Detection method of potassium di-tert-butyl phosphate

Abstract

The invention provides a method for detecting potassium di-tert-butyl phosphate. According to the method, the impurity potassium polyphosphate in the potassium di-tert-butyl phosphate is detected by adopting HPLC (High Performance Liquid Chromatography). The method is good in specificity, other impurities (such as degradation product tert-butyl alcohol of the potassium di-tert-butyl phosphate) do not interfere with detection, the content of the impurity potassium polyphosphate in the potassium di-tert-butyl phosphate can be rapidly and accurately separated and detected, the product quality of the potassium di-tert-butyl phosphate can be improved, and the safety and effectiveness of a phosphoric acid prodrug are further guaranteed. The method disclosed by the invention is good in durability, and when chromatographic condition parameters are slightly changed, the separation and measurement results of the impurity potassium polyphosphate are not influenced.

Description

Technical Field

[0001] This invention relates to a method for detecting potassium di-tert-butyl phosphate, belonging to the field of pharmaceutical analysis technology. Background Technology

[0002] In recent years, the research scope of phosphorus-containing organic drugs has gradually expanded from anti-tumor and insecticidal applications to multiple fields such as central nervous system, antibacterial, antiviral, cardiotonic, antihypertensive, and enzyme inhibition. The emergence of phosphate prodrugs such as fosphenytoin sodium, betamethasone sodium phosphate, dexamethasone sodium phosphate, and clindamycin phosphate indicates that phosphorylation has become an important means of prodrug development in the pharmaceutical industry, playing an important role in improving the biological and chemical properties of drug compounds.

[0003] Potassium di-tert-butyl phosphate (DTB) is a key material in the synthesis of phosphate precursor drugs. Its CAS number is 33494-80-3. Due to its ease of deprotection during reaction and good storage stability, it is widely used in the preparation processes of drugs such as phenytoin sodium, betamethasone sodium phosphate, dexamethasone sodium phosphate, and clindamycin phosphate. The structure of potassium di-tert-butyl phosphate is as follows:

[0004]

[0005] Polyphosphates are a class of inorganic compounds with polyphosphate groups. Among them, potassium polyphosphate, with the molecular formula (KPO3)n, is crystalline at room temperature and has high solubility in water.

[0006] The inventors discovered that potassium polyphosphate is a major process impurity formed during the preparation of potassium di-tert-butyl phosphate. High levels of this impurity, if transferred to the finished drug product, may increase the risks associated with medication use (especially for individuals with underlying medical conditions). For example, the alkalinity of this impurity may cause moderate irritation to the gastrointestinal mucosa; furthermore, before being absorbed by the intestines, it may hydrolyze into smaller orthophosphate molecules, inducing metabolic acidosis and increasing the likelihood of cardiovascular disease; there are also reports that high doses of this impurity may lead to kidney stones or induce the initiation and accelerated growth of lung cancer cells. Potassium polyphosphate may also participate in subsequent manufacturing processes of the finished drug product, generating undesirable side effects that could affect the safety and efficacy of the drug.

[0007] Therefore, it is necessary to strictly control the potassium polyphosphate in di-tert-butyl phosphate potassium salt to ensure the quality of di-tert-butyl phosphate potassium salt, thereby ensuring the safety and efficacy of the drug. Summary of the Invention

[0008] The purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for determining the content of potassium polyphosphate impurities in the pharmaceutical intermediate di-tert-butyl phosphate potassium salt. Using this HPLC method, the content of potassium polyphosphate impurities in di-tert-butyl phosphate potassium salt can be rapidly, effectively, accurately and reliably separated and detected, which is beneficial to ensuring the product quality of di-tert-butyl phosphate potassium salt.

