Fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and method for determining contents of 12 bile acids

Abstract

The invention provides a fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and a method for determining the content of 12 bile acids, and belongs to the field of medicine detection. The determination method provided by the invention adopts an HPLC-CAD method. The present invention provides a compound paracetamol and amantadine hydrochloride tablet fingerprint spectrum determination method, the method is an HPLC-CAD method, the determination method can simultaneously detect the contents of 12 bile acids in the compound paracetamol and amantadine hydrochloride tablet, and the 12 bile acids have a good linear relationship (r is greater than or equal to 0.999) in respective detection concentration ranges; meanwhile, the detection limit and the quantitation limit of the determination method are very low, and the detection sensitivity is good; the determination method has good precision, stability and repeatability (RSD is less than or equal to 2.0%). The determination method is simple, feasible, rapid and accurate, can provide reference for quality evaluation of the compound paracetamol and amantadine hydrochloride tablets, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of drug detection, and particularly relates to a fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and a method for determining the content of 12 bile acids. Background Art

[0002] Bile acids are derivatives of cholanic acids with a steroid structure, composed of a steroid ring and a fatty side chain, and have the dual characteristics of lipophilicity and hydrophilicity. Bile acids are a kind of bioactive components and have a variety of important physiological and pharmacological effects, such as regulating metabolism, treating hepatobiliary diseases, regulating immunity and inflammatory responses, and so on.

[0003] Compound paracetamol and amantadine hydrochloride tablets are a commonly used over-the-counter (OTC) drug, mainly used to relieve symptoms such as nasal congestion, runny nose, sneezing, sore throat, headache, fever, etc. caused by colds. Its main components include paracetamol, amantadine hydrochloride, artificial bezoar, caffeine, and chlorpheniramine maleate. Artificial bezoar contains various bile acid components, which are also the main active components in artificial bezoar and are closely related to pharmacological effects such as anti-inflammatory, anti-pathogenic microorganisms, antiviral, and anti-asthmatic and cough-suppressing effects. Therefore, detecting the bile acid components in compound paracetamol and amantadine hydrochloride tablets helps to evaluate the effectiveness of the drug.

[0004] There are no conjugated double bonds in the molecular structure of bile acid components, and they have almost no absorption in the ultraviolet-visible region. The determination of their content mainly uses colorimetry, thin-layer scanning method, high-performance liquid chromatography (HPLC) method, etc. In HPLC method, it is often combined with various universal detectors such as charged aerosol detector (CAD), evaporative light scattering detector (ELSD), refractive index detector (RID), mass spectrometry detector (MS), etc. At present, the methods for detecting bile acid components not only have problems such as high instrument cost and complex operation, but more importantly, it is difficult to achieve chromatographic separation of various bile acid components.

[0005] In addition, the fingerprint spectrum can comprehensively reflect the chemical composition characteristics of the sample, and by analyzing the overall information of multiple components, it provides a more comprehensive quality evaluation. Therefore, providing the fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets is of great significance for its quality control and evaluation.

[0006] How to provide a detection method that can reflect the fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and can detect multiple bile acid components simultaneously as much as possible needs further research. Summary of the Invention

[0007] The purpose of the present invention is to provide a fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and a method for determining the content of 12 bile acids.

[0008] The present invention provides a fingerprint of compound paracetamol and amantadine hydrochloride tablets and a method for determining the content of 12 bile acids. It is detected by HPLC-CAD method, and specifically includes the following steps:

[0009] 1) Preparation of test solution: Take the compound paracetamol and amantadine hydrochloride tablets to be tested, add an alcohol organic solvent for extraction, filter, and take the subsequent filtrate to obtain;

[0010] 2) Inject the test solution into a high performance liquid chromatograph for detection, and record the chromatogram. The chromatographic conditions are as follows:

[0011] Chromatographic column: Packed with octadecylsilane chemically bonded silica gel; Mobile phase: Take trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B.

[0012] Furthermore,

[0013] In the step 1), the volume-mass ratio of the alcohol organic solvent to artificial bezoar in the compound paracetamol and amantadine hydrochloride tablets is (0.5 - 5) mL: 1 mg;

[0014] And / or, in the step 1), the extraction method is ultrasonic extraction, the ultrasonic extraction time is 15 - 60 min, the ultrasonic extraction frequency is 50 - 80 kHz, and the ultrasonic extraction power is 200 - 500 W;

[0015] And / or, in the step 1), before filtration, add water to dilute to the concentration of artificial bezoar in the compound paracetamol and amantadine hydrochloride tablets being 0.1 - 1 mg / mL.

