A quality control method for calcium folinate

Abstract

The present invention provides a quality control method for calcium folinate, and relates to the field of drug quality control. The present invention first provides calcium folinate, wherein the weight percentage content of impurity piperazine in the calcium folinate is ≤0.1%. The present invention also provides a method for qualitatively and / or quantitatively detecting impurities in calcium folinate. The method of the present invention performs a simple pre-treatment on the test solution and then directly injects the sample, and uses gas chromatography for detection. The detection method is easy to operate, has few influencing factors, good reproducibility, short running time, and low cost; and the reagents used have little damage to the environment and little impact on the health of operators; in addition, the detection method of the present invention has high sensitivity and can accurately detect the content of impurities in calcium folinate, especially the content of piperazine. The detection method of the present invention can be used to control the quality of calcium folinate, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the field of drug quality control, and in particular to a quality control method for calcium folinate. Background Art

[0002] Calcium folinate, also known as calcium folinate, often exists in the form of pentahydrate, with a molecular formula of C 20 H 21 CaN7O7·5H2O, molecular weight 601.58, chemical name 6S(-)-N-[4-[(2-amino-5-formyl-1,4,5,6,7,8-hexahydro-4-oxo-6-pterydinyl)methyl]amino]benzoyl-L-glutamic acid calcium salt pentahydrate, white to light yellow crystals or amorphous powder. Calcium folinate is an adjuvant drug for tumors, used to rescue poisoning after high-dose methotrexate treatment of osteosarcoma; combined with 5-fluorouracil, it is used to treat gastric cancer and colorectal cancer. Its structural formula is:

[0003]

[0004] The production process of calcium folinate is chemical synthesis. In the last step of preparing crude calcium folinate and purifying calcium folinate from the crude product, the existing synthesis process often requires the use of the reagent piperazine, which easily leads to piperazine residues in the final product. On the one hand, calcium folinate is prepared by alkaline hydrolysis and ring opening of 5,10-methylenetetrahydrofolate, and piperazine is a commonly used alkaline reagent in this step. The reaction route is as follows, for example, see the patent application with publication number CN104045640A:

[0005]

[0006] On the other hand, the use of piperazine is also involved in the purification of calcium folinate (separation from its optical isomer calcium folinate) from crude calcium folinate, for example, see patent applications with publication numbers WO1993017022A1 and WO1995033749A1. Therefore, it is necessary to detect whether piperazine exists in calcium folinate. Piperazine, also known as p-diazole, is a white or light yellow solid with a molecular formula of C4H 10 N2, molecular weight 86.14, structural formula:

[0007]

[0008] The piperazine content directly affects the quality of calcium folinate as a raw material, so the control of piperazine content is of great significance for the product quality control of calcium folinate.

[0009] However, there is no report on the detection and quality control of piperazine in calcium folinate. After searching, the patent application with publication number CN110927091A discloses a method for detecting the content of piperazine in a piperazine aqueous solution, which uses benzoquinone and piperazine molecules to produce an oxidation-reduction reaction, develops color on benzoquinone, and determines the content of piperazine molecules by colorimetry. This detection method requires chemical reaction, and the process is complicated. The reaction conversion rate affects the detection result, and it is only applicable to the detection of piperazine as the main component, and is not applicable to the detection of piperazine as the impurity content in calcium folinate, because the content of piperazine in calcium folinate is low, resulting in excessive concentration of the test sample, and it is impossible to directly inject the sample, and the oxidation-reduction reaction is produced by benzoquinone and a small amount of piperazine molecules. When developing color on benzoquinone, it will be interfered by the color (light yellow) of calcium folinate, and when benzoquinone and piperazine react, benzoquinone and calcium folinate may react, affecting the detection result. The patent application with publication number CN113514565A discloses a method for detecting residual piperazine in high piperazine, but the detection limit of the detection method is 6.25 μg / ml, and the test sample concentration is 100 mg / ml. The inventors of the present invention found in exploring the detection method that the test sample concentration is too high and is not suitable for the detection of piperazine in calcium folinate, which will interfere with the peak of piperazine and even cause chromatographic column blockage, and the sensitivity of the detection method needs to be further improved. In short, the existing piperazine detection method is not suitable for the detection of piperazine in calcium folinate. Summary of the invention

[0010] In view of the deficiencies of the prior art, the present invention provides a quality control method for calcium folinate, and specifically relates to a quality control standard for calcium folinate and a method for detecting impurities in calcium folinate, which is used to detect the content of impurities in calcium folinate and further perform quality control on calcium folinate.

