Method for separating and detecting calcium folinate intermediates and related substances

Abstract

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and detecting a calcium folinate intermediate and related substances by a high performance liquid chromatography method. The method adopts a chromatographic column with octyl bonded silica gel as a filler, adopts an ammonium acetate buffer solution and an organic solution for gradient elution, separates the calcium folinate intermediate and related substances, and passes them into a detector for detection; the related substances are impurities A, impurity C, impurity G, p-toluenesulfonic acid, impurity Z1b, impurity Z1d-1, impurity Z1d-2, impurity SM1f, impurity SM1g, impurity SM1i, and impurity SM1j; the mobile phase flow rate is 0.8-1.2 ml / min; the column temperature of the chromatographic column is 35-45 DEG C; the wavelength of the detector is 280±10 nm; the method can simultaneously detect the calcium folinate intermediate and 11 substances, the p-toluenesulfonic acid in the product has no interference with the impurity detection, the impurity detection can be calculated by an area normalization method, and the method is simple, fast and effective.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for separating and detecting calcium folinate intermediates and related substances by using an HPLC method. Background Art

[0002] Calcium folinate, with a molecular formula of C20H21CaN7O7, is currently mainly used for the treatment of gastric cancer and colorectal cancer. In the preparation of calcium folinate, there is an intermediate: (2S)-2-[4-[[[(6S)-2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl]methyl]amino]benzamide]glutaric acid p-toluenesulfonate, with a molecular formula of C26H31N7O9S, a molecular weight of 617.63, and a structural formula of:

[0003]

[0004] In addition, 11 related substances may be present in the preparation of calcium folinate intermediates; and the current related technical solutions, such as Chinese patent CN201210294425.9, disclose a high performance liquid chromatography analysis method for calcium folinate, specifically using octadecylsilane bonded silica gel (250×4.0 mm, 5 μm) as a filler, phosphate buffer as a mobile phase A, phosphate buffer-methanol (40:60) as a mobile phase B, and gradient elution; taking calcium folinate or a calcium folinate preparation, adding water to prepare a solution containing 1 mg / ml of calcium folinate as a test solution, and diluting with water to prepare a solution containing about 0.01 mg per 1 ml as a control solution, which can analyze and detect calcium folinate and other related four impurities; However, this method cannot fully detect the calcium folinate intermediate and related substances in the present application, and is not suitable for the quality control of calcium folinate. Summary of the invention

[0005] In view of this, one of the objects of the present invention is to provide a method for separating a calcium folinate intermediate and related substances, which can effectively separate a calcium folinate intermediate and 11 related substances.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] The method for separating calcium folinate intermediates and related substances is characterized in that the chromatographic column used in the method is filled with octyl bonded silica gel, and mobile phase A and mobile phase B are used for gradient elution to separate the calcium folinate intermediates and related substances; the related substances are one or more of impurities A, impurity C, impurity G, p-toluenesulfonic acid, impurity Z1b, impurity Z1d-1, impurity Z1d-2, impurity SM1f, impurity SM1g, impurity SM1i, and impurity SM1j; the impurity Z1d-1 and impurity Z1d-2 are a pair of isomers; the mobile phase A is an ammonium acetate buffer solution; the mobile phase B is an organic solution; the structural formula of the calcium folinate intermediates and related substances is as follows:

[0008]

[0009]

[0010] Further, the concentration of the ammonium acetate buffer solution is 0.015 to 0.025 mol / L;

[0011] Specifically, the pH of the ammonium acetate buffer solution is 3.3-4.0, and acetic acid is used to adjust the pH;

[0012] Further, the organic solution is one or more of methanol, ethanol, and acetonitrile;

[0013] Further, the gradient elution procedure is:

[0014] Time / minute Mobile phase A / % Mobile phase B / % 0 93～97 3～7 5 93～97 3～7 20 80～90 10～20 30 70～80 20～30 35 50～60 40～50 36 93～97 3～7 45 93～97 3～7 ;

[0015] Further, the mobile phase flow rate of the method is 0.8 to 1.2 ml / min;

[0016] Furthermore, the column temperature of the chromatographic column in the method is 35-45°C.