[0016] Furthermore, in the step 1), the alcohol organic solvent is ethanol or an ethanol solution containing acetic acid.

[0017] Preferably, the acetic acid concentration in the ethanol solution containing acetic acid is 0.1 - 1%.

[0018] Furthermore, in the determination method, it also includes preparing a reference solution and injecting the reference solution into a high performance liquid chromatograph for detection; the preparation method of the reference solution is to take a reference substance and dissolve it with an alcohol organic solvent to obtain.

[0019] Furthermore, the reference substances are taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy-cholanic acid, hyodeoxycholic acid, sodium glycocholate, glycoursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, sodium taurocholate, glycocholic acid, cholic acid, and hyodeoxycholic acid; the reference solution is a reference solution containing taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy-cholanic acid, hyodeoxycholic acid, glycoursodeoxycholic acid, glycoursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, sodium taurocholate, glycocholic acid, cholic acid, and hyodeoxycholic acid.

[0020] Further,

[0021] the concentrations of taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy cholanic acid, hyodeoxycholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, chenodeoxycholic acid, and deoxycholic acid are 0.5 to 5 μg·mL -1 ;

[0022] the concentrations of taurocholic acid and glycine cholic acid are 2.5 to 25 μg·mL -1 ;

[0023] the concentrations of cholic acid and hyodeoxycholic acid are 5 to 50 μg·mL -1 。

[0024] Further, when preparing the reference substance solution, the alcoholic organic solvent is methanol, methanol aqueous solution, ethanol, or ethanol aqueous solution;

[0025] Preferably, when preparing the reference substance solution, it is first dissolved in methanol and then diluted with an ethanol aqueous solution.

[0026] Further, in step 2), the elution mode of high performance liquid chromatography is gradient elution, and the elution conditions are:

[0027]

[0028]

[0029] Further, in step 2), the high performance liquid chromatography conditions are:

[0030] Chromatographic column: C 18 chromatographic column; column temperature: 25 to 35 °C; flow rate: 0.8 to 1.5 ml / min; injection volume: 5 to 20 μL; mobile phase A: aqueous solution containing 0.05% trifluoroacetic acid; mobile phase B: acetonitrile;

[0031] The CAD detector acquisition frequency is 5 to 10 Hz, the filter constant is 1 to 5 s, and the atomization temperature is 35 to 60 °C.

[0032] Further, the chromatographic column is Agilent InfinityLab Poroshell 120 EC-C18 or HPH-C18;

[0033] and / or, the column temperature is 30 °C;

[0034] and / or, the flow rate is 1 ml / min;

[0035] and / or, the injection volume is 10 μL;

[0036] And / or, the acquisition frequency of the CAD detector is 5 Hz, the filtering constant is 3.6 s, and the atomization temperature is 50 °C.

[0037] The present invention has achieved the following beneficial effects:

[0038] The present invention provides a method for determining the fingerprint of compound paracetamol and amantadine tablets. This method is HPLC-CAD. This determination method can also simultaneously detect the contents of 12 bile acids in compound paracetamol and amantadine tablets. These 12 bile acids all have good linear relationships (r≥0.999) within their respective detection concentration ranges; at the same time, the detection limit and quantification limit of this determination method are both very low, and the detection sensitivity is good; the precision, stability, and repeatability of this determination method are also very good (RSD≤2.0%). The determination method of the present invention is simple, feasible, fast, and accurate, can provide a reference for the quality evaluation of compound paracetamol and amantadine tablets, and has good application prospects.

[0039] Obviously, based on the above content of the present invention, according to the common general knowledge and conventional means in the art, without departing from the above basic technical idea of the present invention, various other forms of modification, substitution, or variation can also be made.

[0040] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Description of the Drawings

[0041] Figure 1 Chromatograms of the standard curve S5 (A), the test sample without SDS (B), the test sample with SDS (C), and the negative sample (D) solutions; in the figure, 1 is TCA, 2 is TCDCA, 3 is TDCA, 4 is GCA, 5 is KDHCA, 6 is HCA, 7 is CA, 8 is GCDCA, 9 is HDCA, 10 is GDCA, 11 is CDCA, and 12 is DCA.