[0011] The invention provides calcium folinate, wherein the weight percentage content of impurity piperazine in the calcium folinate is less than or equal to 0.1%.

[0012] The present invention also provides a method for qualitatively and / or quantitatively detecting impurities in calcium folinate, which comprises the following steps:

[0013] (1) Prepare the test solution: weigh the calcium folinate test sample, add water and acetone, mix well, centrifuge, and take the supernatant;

[0014] (2) Detection by gas chromatography.

[0015] Furthermore, the impurity is piperazine.

[0016] The inventors of the present invention found in the exploration of detection methods that if the existing piperazine detection method is used to dissolve the calcium folinate sample in a single solvent and dilute the prepared test solution, piperazine often does not peak after injection. The inventors therefore improved the preparation method of the test solution, and after investigation, found that pretreatment in a water and acetone mixed system can effectively improve the interference with the piperazine peak, providing the possibility for accurate detection of piperazine in calcium folinate.

[0017] Furthermore, in step (1), the method for preparing the test solution comprises the following steps:

[0018] 1) Weigh the calcium folinate test sample and add water until it is completely dissolved;

[0019] 2) Add acetone and mix evenly, centrifuge, and take the supernatant.

[0020] Further, in step (1), the volume ratio of water:acetone is 2:(2.4-3.6);

[0021] And / or, in step (1), the concentration of the test solution is 4-6 mg / ml.

[0022] Further, in step (1), the volume ratio of water:acetone is 2:3;

[0023] And / or, in step (1), the concentration of the test solution is 5 mg / ml.

[0024] In the present invention, the concentration of the test solution is measured by the amount of calcium folinate test sample taken.

[0025] Furthermore, in step (2), the gas chromatography method uses a 5% phenyl-95% methyl-polysiloxane stationary phase chromatography column;

[0026] The temperature raising program of the gas chromatography method is as follows: the initial column temperature is 105-115°C, maintained for 2-4 minutes, and then raised to 195-205°C at a rate of 20-30°C / min, and maintained for 2-4 minutes.

[0027] Further, the chromatographic column is CP-Sil 8CB;

[0028] And / or, the temperature rising program is as follows: initial column temperature is 110°C, maintained for 2 min, then heated to 200°C at a rate of 30°C / min, and maintained for 4 min.

[0029] Further, the length of the CP-Sil 8CB is 30 m; and / or, the inner diameter of the CP-Sil 8CB is 0.32 mm; and / or, the film thickness of the CP-Sil 8CB is 1 μm.

[0030] Furthermore, in step (2), the injection volume of the gas chromatography method is 1 to 2 μl, and the split ratio is 2:1;

[0031] And / or, the carrier gas of the gas chromatography is nitrogen with a flow rate of 0.8 to 1.2 ml / min;

[0032] and / or, the injection port temperature of the gas chromatography is 220-240° C.;

[0033] and / or, the detector temperature of the gas chromatography is 230-250° C.;

[0034] And / or, the gas chromatography method adopts an external standard method to calculate the impurity content;

[0035] Preferably, the injection volume is 2 μl and the split ratio is 2:1;

[0036] And / or, the flow rate is 1.0 ml / min;

[0037] and / or, the injection port temperature of the gas chromatography is 240°C;

[0038] And / or, the detector temperature of the gas chromatography is 250°C.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) The method of the present invention is used to pre-treat the sample to be tested, which can effectively reduce the interference of sample components on the detection of impurity piperazine. The sample solution after treatment is not easy to block and damage the chromatographic column and the instrument. It can also increase the concentration of the sample, thereby increasing the detection ability of the impurity piperazine. The limit of quantification of piperazine is 1.6472 μg / ml, accounting for 0.033% of the concentration of the sample, and the detection limit is 0.8236 μg / ml, accounting for 0.016% of the concentration of the sample. It has high sensitivity and can effectively detect piperazine.

[0041] (2) The existing technology uses liquid chromatography to detect piperazine, which requires derivatization of the sample, and the process is complicated. The reaction conversion rate affects the detection result. The gas chromatography method of the present invention does not require derivatization reaction. The sample solution is directly injected after simple pretreatment. It is easy to operate, has few influencing factors, good reproducibility, high impurity piperazine detection ability, short operation time, and low cost. The reagents used have little damage to the environment and little impact on the health of operators.