[0009] This invention provides a method for detecting potassium di-tert-butyl phosphate, which uses HPLC to detect the impurity potassium polyphosphate in potassium di-tert-butyl phosphate. The chromatographic conditions are as follows:

[0010] The chromatographic column is packed with adamantylsilane-bonded silica gel. The mobile phase consists of phase A (aqueous) and phase B (methanol), and isocratic elution is performed. The volume ratio of phase A to phase B is 80–90:20–10. The flow rate is 0.8–1.2 mL / min. The column temperature is 25–45 °C. The detector is a differential refractive index detector, an evaporative light scattering detector, or a conductivity detector. The temperature of the differential refractive index detector is 25–45 °C.

[0011] The chromatographic column is packed with OSAKA SODA CAPCELL PAK ADME, with a size of 4.6 mm × 250 mm and a particle size of 5 μm.

[0012] The volume ratio of phase A to phase B is 83–87:17–13; the flow rate is 0.8 mL / min, 1.0 mL / min, or 1.2 mL / min; the column temperature is 30–40 °C; and the differential refractive index detector temperature is 30–40 °C.

[0013] More preferably, the volume ratio of phase A to phase B is 85:15; the column temperature is 30°C, 35°C or 40°C.

[0014] Specifically, it includes the following steps:

[0015] a. Prepare the potassium ditert-butyl phosphate solution and the potassium polyphosphate impurity reference solution for the test sample.

[0016] b. Set the high-performance liquid chromatography detection conditions:

[0017] A chromatographic column packed with adamantylsilane-bonded silica gel was used. The mobile phase consisted of phase A (aqueous) and phase B (methanol), with isocratic elution. The volume ratio of phase A to phase B was 80–90:20–10. The flow rate was 0.8–1.2 mL / min. The column temperature was 25–45 °C. The detector was a differential refractive index detector, an evaporative light scattering detector, or a conductivity detector. The temperature of the differential refractive index detector was 25–45 °C.

[0018] The chromatographic column packing material is OSAKA SODA CAPCELL PAK ADME, with dimensions of 4.6 mm × 250 mm and a particle size of 5 μm.

[0019] The volume ratio of phase A to phase B is 83–87:17–13; the flow rate is 0.8 mL / min, 1.0 mL / min, or 1.2 mL / min; the column temperature is 30–40 °C; and the differential refractive index detector temperature is 30–40 °C.

[0020] More preferably, the volume ratio of phase A to phase B is 85:15; the column temperature is 30°C, 35°C, or 40°C.

[0021] c. Accurately pipette the test solution and the impurity reference solution into the liquid chromatograph, start the detection and record the chromatogram;

[0022] d. Calculate the content of impurity potassium polyphosphate in di-tert-butyl phosphate potassium salt using the external standard method based on peak area.

[0023] The preparation method of the test solution in step a is as follows: weigh the test sample potassium ditert-butyl phosphate, add diluent to dissolve and dilute to prepare a solution containing 20 mg of potassium ditert-butyl phosphate per 1 mL.

[0024] The method for preparing the impurity reference solution is as follows: accurately weigh potassium polyphosphate reference standard, add diluent to dissolve and dilute to prepare a solution containing 40 μg of potassium polyphosphate per 1 mL.

[0025] Furthermore, the diluent is selected from water.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] (1) The method of the present invention has good specificity. Other impurities (such as tert-butanol, a degradation product of potassium di-tert-butyl phosphate) do not interfere with the detection. It can quickly and accurately separate and detect the content of impurity potassium polyphosphate in potassium di-tert-butyl phosphate, which is beneficial to improve the product quality of potassium di-tert-butyl phosphate and thus ensure the safety and effectiveness of phosphate precursor drugs.

[0028] (2) The method of the present invention has good durability. When the chromatographic conditions and parameters change slightly, the separation and determination results of the impurity potassium polyphosphate are not affected. Attached Figure Description

[0029] Figure 1 This is a high-performance liquid chromatogram of a blank solution (solvent);

[0030] Figure 2 This is a high-performance liquid chromatogram of a tert-butanol solution;

[0031] Figure 3This is a high-performance liquid chromatogram of potassium polyphosphate reference solution;

[0032] Figure 4 High-performance liquid chromatogram of potassium di-tert-butyl phosphate test solution;

[0033] Figure 5 The high performance liquid chromatogram of potassium di-tert-butyl phosphate (batch number: NP1105-2101014) is shown.