[0042] Figure 2 It is the cluster analysis chart of the samples.

[0043] Figure 3 It is the score chart of the samples.

[0044] Figure 4 It is the chromatogram of 33 batches of samples and the reference fingerprint (R).

[0045] Figure 5 It is the influence of different preparation methods of the test sample solutions on the total bile acid content (calculated by the total bile acid content per tablet, mg).

[0046] Figure 6Chromatogram after detection by 0.2% acetic acid - methanol system.

[0047] Figure 7 Chromatogram after detection by 0.1% formic acid - methanol system.

[0048] Figure 8 Chromatogram after detection by 0.05% trifluoroacetic acid - methanol system.

[0049] Figure 9 Chromatogram after detection by 0.35% formic acid - methanol system.

[0050] Figure 10 Chromatogram after detection by 0.35% formic acid - acetonitrile system.

[0051] Figure 11 Chromatogram of 0.05% trifluoroacetic acid - acetonitrile (65:35) isocratic elution.

[0052] Figure 12 Chromatogram of the first gradient elution mode of 0.05% trifluoroacetic acid - acetonitrile.

[0053] Figure 13 Chromatogram of the second gradient elution mode of 0.05% trifluoroacetic acid - acetonitrile.

[0054] Figure 14 Chromatogram with the chromatographic column being Agilent InfinityLab Poroshell 120EC - C18.

[0055] Figure 15 Chromatogram with the chromatographic column being Waters CORTECS C18.

[0056] Figure 16 Chromatogram with the chromatographic column being NanoChrom ChromCore C18.

[0057] Figure 17 Chromatogram with the chromatographic column being Agilent InfinityLab Poroshell 120HPH - C18.

[0058] Figure 18 Chromatogram with the chromatographic column being Agilent InfinityLab Poroshell 120Aq - C18. Detailed implementation mode

[0059] The raw materials and equipment used in the present invention are all known products and are obtained by purchasing commercially available products.

[0060] Vanquish UHPLC high performance liquid chromatograph (Thermo Fisher Scientific, USA), Corona Veo RS charged aerosol detector (Thermo Fisher Scientific, USA), CPA225D electronic balance (Sartorius, Germany), SK5200HP ultrasonic cleaner (Shanghai Kedao), Milli-Q deionized water generator (Millipore, USA).

[0061] Sodium taurocholate (batch number 110815 - 202412, purity 98.1%), chenodeoxycholic acid (batch number 110806 - 202310, purity 97.9%), paracetamol (batch number 100018 - 202312, purity 100.0%), caffeine (batch number 171215 - 202013, purity 99.7%), chlorpheniramine maleate (batch number 100047 - 202008, purity 99.7%) reference substances and artificial bezoar (batch number 121197 - 201905) reference medicinal materials were all purchased from the National Institutes for Food and Drug Control; 7-keto-3α,12-α-dihydroxy cholanic acid (batch number F23301152, purity 99.82%) reference substance was purchased from aladdin company; hyocholic acid (batch number 0422 - RF - 0085, purity 96.9%), glycocholic acid (batch number 0517 - RC - 0009, purity 95.1%), hyodeoxycholic acid (batch number 0825 - RC - 0003, purity 100.0%), cholic acid (batch number 1125 - RD - 0062, purity 100.0%), amantadine hydrochloride (batch number 0718 - RD - 0016, purity 99.6%) reference substances were all purchased from Guangzhou Jiantu Technology Co., Ltd.; deoxycholic acid (batch number 22 - 04 - 0301, purity 96.53%), glycochenodeoxycholic acid (batch number 22 - 04 - 2006, purity 95.48%), sodium taurochenodeoxycholate (batch number 23 - 06 - 0501, purity 93.87%), sodium glycochenodeoxycholate (batch number 23 - 12 - 1106, purity 89.21%) reference substances were all purchased from SINCO PHARMACHEM company; sodium taurodeoxycholate (batch number YT230819 - 22, purity 96.07%) reference substance was purchased from stanford Chemicals company. Methanol, acetonitrile, ethanol, and trifluoroacetic acid were of chromatographic grade; water was Millipore water; the rest of the reagents were of analytical grade. There were 33 batches of compound paracetamol and amantadine tablets, and the specific batch numbers are shown in Table 5.