[0042] (3) The column temperature program set by the present invention can reduce the running time, save carrier gas, and reduce the instrument chromatographic column loss cost.

[0043] (4) The detection method of the present invention has good system applicability, specificity, linearity and accuracy: the RSD of the peak area of ​​the reference substance for 6 consecutive times was 3.5%, the separation degree of piperazine and adjacent peaks was 2.6, the linear correlation coefficient r was 0.9997, and the recovery rate of high, medium and low concentrations in the method validation was between 92% and 104%.

[0044] In summary, the present invention provides a method for detecting impurities in calcium folinate, and can especially accurately detect the impurity piperazine therein. The detection method of the present invention can be used to control the quality of calcium folinate, and has good application prospects.

[0045] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0046] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The chromatogram obtained by detecting the blank solution in Example 1.

[0048] Figure 2 This is the chromatogram obtained by detecting the reference solution in Example 1.

[0049] Figure 3 This is the chromatogram obtained by detecting the test solution in Example 1.

[0050] Figure 4 This is the chromatogram obtained by detecting the spiked test solution in Example 1.

[0051] Figure 5 This is the chromatogram of the blank solution in the experiment of investigating the system suitability, specificity and solution stability in Example 1.

[0052] Figure 6 This is the chromatogram of the reference solution in the experiment of investigating the system suitability, specificity and solution stability of Example 1.

[0053] Figure 7 This is the chromatogram of the spiked test solution in the experiment of investigating the system suitability, specificity and solution stability in Example 1.

[0054] Figure 8 This is the chromatogram obtained from the stability study solution of the reference substance in Example 1.

[0055] Fig. 9The quantitative limit chromatogram obtained in Example 1 is shown.

[0056] Fig.10 The detection limit chromatogram obtained in Example 1 is shown.

[0057] Fig.11 This is a standard curve diagram obtained from the linear investigation experiment in Example 1.

[0058] Fig.12 It is the detection chromatogram of each linear concentration in the linear investigation experiment of Example 1.

[0059] Fig.13 This is the chromatogram obtained from the accuracy investigation experiment of Example 1.

[0060] Fig.14 This is the chromatogram obtained by detecting the chromatographic condition 1 in Comparative Example 1.

[0061] Fig.15 This is the chromatogram obtained by detecting the chromatographic condition 2 in Comparative Example 1.

[0062] Fig.16 This is the chromatogram obtained by detecting under chromatographic condition 3 in Comparative Example 1.

[0063] Fig.17 This is the chromatogram obtained by detecting the chromatographic condition 4 in Comparative Example 1.

[0064] Fig.18 This is the chromatogram obtained by detecting the LOQ solution under chromatographic condition 5 in Comparative Example 1.

[0065] Fig.19 This is the chromatogram obtained by detecting the test solution under chromatographic condition 5 in Comparative Example 1.

[0066] Fig. 20 This is the chromatogram obtained by detecting the PIP solution under chromatographic condition 5 in Comparative Example 1.

[0067] Fig.21 The chromatogram obtained by detecting the blank solution under chromatographic condition 6 in Comparative Example 2.

[0068] Fig. 22 This is the chromatogram obtained by detecting the reference solution under chromatographic condition 6 in Comparative Example 2.

[0069] Fig.23 This is the chromatogram obtained by the chromatographic condition 7 in Experimental Example 1.

[0070] Fig.24 This is the chromatogram obtained by the chromatographic condition 8 in Experimental Example 2.

[0071] Fig.25 This is the chromatogram obtained by the chromatographic condition 9 in Experimental Example 2. DETAILED DESCRIPTION

[0072] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0073] Example 1: Detection method of piperazine in calcium folinate of the present invention

[0074] The present invention adopts gas chromatography to detect the content of piperazine in calcium folinate, and the detection conditions are as follows:

[0075] Chromatographic column: A 5% phenyl-95% methyl-polysiloxane stationary phase chromatographic column is used. In this embodiment, a CP-Sil 8CB chromatographic column is specifically selected, and the specific specifications are 30m*0.32mm, 1μm;

[0076] The carrier gas was nitrogen with a flow rate of 1.0 ml / min;

[0077] The initial column temperature was 110 °C, maintained for 2 min, then raised to 200 °C at a rate of 30 °C / min and maintained for 4 min;

[0078] The injection volume was 2 μl, and the split ratio was 2:1;

[0079] The injection port temperature was 240°C; the detector temperature was 250°C.