[0017] The second object of the present invention is to provide a method for identifying whether a calcium folinate intermediate and related substances exist in a test object by HPLC, which can effectively monitor whether a calcium folinate intermediate and related substances exist in a test object.

[0018] In order to achieve the above object, the technical solution of the present invention is:

[0019] The invention discloses a method for identifying calcium folinate intermediates and related substances, comprising separating the calcium folinate intermediates and related substances in a test object by the method described in the first objective, passing the separated substances into a detector for detection, and comparing the chromatogram obtained by the detection with the chromatogram of a known reference substance to determine whether the test object contains the calcium folinate intermediates and related substances; the wavelength of the detector is set to 280±10 nm.

[0020] The third object of the present invention is to provide a method for determining the content of calcium folinate intermediates and related substances by HPLC, which can accurately determine the content of calcium folinate intermediates and related substances.

[0021] In order to achieve the above object, the technical solution of the present invention is:

[0022] A method for determining the content of calcium folinate intermediates and related substances, the method specifically comprising the following steps:

[0023] 1) Prepare test solution and reference solution;

[0024] The test product is dissolved in a diluent to obtain a test product solution; the standard product of the related substance is dissolved in a diluent to obtain a reference product solution;

[0025] 2) Separation;

[0026] Using the method described in the first objective, the calcium folinate intermediate and related substances are separated;

[0027] 3) Identification;

[0028] Using the method described in the second objective, identifying the calcium folinate intermediate and related substances;

[0029] 4) Content calculation;

[0030] According to the chromatogram obtained during the identification process, the content of the calcium folinate intermediate and related substances in the test solution is calculated according to the peak area and the peak area normalization method;

[0031] Furthermore, in step 1), the diluent is a mixed solution of 0.025 mol / L sodium phosphate and 10 g / L ascorbic acid;

[0032] Specifically, the pH of the diluent is 7.0, which is adjusted using phosphoric acid or sodium hydroxide.

[0033] The fourth object of the present invention is to provide a method for evaluating the quality of a calcium folinate intermediate, which can accurately determine the content of the calcium folinate intermediate and related substances.

[0034] In order to achieve the above object, the technical solution of the present invention is:

[0035] The method for evaluating the quality of the calcium folinate intermediate is to determine the mass percentage of the related substances in the calcium folinate or the mass percentage of the calcium folinate intermediate by the method described in the third purpose, and compare the mass percentage with the specified qualified standard to determine the quality of the calcium folinate intermediate.

[0036] The invention has the beneficial effects of: the calcium folinate intermediate (dihydrofolic acid p-toluenesulfonate), dihydrofolic acid is unstable, a degradation platform peak will appear in a liquid phase system, octyl bonded silica gel is used as a separation chromatographic column, an ammonium acetate-methanol mobile phase elution system is used, the retention time of dihydrofolic acid and its impurities is moderate, the peak shape is symmetrical, the separation degree between each impurity can meet the requirements, the product and 11 substances can be detected at the same time, the p-toluenesulfonic acid in the product has no interference with the impurity detection, the area normalization method can be used for calculation, and the method is simple, fast and effective. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 : HPLC chromatogram of this method;

[0038] Figure 2 : HPLC graph of detection limits of calcium folinate intermediate Z1, impurity A, impurity Z1b, impurity C and impurity G;

[0039] Figure 3 : HPLC graphs of detection limits of impurity SM1f, impurity SM1g, impurity SM1i and impurity SM1j;

[0040] Figure 4 : Durability - Normal conditions - HPLC chart of mixed solution;

[0041] Figure 5 : Durability-mobile phase A (pH=3.4)-mixed solution HPLC chart;

[0042] Figure 6 : Durability-mobile phase A (pH=3.6)-mixed solution HPLC chart;