[0062] Example 1, Fingerprint spectrum of the compound paracetamol and amantadine tablets of the present invention and determination method for the content of 12 bile acids (HPLC - CAD method)

[0063] 1. Chromatographic conditions

[0064] Chromatographic column: InfinityLab Poroshell 120 EC-C18 (4.6 mm × 150 mm, 2.7 μm);

[0065] An aqueous solution containing 0.05% trifluoroacetic acid was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient elution conditions are shown in Table 1;

[0066] Column temperature was 30 °C; CAD detector, acquisition frequency was 5 Hz, filter constant was 3.6 s, and nebulization temperature was 50 °C; flow rate was 1.0 mL·min -1 ; injection volume was 10 μL.

[0067] Table 1. Gradient elution conditions

[0068] Time (min) <![CDATA[Flow rate (1.0 mL·min -1 )]]> Mobile Phase A (%) Mobile Phase B (%) 0 1.0 65 35 8 1.0 60 40 18 1.0 50 50 22 1.0 25 75 25 1.0 25 75 25.1 1.0 65 35 32 1.0 65 35

[0069] 2. Solution preparation

[0070] (1) Single reference stock solution: Weigh an appropriate amount of 12 bile acid reference substances accurately, dissolve them in methanol respectively, and then quantitatively dilute them with water to prepare single reference stock solutions with a concentration of 1 mg·mL -1 .

[0071] (2) Standard curve solution (mixed reference solution): Accurately pipette appropriate amounts of each single reference stock solution of bile acids respectively, mix them, and then quantitatively dilute them with 40% ethanol aqueous solution to prepare a series of standard curve solutions S1 - S10. In the standard curve solutions S1 - S10, taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), 7-keto-3α,12-α-dihydroxy-cholanic acid (KDHCA), hyocholic acid (HCA), glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GDCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA) were contained at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5 μg·mL -1 , taurocholic acid (TCA), glycine cholic acid (GCA) were contained at 2.5, 5.0, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 μg·mL -1 , and cholic acid (CA), hyodeoxycholic acid (HDCA) were contained at 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 μg·mL -1 .

[0072] (3) Test sample solution: Weigh accurately an appropriate amount of the finely powdered test sample (equivalent to about 1 tablet of compound paracetamol and amantadine hydrochloride tablets, containing 10 mg of artificial bezoar), place it in a 25 mL volumetric flask, add 10 mL of ethanol, extract ultrasonically (53 kHz, 200 W) for 30 minutes, add 10 mL of water, cool to room temperature, dilute to the mark with water, shake well, filter, and take the subsequent filtrate as the test sample solution.

[0073] (4) Negative sample solution: Take the self - prepared negative sample without artificial bezoar (prepared according to the formula for compound paracetamol and amantadine hydrochloride tablets, but without adding artificial bezoar), and prepare the negative sample solution according to the preparation method of the test sample solution.

[0074] 3. Detection method

[0075] Respectively take the standard curve solution, the test sample solution, and the negative sample solution, and inject them for detection according to the conditions under "1. Chromatographic conditions". Finally, obtain the fingerprint chromatogram of the test sample (compound paracetamol and amantadine hydrochloride tablets), and conduct qualitative and quantitative analysis of 12 bile acids in the test sample based on the chromatogram.

[0076] 4. Specificity test

[0077] Respectively take the standard curve solution, the test sample solution, and the negative sample solution, and inject them for detection according to the conditions under "1. Chromatographic conditions". The chromatograms of each sample are as Figure 1 shown. Figure 1 They are the chromatograms of the standard curve S5 (A), the test sample without SDS (B), the test sample with SDS (C), and the negative sample (D) solution. The test sample without SDS is the sample without SDS in Table 5, and the test sample with SDS is the sample with SDS in Table 5.

[0078] It can be seen from Figure 1 that: The resolution of each component chromatographic peak is greater than 1.5, and the negative sample has no interference. In addition, the test sample containing sodium dodecyl sulfate (SDS) shows SDS peaks at about 18 min and 24 min, which have no interference on the 12 bile acid components. This method has good specificity.