[0080] The content of piperazine in calcium folinate was calculated by external standard method.

[0081] Sample preparation:

[0082] (1) Blank solution: Take 4 ml of water and put it into a 10 ml volumetric flask. Add acetone to dilute to the mark and shake well.

[0083] (2) Reference substance stock solution: Dissolve 11.61 mg of piperazine reference substance in a 10 ml volumetric flask, add 4 ml of water, add acetone to make up to volume, and shake well.

[0084] (3) Reference substance solution: Accurately measure 0.1 ml of the reference substance stock solution into a 20 ml volumetric flask, add 4 ml of water, add acetone to make up to volume, shake well, and the solution is ready.

[0085] (4) The concentration of the test solution is 5 mg / ml. The preparation method is as follows: take 100 mg of the calcium folinate test sample, accurately weigh it, put it in a 20 ml volumetric bottle, add 8 ml of water, sonicate it in 40°C water until it is completely dissolved, let it cool, dilute it to the scale with acetone, shake it well, let it stand for 3 minutes, centrifuge it, and take the supernatant. The above test solution concentration is calculated based on the amount of calcium folinate test sample taken.

[0086] (5) Spiked test solution: weigh 100.21 mg of calcium folinate test sample into a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, let cool, add 0.1 ml of reference stock solution, add acetone to make up to volume, shake well, let stand for 3 minutes, centrifuge, and take the supernatant.

[0087] Each solution was tested according to the above test conditions, and the test results were as follows: Figure 1-Figure 4 As shown. The peak areas of the six-point reference were 18.022, 18.623, 18.810, 17.361 and 17.940, respectively, with an RSD of 4.2%. The theoretical plate number of the piperazine peak in the reference solution was 22977, with a tailing factor of 1.8, and the RSD of the six-point reference solution was 4.2%, which met the detection requirements.

[0088] 1. System applicability, specificity and solution stability of the above detection methods

[0089] The preparation methods of each solution are as follows:

[0090] Step (1): Take 4 ml of water in a 10 ml volumetric flask, dilute to the mark with acetone, and shake well to serve as the blank solution;

[0091] Step (2): Take 5.580 mg of piperazine reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add 4 ml of water to dissolve it, dilute it to the mark with acetone, shake well, accurately measure 0.2 ml into a 20 ml volumetric flask, add 8 ml of water, dilute it to the mark with acetone, shake well, and use it as the reference substance solution;

[0092] Step (3): Take 5.580 mg of piperazine reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add 4 ml of water to dissolve it, then dilute it to the mark with acetone, shake well, and use it as the reference substance stock solution;

[0093] Step (4): Accurately measure 0.2 ml of the reference substance stock solution into a 20 ml volumetric flask, add 8 ml of water, dilute to the mark with acetone, and shake well to serve as the reference substance stability test solution;

[0094] Step (5): Weigh 100.100 mg of calcium folinate test sample into a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, cool, add 0.2 ml of reference substance stock solution, make up to volume with acetone, shake well, let stand for 3 minutes, centrifuge, and take the supernatant as the spiked test solution.

[0095] The blank solution was injected once, the reference solution was injected 6 times, and the spiked test solution was injected once. Figure 5 , the chromatogram of the reference solution is shown in Figure 6 , the chromatogram of the spiked test solution is shown in Figure 7 The chromatogram obtained from the stability test solution of the reference substance is shown in Figure 8 The results of the system suitability, specificity and solution stability are listed in Table 1.

[0096] Table 1. Results of system suitability, specificity and solution stability studies

[0097]

[0098]

[0099] The results of the system suitability investigation showed that the blank solution did not interfere with the peak of the test sample; the separation degree between piperazine and the adjacent peak in the spiked test solution was 2.6, which was a good separation degree; the tailing factor of the piperazine peak was 1.9, and the number of theoretical plates was 26651; the RSD of the peak area of ​​the piperazine reference solution after 6 consecutive injections was 3.5%. The above results showed that the detection method of the present invention had good system applicability.