[0043] Figure 7 : Durability - flow rate 1.1ml / min - HPLC chart of mixed solution;

[0044] Figure 8 : Durability - flow rate 0.9ml / min - HPLC chart of mixed solution;

[0045] Fig. 9 : Durability - column temperature 42°C - HPLC chart of mixed solution;

[0046] Fig.10 : Durability - column temperature 38°C - HPLC chart of mixed solution;

[0047] Fig.11 : Durability - initial ratio of gradient (mobile phase B is 5.5%) - HPLC graph of mixed solution;

[0048] Fig.12 : Durability - initial ratio of gradient (mobile phase B is 4.5%) - HPLC graph of mixed solution;

[0049] Fig.13 : Durability-different batches of chromatographic columns-mixed solution HPLC graph. DETAILED DESCRIPTION

[0050] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0051] Example 1

[0052] Main instrument: Shimadzu LC-20A high performance liquid chromatograph.

[0053] Mobile Phase:

[0054] Mobile phase A: 0.015 mol / L to 0.025 mol / L ammonium acetate buffer solution (dissolve ammonium acetate in 1000 ml of water and adjust the pH to 3.3 to 4.0 with acetic acid);

[0055] Mobile phase B: methanol;

[0056] Gradient elution program (Table 1):

[0057] Table 1 Gradient elution program

[0058] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 93-97 3-7 5 93-97 3-7 20 80-90 10-20 30 70-80 20-30 35 50-60 40-50 36 93-97 3-7 45 93-97 3-7

[0059] Chromatographic column: octyl bonded silica gel, Agilent SB-C8 4.6mm×250mm, 5μm;

[0060] Flow rate: 0.8~1.2ml / min;

[0061] Detection wavelength: 280nm;

[0062] Column temperature: 35-45°C;

[0063] Diluent: 0.025 mol / L sodium phosphate (containing 10 g / L ascorbic acid), adjust the pH to 7.0 with phosphoric acid or sodium hydroxide (use on the same day, store away from light);

[0064] Injection volume: 20 μl;

[0065] Test solution: 0.2 mg / ml;

[0066] Quantitative method: peak area normalization method.

[0067] Example 2

[0068] Solution preparation:

[0069] Avoid light operation

[0070] Test solution: Keep away from light and prepare freshly before use. Take an appropriate amount of this product, weigh accurately, add diluent (take 9.5g of sodium phosphate dodecahydrate and 10g of ascorbic acid, add 1000ml of water to dissolve, adjust pH to 7.0 with phosphoric acid or sodium hydroxide test solution) and dissolve and dilute to make a solution containing about 0.2mg per 1ml, shake well, and you have it.

[0071] System suitability solution: Take appropriate amounts of calcium folinate intermediate, impurity A, impurity C, impurity G, impurity Z1d, impurity Z1b, impurity SM1f, impurity SM1g, impurity SM1i, and impurity SM1j reference substances, dissolve and dilute them with a diluent to prepare a solution containing approximately 2 μg of each impurity and 0.2 mg of calcium folinate intermediate per 1 ml.

[0072] Example 3

[0073] Test method:

[0074] Accurately measure 20 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The order of peaks is impurity A, p-toluenesulfonic acid, the main component of this product, impurity Z1d-1, impurity Z1d-2, impurity Z1b, impurity SM1f, impurity C, impurity G, impurity SM1g, impurity SM1i, impurity SM1j. The separation between impurity C and impurity G should meet the requirements.

[0075] Accurately measure 20 μl of the test solution and inject it into the liquid chromatograph. Record the chromatogram. Except for the solvent peak (not integrated before the retention time of 5 minutes) and the gradient elution peak, calculate the content of each impurity by the peak area normalization method (see Appendix Figure 1 ).

[0076] Example 4

[0077] Exclusive properties:

[0078] Diluent: 0.025 mol / L sodium phosphate, containing 10 g / L vitamin C, phosphoric acid or sodium hydroxide to adjust pH to 7.0.