[0079] 5. Investigation of linear relationship

[0080] Take a series of standard curve solutions S1 - S10, inject them for determination according to the conditions under "1. Chromatographic conditions", take the mass concentration of the reference substance as the abscissa (X), the peak area as the ordinate (Y), and perform linear regression with a weight of 1 / X. The results are shown in Table 2, indicating that each component has a good linear relationship within its respective range.

[0081] Table 2. Linear regression equations, correlation coefficients, linear ranges, detection limits, and quantification limits of 12 components

[0082]

[0083]

[0084] 6. Detection Limit and Quantitation Limit

[0085] Appropriately take a single reference substance stock solution, quantitatively dilute it with 40% ethanol aqueous solution to prepare a mixed reference substance stock solution containing 2 μg·mL -1 of each component. After successive dilutions, inject samples for determination according to the conditions under "1. Chromatographic Conditions". Take the mass concentrations corresponding to 3 and 10 times the signal-to-noise ratio as the detection limit and quantitation limit respectively. The results are shown in Table 2. It can be seen from Table 2 that the detection limit and quantitation limit of the detection method of the present invention are both very low, indicating good detection sensitivity.

[0086] 7. Tests on Precision, Repeatability and Stability

[0087] Take the standard curve solution S5 and inject samples continuously 6 times according to the conditions under "1. Chromatographic Conditions" to examine the precision of the method.

[0088] Take the No. 14 sample (shown in Table 5), prepare 6 portions of test solution in parallel according to the preparation method of test solution under "2. Solution Preparation", and inject samples for determination respectively according to the conditions under "1. Chromatographic Conditions" to examine the repeatability of the method.

[0089] Take the standard curve solution S5 and the No. 14 test solution, inject samples at 0, 3, 6, 10, 24, 48 h and 0, 1, 2, 4, 8, 12 h respectively to examine the stability.

[0090] The results are shown in Table 3. It can be seen from Table 3 that when the detection method of the present invention determines each bile acid, the RSDs of precision, repeatability and stability are all ≤2.0%, indicating that the precision, repeatability and stability of the detection method of the present invention are all good.

[0091] Table 3. Results of Precision, Repeatability and Stability Tests (n = 6)

[0092]

[0093] 8. Recovery Test of Added Samples

[0094] Take the No. 14 sample (shown in Table 5), add reference substances respectively to prepare recovery test solutions of added samples, inject samples for determination respectively according to the conditions under "1. Chromatographic Conditions", and calculate the recovery rates of added samples. The recovery rates and RSDs of 12 components are shown in Table 4.

[0095] Table 4. Results of Recovery Tests of 12 Components (μg·mL -1 , n = 9)

[0096]

[0097]

[0098]

[0099]

[0100] As can be seen from Table 4, the average sample addition recovery rate is 95.4% - 104.5%, and the RSDs are all ≤ 3%. This indicates that the detection method of the present invention has high accuracy, good repeatability and stability, and reliable detection results.

[0101] 9. Determination of sample content

[0102] Take 33 batches of samples, prepare the test solution according to the test solution preparation method under "2. Solution Preparation", inject and determine under the chromatographic conditions in "1. Chromatographic Conditions", and calculate the content by the standard curve method. The results are shown in Table 5.

[0103] Table 5. Determination results of 12 components' contents (mg·tablet -1 )

[0104]

[0105]

[0106] 10. Cluster analysis

[0107] Import the content determination results into the OriginPro2021 software. After data standardization, select the Ward connection and perform a systematic classification of the similarity degree of the samples with the Euclidean square distance. As Figure 2 can be seen: when the distance is 100, the 33 batches of samples can be clustered into 3 major categories, indicating that the content differences of each component in the samples may be related to the quality of the raw materials.

[0108] 11. Principal component analysis

[0109] Perform principal component analysis on the content determination results using SPSS22 and OriginPro2021 software. The variance ratios of the first principal component (PC1), the second principal component (PC2), the third principal component (PC3), and the fourth principal component (PC4) are 32.75%, 26.73%, 14.63%, and 8.42% respectively, and the cumulative variance reaches 82.54%; it can be seen from the rotated component matrix (Table 6) that TDCA, CA, TCA, and CDCA contribute greatly to PC1, GDCA, GCDCA, and GCA contribute greatly to PC2, DCA, KDHCA, HDCA, and HCA contribute greatly to PC3, and TCDCA contributes greatly to PC4. From the score plot ( Figure 3 ), it can be seen that there are no abnormal samples that deviate significantly from other samples, indicating that the product quality is relatively consistent.