[0100] The results of the specificity investigation showed that the difference in retention time of the piperazine peak in the reference solution and the spiked test solution was 0.005 min, which was much smaller than the standard of ±0.1 min, indicating that the detection method of the present invention had better specificity.

[0101] The results of the solution stability test showed that the percentage of the piperazine peak area at each time point to the 0-hour peak area of ​​the piperazine reference solution and the spiked test solution was in the range of 95% to 105%, indicating good stability.

[0102] Based on the above experimental results, it can be seen that the detection method of the present invention is easy to operate, has few influencing factors, good reproducibility, short operating time and low cost; the reagents used have little damage to the environment and little impact on the health of operators; the system applicability, specificity and solution stability all meet the detection requirements.

[0103] 2. Limit of Quantitation and Limit of Detection of the above-mentioned Detection Methods

[0104] The preparation methods of each solution are as follows:

[0105] Step (1): Take 5.580 mg of piperazine reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add 4 ml of water to dissolve it, then dilute it to the scale with acetone, shake it well, and use it as the reference substance stock solution.

[0106] Step (2): Take 0.2 ml of the solution in step (1) in a 20 ml volumetric flask, add 8 ml of water, dilute to volume with acetone, shake well, take 3 ml in a 10 ml volumetric flask, add 1 ml of water, dilute to volume with acetone, shake well. Accurately measure 2 μl and inject it into the gas chromatograph. Detect according to the above detection conditions. At this time, the S / N is greater than or equal to 10:1, which is taken as the quantification limit of piperazine. The quantification limit spectrum is as follows: Fig. 9As shown, the solution concentration is 1.6472μg / ml and the limit of quantification is 3.2944ng, which is equivalent to a test sample concentration ratio of 0.033%.

[0107] Step (3): Take 5 ml of the solution from step (2) and place it in a 10 ml volumetric flask, dilute with acetone, and shake well. Accurately measure 2 μl and inject it into the gas chromatograph. Perform the test according to the above test conditions. At this time, the S / N is greater than or equal to 3:1, which is taken as the detection limit of piperazine. The detection limit spectrum is as follows: Fig.10 As shown, the solution concentration is 0.8236 μg / ml and the detection limit is 1.6472 ng, which is equivalent to a test sample concentration ratio of 0.016%.

[0108] 3. Linearity and range of the above detection methods

[0109] Preparation of each solution in the research method:

[0110] Step (1): Take 5.580 mg of piperazine reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add 4 ml of water to dissolve it, dilute it to the mark with acetone, and shake it well.

[0111] Step (2): Accurately measure 0.1 ml, 0.16 ml, 0.2 ml, 0.24 ml and 0.3 ml of the solution in step (1), respectively, and place them in 20 ml volumetric flasks, add 8 ml of water to each, dilute to the scale with acetone, shake well, and use 50%, 80%, 100%, 120% and 150% solutions with a limit concentration of 0.1% to examine the linearity and range.

[0112] Accurately measure 2 μl of each linear solution, inject it into the gas chromatograph, and test it according to the above test conditions. Record the chromatogram and perform linear regression with concentration C (μg / ml) as the abscissa and peak area A as the ordinate. The standard curve is as follows: Fig.11 The detection spectrum of each linear concentration is shown in Fig.12 shown.

[0113] The above results indicate that the detection method of the present invention has good linearity in detecting piperazine content within the range of 50% to 150% (2.5 μg / ml to 7.5 μg / ml) of the limit concentration.

[0114] 4. Accuracy of the above detection methods

[0115] Preparation of each solution in the research method:

[0116] Step (1): Take 5.580 mg of piperazine reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add 4 ml of water to dissolve it, dilute it to the mark with acetone, and shake it well.

[0117] Step (2): Accurately measure 0.2 ml of step (1) into a 20 ml volumetric flask, add 8 ml of water, dilute to the mark with acetone, and shake well.

[0118] Step (3): Take 100.16 mg of the test sample, accurately weigh it, place it in a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, cool, dilute to the scale with acetone, shake well, let stand for 3 minutes, centrifuge, and take the supernatant.

[0119] Step (4): 50% recovery solution: weigh 100.13 mg of the test sample into a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, let cool, add 0.1 ml of step (1), make up to volume with acetone, shake well, let stand for 3 minutes, centrifuge, take the supernatant, and prepare 3 portions in the same way.