[0079] Impurity A positioning solution: Accurately weigh 2.28 mg of impurity A, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the impurity A stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0080] Impurity C localization solution: accurately weigh 2.20 mg of impurity C, place it in a 25 ml volumetric flask, add 2.0 ml of 28.6 g / L sodium carbonate aqueous solution to dissolve, add diluent to dilute to the scale, shake well, and use it as the impurity C stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0081] Impurity G localization solution: Accurately weigh 2.43 mg of impurity G, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the impurity G stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0082] Impurity SM1f positioning solution: Accurately weigh 2.49 mg of impurity SM1f, place it in a 25 ml volumetric flask, add 2.0 ml of 28.6 g / L sodium carbonate aqueous solution to dissolve, add diluent to dilute to the scale, shake well, and use it as the impurity SM1f stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0083] Impurity SM1g positioning solution: Accurately weigh 2.26 mg of impurity SM1g, place it in a 25 ml volumetric flask, add 2.0 ml of 28.6 g / L sodium carbonate aqueous solution to dissolve, add diluent to dilute to the scale, shake well, and use it as the impurity SM1g stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0084] Impurity SM1i positioning solution: Accurately weigh 2.36 mg of impurity SM1i, place it in a 25 ml volumetric flask, add 2.0 ml of 28.6 g / L sodium carbonate aqueous solution to dissolve, add diluent to dilute to the scale, shake well, and use it as the impurity SM1i stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0085] Impurity SM1j positioning solution: Accurately weigh 2.55 mg of impurity SM1j, place it in a 25 ml volumetric flask, add 2.0 ml of 28.6 g / L sodium carbonate aqueous solution to dissolve, add diluent to dilute to the scale, shake well, and use it as the impurity SM1j stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0086] Impurity Z1b localization solution: Accurately weigh 2.13 mg of impurity Z1b, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the impurity Z1b stock solution; then transfer 0.5 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0087] Impurity Z1d localization solution: Accurately weigh 3.25 mg of impurity Z1d, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use as the impurity Z1d stock solution; then transfer 1.0 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0088] p-Toluenesulfonic acid positioning solution: Accurately weigh 10.05 mg of p-toluenesulfonic acid, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the impurity p-toluenesulfonic acid stock solution; then transfer 1.0 ml to a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0089] Test solution: Accurately weigh about 20 mg of calcium folinate intermediate Z1, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and the test solution is obtained.

[0090] Mixed solution: Accurately weigh 11.00 mg of calcium folinate intermediate Z1, and place it in a 50 ml volumetric flask. Accurately pipette 1.0 ml each of impurity Z1d and p-toluenesulfonic acid stock solutions, and accurately pipette 0.5 ml each of impurity A, impurity C, impurity G, impurity SM1f, impurity SM1g, impurity SM1j, impurity SM1i, and impurity Z1b stock solutions, and place them in the same volumetric flask mentioned above. Add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0091] Take 20 μl of blank diluent, test solution, mixed solution and each impurity location solution respectively and inject according to the method. The structural data are shown in Table 2.

[0092] Table 2 Specificity test results

[0093]

[0094] Detection Limit:

[0095] ACG impurity stock solution: Accurately weigh 2.100 mg of impurity A, 2.099 mg of impurity C, and 2.192 mg of impurity G and place them in the same 250 ml volumetric flask. Add diluent to dissolve them and dilute to the scale. Shake well to prepare the ACG impurity stock solution.

[0096] Z1b-Z1 impurity stock solution: Accurately weigh 2.254 mg of impurity Z1b and 2.294 mg of intermediate Z1 and place them in the same 100 ml volumetric flask. Add diluent to dissolve them and dilute to the scale. Shake well to prepare the Z1b-Z1 impurity stock solution.

