45 70 95 5 75 2 98

TABLE-US-00011 TABLE 10 Time (min) A % (acetonitrile) B % (water) 0 25 75 5 25 75 30 55 45 35 55 45 40 90 10 45 90 10 50 25 75 60 25 75

TABLE-US-00012 TABLE 11 A % Time (min) (acetonitrile) B % (water) 0 12 88 30 25 75 40 32 68 55 55 45 68 90 10 72 90 10

[0192] It can be seen from the results that the condition shown in table 11 is desirable for good peak shape and flat baseline.

[0193] b. Determination of Chromatographic Column

[0194] The effect of different types of column (Kromasil column, Diamonsil column and Discovery column) were evaluated was evaluated respectively. The results were shown in FIG. 9-11. It can be seen from the results that Discovery column was superior to other columns in separating degree and peak shape.

[0195] c. Determination of Flow Rate

[0196] The effect of different flow rate (0.8, 1.0 and 1.2 ml/min) was evaluated respectively. The results were shown in FIG. 12-14. It can be seen from the results that the separating degree and peak number were desirable under the flow rate of 1.0 ml/min.

[0197] d. Determination of Injection Volume

[0198] The effect of different injection volume (10 l and 20 l) was evaluated. The results were shown in FIG. 15-16. It can be seen from the results that 20 l of injection volume can meet the requirement.

[0199] e. Determination of Pretreatment Method

[0200] The effect of different pretreatment method (extraction with n-butanol, purification on D.sub.101 macroporous resin directly and purification on D.sub.101 macroporous resin after concentration) was evaluated. The results were shown in FIG. 17-19. It can be seen from the results that the chromatogram obtained by purification on D.sub.101 macroporous resin directly was better, and the method was simple and easy to operate.

[0201] f. Determination of Collecting Time

[0202] 20 l of the test sample solution was accurately taken, and then injected into the high performance liquid chromatography to detect, the chromatogram for 120 minutes was recorded (see FIG. 20). It can be seen from the results that there were no chromatographic peaks after 70 minutes.

[0203] g. Chromatogram of Control Samples

[0204] The chromatogram of control samples were shown in FIG. 21-25.

[0205] h. Methodological Investigation

[0206] Precision assay: The test sample solution from the same batch of Shenqi Fuzheng injection (1310907) was prepared according to the method of preparing the test sample solution, injected for continuously 6 times (20 l each time), the chromatogram of each time was evaluated. The results were shown in table 12. The results showed that the RSD values of the peak area of each characteristic peak were all less than 3%, which meet the requirements of fingerprint.

TABLE-US-00013 TABLE 12 Peak Nos. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 RSD (%) 1 106726 107456 105375 105299 106357 103779 1.23 2 47169 48259 48067 46995 49608 48083 1.94 3 676756 678533 697947 695351 680992 681028 1.33 4 111327 114908 112780 115608 110807 114194 1.72 5 32065 31124 32754 31971 31653 31742 1.69 6 32526 33830 34814 34093 34722 33257 2.59

[0207] Stability Assay:

[0208] This experiment is used to evaluate the Stability of the test samples. The test sample solution from the same batch of Shenqi Fuzheng injection (1310907) was prepared according to the method of preparing the test sample solution, the detection were performed at 0 hour, 4 hour, 12 hours, 16 hours and 24 hours respectively. The results were shown in table 13. The results showed that the RSD values of the peak area of each characteristic peak were all less than 3%, which meet the requirements of fingerprint.

TABLE-US-00014 TABLE 13 Peak Nos. 0 h 4 h 12 h 16 h 24 h RSD (%) 1 103110 103915 104753 104582 100935 1.50 2 47487 46044 45048 47213 45963 2.15 3 674093 682926 674569 678924 694969 1.25 4 105154 108038 105012 103491 108602 2.05 5 31502 31883 31634 30861 31443 1.20 6 32204 32363 32467 31785 33315 1.73

[0209] Repeatability Assay

[0210] Six Shenqi Fuzheng injection of the same batch were taken (1310907), prepared according to the method of preparing the test sample solution, and then injected, respectively, the respective chromatogram was recorded. The results were shown in table 14. The results showed that the RSD values of the peak area of each characteristic peak were all less than 3%, which meet the requirements of fingerprint.

TABLE-US-00015 TABLE 14 Peak No. Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 RSD (%) 1 103915 105463 105785 102326 101069 106726 2.10 2 46044 48468 47416 46653 45984 47169 2.00 3 682926 688582 668801 675287 679817 676756 1.00 4 108038 108716 111045 107912 108836 111327 1.37 5 31883 31708 31019 31492 30739 32065 1.63 6 32363 32557 33979 32916 33906 32526 2.18

[0211] 4. Fingerprint Spectrum and Technical Parameters

[0212] Similarity

[0213] Six chromatographic peaks of 1, 2, S, 3, 4 and 5 in the control sample and test sample were corrected, and then the similarity was calculated, which should not be less than 0.95.

[0214] Setting of Parameters for Similarity

[0215] (1) Software: Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004A) compiled by the National Pharmacopoeia Society was used.

[0216] (2) Reference spectrum: Referring to document of Reference Spectrum for Calculation. Scp.

[0217] (3) Time window width: 0.10

[0218] (4) Data cutting: the chromatographic peaks before 38 minutes and after 65 minutes in the fingerprint spectrum were cut.

[0219] (5) Correction of chromatographic peaks: Multi-point correction between the reference spectrum and six peaks of 1, 2, S, 3, 4 and 5 was performed.

[0220] Identification of main chromatographic peaks Six main saponins in Shenqi Fuzheng Injection were identified by HPLC-ELSD and HPLC-MS.sup.n.

[0221] 5. Determination of Limit Value of Similarity

[0222] The fingerprints of 15 batches of Shenqi Fuzheng injections were investigated. The results of calculation for similarity were shown in Table 15. Considering the actual condition and the statistical result of table 15 and other many factors, the limit value of similarity was provisionally set as 0.85.

TABLE-US-00016 TABLE 15 Batch Nos. Similarity 121201 1.000 121202 0.996 121203 0.998 130101 0.999 130102 0.999 130803 0.999 130804 0.993 130805 0.990 1305443 0.998 1310025 0.999 1310026 0.998 1310027 0.997 1310907 0.995 1310908 0.995 1310909 0.996 Minimum: 0.990 Maximum: 1.000 Average: 0.997

[0223] The method provided by the present invention for establishing Shenqi Fuzheng injection fingerprint profile was described in details hereinbefore. The principles and embodiments of the present invention are described herein with reference to some specific examples, however, the examples described above are only intended to help understand the method and the core idea of the present invention. It should be noted that, for those skilled in the art, a number of improvements and modifications can be introduced to the present invention, without departing from the principles of the present invention, and these improvements and modifications shall fall into the scope defined by the appended claims.