[0120] Step (5): 100% recovery solution: weigh 100.13 mg of the test sample into a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, cool, add 0.2 ml of step (1), make up to volume with acetone, shake well, let stand for 3 minutes, centrifuge, take the supernatant, and prepare 3 portions in the same way.

[0121] Step (6): 150% recovery solution: weigh 100.13 mg of the test sample into a 20 ml volumetric flask, add 8 ml of water, sonicate in 40°C water until completely dissolved, cool, add 0.3 ml of step (1), make up to volume with acetone, shake well, let stand for 3 minutes, centrifuge, take the supernatant, and prepare 3 portions in the same way.

[0122] Accurately measure 2 μl of each of the solutions prepared in steps (2), (3), (4), (5), and (6) above, inject them into the liquid chromatograph, perform the test according to the above test conditions, and record the chromatogram ( Fig.13 ). The test results are shown in Table 2.

[0123] Table 2. Test results of each group

[0124]

[0125]

[0126] The above results show that the detection method of the present invention has a detection result recovery rate in the range of 92.2% to 104.4% at a concentration of 50% to 150% of the limit, has high detection accuracy, a wide accuracy range, and is more effective and accurate in detecting piperazine in calcium folinate.

[0127] The beneficial effects of the present invention are demonstrated below through comparative examples and test examples.

[0128] Comparative Example 1: Effect of the Preparation Method of Chromatographic Column and Test Solution on the Detection Results

[0129] 1. Chromatographic conditions 1:

[0130] Chromatographic column: DB-1, 30m×0.53mm, 1.00μm;

[0131] Inlet temperature: 250°C; Detector temperature: 250°C;

[0132] Temperature program: initial column temperature 50°C, maintained for 9 min, increased to 180°C at a rate of 8°C / min, then increased to 240°C at a rate of 15°C / min, maintained for 10 min;

[0133] Constant flow rate: 3.0ml / min;

[0134] Split ratio: 15:1; diluent: water; injection volume: 1 μl; carrier gas: high-purity nitrogen;

[0135] Sample preparation: Weigh 19.55 mg of piperazine reference substance into a 100 ml volumetric flask, add appropriate amount of water to dissolve, dilute with water to the scale, and shake well.

[0136] The chromatogram of the test is as follows Fig.14 When the detection conditions were used, no peak of piperazine was observed.

[0137] 2. Chromatographic conditions 2:

[0138] Chromatographic column: DB-1, 30m×0.32mm, 1.00μm; other chromatographic conditions are the same as chromatographic condition 1.

[0139] Sample preparation: Weigh 0.8008 g of piperazine reference substance into a 10 ml volumetric flask, add water to dissolve, dilute to the mark with water, shake well, and take an appropriate amount into a sample vial for testing.

[0140] The chromatogram of the test is as follows Fig.15 When the detection conditions were used, the piperazine peak was bifurcated and the peak shape was poor.

[0141] 3. Chromatographic conditions 3:

[0142] Chromatographic column: CP-Volamine, 30m*0.32mm; other chromatographic conditions are the same as chromatographic condition 2.

[0143] Sample preparation: Weigh 19.55 mg of piperazine reference substance into a 100 ml volumetric flask, add appropriate amount of water to dissolve, dilute to the scale with water, and shake well.

[0144] The chromatogram of the test is as follows Fig.16 When the detection conditions were used, the piperazine peak area was too small and the peak elution time was too late.

[0145] 4. Chromatographic conditions 4:

[0146] The temperature program was as follows: initial column temperature 110°C, maintained for 2 min, then heated to 130°C at a rate of 8°C / min, maintained for 10 min, then heated to 180°C at a rate of 10°C / min, maintained for 2 min. The remaining chromatographic conditions were the same as those of chromatographic condition 3.

[0147] Sample preparation: Weigh 19.55 mg of piperazine reference substance into a 100 ml volumetric flask, add appropriate amount of water to dissolve, dilute to the scale with water, and shake well.

[0148] The chromatogram of the test is as follows Fig.17 When using this detection condition, although it helps to improve the peak shape, response and late peak time of piperazine, the running time is long, which increases the nitrogen loss and instrument chromatographic column loss cost, and is not conducive to improving the detection efficiency.