[0110] Table 6. Rotation Component Matrix

[0111]

[0112]

[0113] 12. Establishment of Fingerprint and Evaluation of Similarity

[0114] The HPLC chromatograms of 33 batches of samples were imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)". The chromatogram of No. 14 sample was set as the reference chromatogram, the time window was set to 0 - 23 min, the time width was set to 0.1 min. After multi-point calibration of chromatographic peaks, Mark peak matching was adopted, and the HPLC superposition chromatogram and reference chromatogram (R) of bile acid components in compound paracetamol and amantadine hydrochloride tablets were generated by the average value method. The results are shown in Figure 4 . The RSD of the peak areas of 12 common peaks was 12.1% - 85.9%, and the proportion of non-common peaks was 2.5% - 18.0%. The similarities (Table 7) of the HPLC fingerprints of 33 batches of samples and the reference chromatogram (R) were all greater than 0.95, indicating that the differences between samples from different enterprises and batches were small, and the product quality was relatively stable and controllable. It also shows that the detection method of the present invention has good stability.

[0115] Table 7. Similarities of 33 Batches of Samples

[0116]

[0117] As can be seen from the above: The 12 bile acid components detected by the present invention are: taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), 7-keto-3α,12-α-dihydroxy cholanic acid (KDHCA), hyocholic acid (HCA), glycochenodeoxycholic acid (GCDCA), glycoursodeoxycholic acid (GDCA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), taurocholic acid (TCA), glycine cholic acid (GCA), cholic acid (CA), hyodeoxycholic acid (HDCA). The present invention can simultaneously detect the contents of the above 12 bile acids in compound paracetamol and amantadine hydrochloride tablets, and these 12 bile acids all have good linear relationships (r≥0.999) within the detection concentration range. The detection limit and quantification limit of the detection method of the present invention are very low, and the detection sensitivity is good; moreover, the precision, stability and repeatability of the detection are good (RSD≤2.0%), and the detection results are reliable.

[0118] The beneficial effects of the present invention are demonstrated by the following specific test examples.

[0119] Test Example 1. Screening of Sample Extraction Methods

[0120] Referring to the preparation method of the test solution under "2. Solution Preparation" in Example 1, only replace ethanol with methanol, methanol containing 0.1% acetic acid, and ethanol containing 0.1% acetic acid respectively. Study the detection effects of the test solutions prepared with the same amount of different extraction solvents.

[0121] Referring to the preparation method of the test solution under "2. Solution Preparation" in Example 1, only change the amount of ethanol (10 mL) to 15 mL and 20 mL. Study the detection effects of the test solutions prepared with different amounts of extraction solvents.

[0122] Referring to the preparation method of the test solution under "2. Solution Preparation" in Example 1, only change the ultrasonic time (30 min) to 10, 15, 45, and 60 min, and study the detection effects of the test solutions prepared with different ultrasonic times.

[0123] The test sample in this test example is compound paracetamol and amantadine hydrochloride tablets, and injection and determination are carried out according to the conditions under "1. Chromatographic Conditions" in Example 1. After determination, calculate the total bile acid content of each group (calculated as the total bile acid content per tablet, mg).

[0124] The results are as Figure 5 shown: It was found that the extraction effects of ethanol and ethanol (containing 0.1% acetic acid) were slightly better than those of methanol and methanol (containing 0.1% acetic acid), and the effects of ethanol and ethanol (containing 0.1% acetic acid) were equivalent. The extraction effects were equivalent when the ethanol amounts were 10, 15, and 20 mL. The ultrasonic times were investigated for 10, 15, 30, 45, and 60 min, and it was found that the extraction effects were equivalent after 15 min (see Figure 5 ). In summary, 10 mL of ethanol was selected for ultrasonic extraction for 30 min as the sample extraction method.

[0125] Test Example 2. Screening of Chromatographic Conditions

[0126] 1. Using artificial bezoar as the sample, compare the influence of different mobile phases on the separation effect. The different mobile phases are 0.1% formic acid - methanol system, 0.2% acetic acid - methanol system, and 0.05% trifluoroacetic acid - methanol system.