[0097] Detection limit solution (ACG-Z1b-Z1): Accurately weigh 0.5 ml of ACG impurity stock solution and Z1b-Z1 impurity stock solution and place them in the same 100 ml volumetric flask. Add diluent and dissolve by ultrasonication, then dilute to the scale and shake well to obtain.

[0098] Detection limit solution (SM1f-SM1g-SM1i-SM1j): Accurately weigh 2.114 mg of impurity SM1g, 1.948 mg of impurity SM1f, 1.306 mg of impurity SM1j, and 2.499 mg of impurity SM1i and place them in the same 250 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, then transfer 0.5 ml of the above solution to a 100 ml volumetric flask, add diluent to dissolve by ultrasonication and dilute to the scale, shake well.

[0099] Determination method: Take the above detection limit solutions and inject them continuously, and record the chromatogram (attached Figure 2-3 , Table 3), and calculate the ratio of the target peak height to the baseline noise (signal-to-noise ratio).

[0100] Table 3 Detection limit determination results

[0101]

[0102] Durability

[0103] Take the mixed solution (preparation method is the same as the mixed solution under the specificity item), use normal mobile phase, different mobile phase initial ratios, different mobile phase pH, different column temperatures, different column flow rates and different batches of chromatographic columns of the same model for testing, and record the separation degree and relative retention time between each peak (see attached Figure 4-13 , Tables 4 and 5).

[0104] Table 4 Chromatographic condition change durability test results (relative retention time)

[0105]

[0106] Table 5 Chromatographic conditions change durability test results (separation)

[0107]

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A method for separating a calcium folinate intermediate and related substances, characterized in that: The chromatographic column used in the method is filled with octyl bonded silica gel, and mobile phase A and mobile phase B are used for gradient elution to separate the calcium folinate intermediate and related substances; the mobile phase A is an ammonium acetate buffer solution; the mobile phase B is methanol; the related substances are impurity A, impurity C, impurity G, p-toluenesulfonic acid, impurity Z1b, impurity SM1f, impurity SM1g, impurity SM1i and impurity SM1j; the structural formula of the calcium folinate intermediate and related substances is as follows: The concentration of the ammonium acetate buffer solution is 0.015-0.025 mol / L; the gradient elution procedure is: 。 2. The method according to claim 1, characterized in that The pH of the ammonium acetate buffer solution is 3.3-4.0, and acetic acid is used to adjust the pH.

3. The method according to claim 1, characterized in that The method has a mobile phase flow rate of 0.8-1.2 ml / min and a chromatographic column temperature of 35-45°C.

4. A method for identifying calcium folinate intermediates and related substances, characterized in that: The method described in claim 1 is used to separate the calcium folinate intermediate and related substances in the test object, pass them into a detector for detection, and compare the chromatogram obtained by detection with the chromatogram of a known reference substance to determine whether the test object contains the calcium folinate intermediate and related substances; the detector wavelength is set to 280±10nm.

5. A method for determining the content of calcium folinate intermediates and related substances, characterized in that: The method specifically comprises the following steps: 1) Prepare test solution and reference solution; The test product is dissolved in a diluent to obtain a test product solution; the standard product of the related substance is dissolved in a diluent to obtain a reference product solution; 2) Separation; Using the method described in claim 1, the calcium folinate intermediate and related substances are separated; 3) Identification; Using the method described in claim 4, identifying the calcium folinate intermediate and related substances; 4) Calculation of content; According to the chromatogram obtained during the identification process, the content of the calcium folinate intermediate and related substances in the test solution was calculated by the peak area normalization method.

6. The method according to claim 5, characterized in that In the step 1), the diluent is a mixed solution of 0.025 mol / L sodium phosphate and 10 g / L ascorbic acid.

7. A method for evaluating the quality of a calcium folinate intermediate, characterized in that: The mass percentage of the related substance in the calcium folinate or the mass percentage of the calcium folinate intermediate is determined by the method of claim 6, and compared with the specified qualified standard to determine the quality of the calcium folinate intermediate.

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