[0149] 5. Chromatographic conditions 5:

[0150] The temperature program was as follows: initial column temperature 110°C, maintained for 2 min, then increased to 130°C at a rate of 8°C / min, and maintained for 10 min. The other chromatographic conditions were the same as those in chromatographic condition 4.

[0151] Sample preparation:

[0152] (1) LOQ solution (limit of quantitation solution): Weigh 2.236 mg of piperazine reference substance into a 10 ml volumetric flask, add water to dissolve and dilute to the mark. Take 1 ml into a 10 ml volumetric flask, add water to dilute to the mark, and shake well. Take 1 ml into a 10 ml volumetric flask, add water to dilute to the mark, and shake well. Take 5 ml into a 10 ml volumetric flask, add water to dilute to the mark, and shake well.

[0153] (2) Test solution: Weigh 100.15 mg of calcium folinate sample into a 10 ml volumetric flask, add water to dissolve and dilute to the mark.

[0154] (3) PIP (piperazine) solution: Weigh 10.51 mg of piperazine reference substance into a 10 ml volumetric flask, add water to dissolve and dilute to the mark, shake well. Take 1 ml into a 100 ml volumetric flask, add water to dilute to the mark, shake well.

[0155] The test results of each solution are as follows Figure 18-Figure 20 As shown, after the test sample was added, no peak appeared in the PIP solution.

[0156] The comprehensive test results of chromatographic conditions 1 to 5 show that the detection of piperazine cannot be achieved by using DB-1 columns of different specifications; even if the heating program is continuously optimized, the ideal detection effect cannot be achieved by using CP-Volamine columns. In addition, chromatographic condition 5 uses a single solvent, water, to dissolve the calcium folinate sample and dilute it to prepare the test solution. After injection, no piperazine peaks appear, indicating that the test solution preparation method is not suitable for the detection of piperazine in calcium folinate.

[0157] Comparative Example 2: Effect of Heating Program on Test Results

[0158] Chromatographic conditions 6:

[0159] Chromatographic column: CP-Sil 8CB, 30m*0.32mm, 1μm;

[0160] Inlet temperature: 240°C; Detector temperature: 250°C;

[0161] Temperature program: initial column temperature 60°C, maintained for 1 min, heated to 110°C at a rate of 5°C / min, then heated to 200°C at a rate of 40°C / min, maintained for 3 min;

[0162] Constant flow rate: 1.0ml / min;

[0163] Split ratio: 2:1; Solvent: water; Diluent: acetone;

[0164] Injection volume: 1 μl; carrier gas: high-purity nitrogen;

[0165] Sample preparation:

[0166] (1) Blank solution: Take 3 ml of water in a 10 ml volumetric flask, dilute to volume with acetone, and shake well to obtain;

[0167] (2) Reference solution: Weigh 9.97 mg of piperazine into a 10 ml volumetric flask, add 3 ml of water to dissolve, dilute to the mark with acetone, shake well, take 0.1 ml into 10 ml, add 3 ml of water, dilute to the mark with acetone, shake well.

[0168] The test results of each solution are as follows Figure 21-22 As shown, the piperazine peak eluted too late and there were many impurity peaks.

[0169] Test Example 1: Method Durability Study - Adjustment of Heating Holding Time and Injection Volume

[0170] Chromatographic conditions 7:

[0171] Temperature program: initial column temperature 110℃, maintain for 2 min, increase to 200℃ at a rate of 30℃ / min, maintain for 2 min. Other chromatographic conditions are the same as chromatographic condition 6.

[0172] Sample preparation: Weigh 10.20 mg of piperazine into a 10 ml volumetric flask, add 4 ml of water to dissolve, dilute to volume with acetone, and shake well. Then take 50 μl into a 10 ml volumetric flask, add 4 ml of water, dilute to volume with acetone, and shake well.

[0173] Test results such as Fig.23As shown. It can be seen that based on the chromatographic conditions of Example 1, the temperature program of Test Example 1 is shortened to 2 minutes at 200°C, and the injection volume is reduced to 1 μl. At this time, effective detection of piperazine in calcium folinate can still be achieved. However, compared with Example 1, the peak area under this condition is smaller and the response is lower.