[0127] In the 0.1% formic acid - methanol system, mobile phase A is an aqueous solution of 0.1% formic acid, and mobile phase B is methanol;

[0128] In the 0.2% acetic acid - methanol system, mobile phase A is an aqueous solution of 0.2% acetic acid, and mobile phase B is methanol;

[0129] In the 0.05% trifluoroacetic acid - methanol system, mobile phase A is an aqueous solution of 0.05% trifluoroacetic acid, and mobile phase B is methanol;

[0130] The gradient elution conditions and other chromatographic conditions are the same.

[0131] The detection results with different mobile phases are as follows Figures 6 - 8 shown: It was found that the separation effects of the 0.1% formic acid - methanol system and the 0.2% acetic acid - methanol system were comparable, and the 0.05% trifluoroacetic acid - methanol system had a higher response value.

[0132] 2. Using compound paracetamol and amantadine tablets as samples, compare the difference in separation effects between methanol and acetonitrile. Specifically, the 0.35% formic acid - methanol system and the 0.35% formic acid - acetonitrile system were used as mobile phases respectively.

[0133] In the 0.35% formic acid - methanol system, mobile phase A was an aqueous solution of 0.35% formic acid, and mobile phase B was methanol;

[0134] In the 0.35% formic acid - acetonitrile system, mobile phase A was an aqueous solution of 0.35% formic acid, and mobile phase B was acetonitrile;

[0135] The gradient elution conditions and other chromatographic conditions were the same.

[0136] The detection results with different mobile phases are as follows Figures 9 - 10 shown, and it was found that the peak shape of acetonitrile was better than that of methanol.

[0137] 3. Using compound paracetamol and amantadine tablets as samples, the gradient elution program was screened as follows:

[0138] ① Using 0.05% trifluoroacetic acid - acetonitrile as the mobile phase, 0.05% trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and a mixture of 0.05% trifluoroacetic acid and acetonitrile in a volume ratio of 65:35 as the mobile phase for isocratic elution.

[0139] ② Using 0.05% trifluoroacetic acid - acetonitrile as the mobile phase, 0.05% trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and the first gradient elution program is shown in Table 8;

[0140] Table 8. Gradient elution program

[0141] Time (min) <![CDATA[Flow rate (1.0 mL·min -1 )]]> 0.05% Trifluoroacetic Acid (%) Acetonitrile (%) 0 1.0 70 30 1 1.0 70 30 1.1 1.0 66 34 18 1.0 64 36 27 1.0 40 60 30 1.0 40 60 30.1 1.0 70 30 36 1.0 70 30

[0142] ③ Using 0.05% trifluoroacetic acid - acetonitrile as the mobile phase, 0.05% trifluoroacetic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and the second gradient elution program is shown in Table 9.

[0143] Table 9. Gradient elution program

[0144]

[0145]

[0146] Other chromatographic conditions were the same.

[0147] The results are as followsFigures 11 - 13 As shown, the result Figure 13 The gradient program used has a shorter time, better separation, and sharper peaks.

[0148] 4. Use a mixed reference solution to screen the chromatographic column. The chromatographic columns are Agilent InfinityLab Poroshell 120 EC-C18, Waters CORTECS C18, NanoChrom ChromCore C18, Agilent InfinityLab Poroshell 120 HPH-C18, and Agilent InfinityLab Poroshell 120 Aq-C18.

[0149] Other chromatographic conditions are the same.

[0150] The result is as Figures 14 - 18 shown. It is found that the Agilent InfinityLab Poroshell 120 EC-C18 and HPH-C18 have better peak elution times and separation effects under the selected gradient elution program.

[0151] In summary, the present invention provides a method for determining the fingerprint of compound paracetamol and amantadine tablets. This method is the HPLC-CAD method. This determination method can also simultaneously detect the contents of 12 bile acids in compound paracetamol and amantadine tablets. These 12 bile acids have good linear relationships (r≥0.999) within their respective detection concentration ranges; at the same time, the detection limit and quantification limit of this determination method are very low, and the detection sensitivity is good; the precision, stability, and repeatability of this determination method are also very good (RSD≤2.0%). The determination method of the present invention is simple, feasible, rapid, and accurate, can provide a reference for the quality evaluation of compound paracetamol and amantadine tablets, and has good application prospects.