[0174] Test Example 2: Method Durability Study - Adjustment of Initial Temperature

[0175] Chromatographic conditions 8:

[0176] Chromatographic column: CP-Sil 8CB, 30m*0.32mm, 1μm;

[0177] Inlet temperature: 240°C; Detector temperature: 250°C;

[0178] Temperature program: initial column temperature 105°C, maintained for 2 min, increased to 200°C at a rate of 30°C / min, maintained for 4 min;

[0179] Constant flow rate: 1.0ml / min;

[0180] Split ratio: 2:1; Solvent: water; Diluent: acetone;

[0181] Injection volume: 2 μl; carrier gas: high-purity nitrogen;

[0182] Test results such as Fig.24 As shown. It can be seen that based on the chromatographic conditions of Example 1, the initial column temperature of Test Example 2 is 105°C, which is consistent with 110°C and can still effectively detect piperazine in calcium folinate.

[0183] Chromatographic conditions 9:

[0184] Chromatographic column: CP-Sil 8CB, 30m*0.32mm, 1μm;

[0185] Inlet temperature: 240°C; Detector temperature: 250°C;

[0186] Temperature program: initial column temperature 115°C, maintained for 2 min, increased to 200°C at a rate of 30°C / min, maintained for 4 min;

[0187] Constant flow rate: 1.0ml / min;

[0188] Split ratio: 2:1; Solvent: water; Diluent: acetone;

[0189] Injection volume: 2 μl; carrier gas: high-purity nitrogen;

[0190] Test results such as Fig.25As shown. It can be seen that based on the chromatographic conditions of Example 1, the initial column temperature of Test Example 2 is 115°C, which is consistent with 110°C and can still effectively detect piperazine in calcium folinate.

[0191] Based on the above experimental results, the method for detecting the content of piperazine in the finished calcium folinate product provided by the present invention was tested for system applicability, specificity, limit of quantification and limit of detection, linearity and range, accuracy, precision and durability (solution stability and adjustment of chromatographic conditions), and the results were all in line with expectations, proving that the detection method is suitable for the detection of piperazine in the finished calcium folinate product.

Claims

1. A method for qualitatively and / or quantitatively detecting impurities in calcium folinate, characterized in that: It includes the following steps: (1) Prepare the test solution: weigh the calcium folinate test sample, add water and acetone, mix well, centrifuge, and take the supernatant; (2) Detection by gas chromatography; the chromatographic column used in the gas chromatography is CP-Sil 8CB, and the temperature program is as follows: the initial column temperature is 105-115°C, maintained for 2-4 minutes, and then increased to 195-205°C at a rate of 20-30°C / min, and maintained for 2-4 minutes; The impurity is piperazine.

2. The method according to claim 1, characterized in that: In step (1), the method for preparing the test solution comprises the following steps: 1) Weigh the calcium folinate test sample and add water until it is completely dissolved; 2) Add acetone and mix evenly, centrifuge, and take the supernatant.

3. The method according to claim 1, characterized in that: In step (1), the volume ratio of water to acetone is 2:(2.4-3.6); And / or, in step (1), the concentration of the test solution is 4-6 mg / ml.

4. The method according to claim 3, characterized in that: In step (1), the volume ratio of water to acetone is 2:3; And / or, in step (1), the concentration of the test solution is 5 mg / ml.

5. The method according to claim 1, characterized in that: The temperature raising program is as follows: the initial column temperature is 110°C, maintained for 2 minutes, raised to 200°C at a rate of 30°C / min, and maintained for 4 minutes.

6. The method according to claim 5, characterized in that: The length of the CP-Sil 8CB is 30 m; and / or, the inner diameter of the CP-Sil 8CB is 0.32 mm; and / or, the film thickness of the CP-Sil 8CB is 1 μm.

7. The method according to any one of claims 1 to 6, characterized in that: In step (2), the injection volume of the gas chromatography method is 1-2 μl, and the split ratio is 2:1; And / or, the carrier gas of the gas chromatography is nitrogen with a flow rate of 0.8 to 1.2 ml / min; and / or, the injection port temperature of the gas chromatography is 220-240° C.; and / or, the detector temperature of the gas chromatography is 230-250° C.; And / or, the gas chromatography method adopts an external standard method to calculate the impurity content.

8. The method according to claim 7, characterized in that: In step (2), the injection volume is 2 μl, and the split ratio is 2:1; And / or, the flow rate is 1.0 ml / min; and / or, the injection port temperature of the gas chromatography is 240°C; And / or, the detector temperature of the gas chromatography is 250°C.

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