Claims

1. Fingerprint spectrum of compound paracetamol and amantadine hydrochloride tablets and determination method for contents of 12 bile acids, characterized in that: It is detected by the HPLC-CAD method, which specifically includes the following steps: 1) Preparation of the test solution: Take the compound paracetamol and amantadine hydrochloride tablets to be tested, add an alcohol organic solvent for extraction, filter, and take the subsequent filtrate to obtain it; 2) Inject the test solution into a high-performance liquid chromatograph for detection, record the chromatogram, and the chromatographic conditions are as follows: Chromatographic column: Filled with octadecylsilane-bonded silica gel; Mobile phase: Take trifluoroacetic acid aqueous solution as mobile phase A and acetonitrile as mobile phase B.

2. According to the determination method described in claim 1, it is characterized in that: In the step 1), the volume-mass ratio of the alcohol organic solvent to artificial bezoar in the compound paracetamol and amantadine hydrochloride tablets is (0.5 - 5) mL: 1 mg; And / or, in the step 1), the extraction method is ultrasonic extraction, the ultrasonic extraction time is 15 - 60 min, the ultrasonic extraction frequency is 50 - 80 kHz, and the ultrasonic extraction power is 200 - 500 W; And / or, in the step 1), before filtration, add water to dilute to the concentration of artificial bezoar in the compound paracetamol and amantadine hydrochloride tablets being 0.1 - 1 mg / mL.

3. The measurement method according to claim 2, wherein: In the step 1), the alcohol organic solvent is ethanol or an ethanol solution containing acetic acid.

4. The measurement method according to claim 1, characterized in that: In the determination method, it also includes preparing a reference solution and injecting the reference solution into a high-performance liquid chromatograph for detection; the preparation method of the reference solution is to take a reference substance and dissolve it with an alcohol organic solvent to obtain it.

5. The measurement method according to claim 4, characterized in that: The reference substances are taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy-cholanic acid, hyodeoxycholic acid, sodium glycocholate, glycodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, sodium taurocholate, glycine cholic acid, cholic acid, and hyodeoxycholic acid; the reference solution is a reference solution containing taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy-cholanic acid, hyodeoxycholic acid, sodium glycocholate, glycodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, sodium taurocholate, glycine cholic acid, cholic acid, and hyodeoxycholic acid.

6. According to the determination method described in claim 5, it is characterized in that: The concentrations of taurochenodeoxycholic acid, taurodeoxycholic acid, 7-keto-3α,12-α-dihydroxy-cholanic acid, hyodeoxycholic acid, glycocholate, glycochenodeoxycholate, chenodeoxycholic acid, and deoxycholic acid are 0.5 to 5 μg·mL -1 ; The concentrations of the taurocholic acid and glycocholic acid are 2.5 to 25 μg·mL -1 ; The concentrations of cholic acid and hyodeoxycholic acid are 5 to 50 μg·mL -1 .

7. The measurement method according to claim 4, characterized in that: When preparing the reference solution, the alcohol organic solvent is methanol, methanol aqueous solution, ethanol, or ethanol aqueous solution; Preferably, when preparing the reference solution, first dissolve it with methanol and then dilute it with an ethanol aqueous solution.

8. The measurement method according to any one of claims 1 to 7, characterized in that: In the step 2), the elution method of the high-performance liquid chromatography is gradient elution, and the elution conditions are: 。 9. The measurement method according to any one of claims 1 to 7, characterized in that: In the step 2), the high-performance liquid chromatography conditions are: Chromatographic column: C 18 Chromatographic column; column temperature: 25 - 35 °C; flow rate: 0.8 - 1.5 ml / min; injection volume: 5 - 20 μL; mobile phase A: aqueous solution containing 0.05% trifluoroacetic acid; mobile phase B: acetonitrile The CAD detector acquisition frequency is 5 - 10 Hz, the filter constant is 1 - 5 s, and the atomization temperature is 35 - 60 °C.

10. The determination method according to claim 9, characterized in that: The chromatographic column is Agilent InfinityLabPoroshell 120 EC-C18 or HPH-C18; And / or, the column temperature is 30 °C; And / or, the flow rate is 1 ml / min; And / or, the injection volume is 10 μL; And / or, the CAD detector acquisition frequency is 5 Hz, the filter constant is 3.6 s, and the atomization temperature is 50 °C.

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