[0034] Figure 6 This is a high-performance liquid chromatogram of potassium di-tert-butyl phosphate (batch number: NP1105-2101015). Detailed Implementation

[0035] Example 1: Method for determining the content of potassium polyphosphate impurities in potassium di-tert-butyl phosphate.

[0036] Determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0037] The test solution is prepared by dissolving 200 mg of potassium di-tert-butyl phosphate in water and diluting it to prepare a solution containing 20 mg per mL.

[0038] For the reference solution, take an appropriate amount of potassium polyphosphate reference standard, dissolve and dilute it with water to prepare a solution containing 40 μg per 1 mL. Chromatographic conditions: Use adamantyl-bonded silica gel as the stationary phase (OSAKA SODA CAPCELL PAK ADME, 4.6 mm × 250 mm, 5 μm or equivalent column); use water-methanol (85:15) as the mobile phase; flow rate is 1.0 mL / min; column temperature is 35℃; differential refractive index detector temperature is 35℃; injection volume is 20 μL.

[0039] For system suitability testing, inject the reference solution into 6 syringes consecutively. The RSD of the peak area of ​​potassium polyphosphate should not exceed 5%, and the theoretical plate number of the potassium polyphosphate peak should be greater than 5000.

[0040] For the assay, accurately measure the reference solution and the test solution, and inject them separately into the liquid chromatograph. Record the chromatograms up to three times the retention time of the main component peak. The potassium polyphosphate content is calculated by peak area using the external standard method.

[0041] Example 2 Methodological Validation

[0042] Verification basis

[0043] This validation was conducted according to the requirements of the 2020 edition of the Chinese Pharmacopoeia, validating the following parameters of the method: specificity, system precision, accuracy, and robustness. All results met the acceptable criteria.

[0044] 1. Exclusivity

[0045] Solution preparation

[0046] Blank solvent: water.

[0047] tert-Butanol solution: Dissolve tert-Butanol in water and dilute to prepare a solution containing 40 μg per 1 mL. Filter and collect the filtrate.

[0048] Potassium polyphosphate reference solution: Take an appropriate amount of potassium polyphosphate reference standard, dissolve and dilute it with water to prepare a solution containing 40 μg per 1 mL, filter it, and take the filtrate.

[0049] Test solution: Dissolve and dilute potassium ditert-butyl phosphate in water to prepare a solution containing 20 mg per 1 mL, filter, and collect the filtrate.

[0050] Measurement

[0051] Take blank solvent, tert-butanol solution, potassium polyphosphate reference solution and test solution in sequence, inject them into the liquid chromatograph and record the chromatograms.

[0052] Results: See the graph. Figures 1-4 The retention time of the potassium polyphosphate reference peak was 2.770 min, and the blank solvent did not interfere with the potassium polyphosphate peak. The retention time of the tert-butanol peak was 12.535 min, and it did not interfere with the potassium polyphosphate peak. The specificity met the validation acceptable standard.

[0053] 2. System precision

[0054] Solution preparation

[0055] Potassium polyphosphate reference solution: Take an appropriate amount of potassium polyphosphate reference standard, dissolve and dilute it with water to prepare a solution containing 40 μg per 1 mL.

[0056] Measurement

[0057] Take a potassium polyphosphate reference solution, inject it into the liquid chromatograph, repeat 6 times, and record the chromatogram.

[0058] Results: The results are shown in Table 1. The relative standard deviation (RSD) of the peak area was 3.65%, and the retention time (t) was... R The RSD of the peak area is 0.13%. The system precision meets the acceptable validation criteria (acceptable criteria: peak area RSD should not exceed 5%).

[0059] Table 1 System precision results

[0060] Number of injection needles <![CDATA[Retention time t R (min)]]> Potassium polyphosphate peak area Theoretical number of plates 1 2.770 2258.704 11457 2 2.771 2150.875 11662 3 2.771 2108.812 11699 4 2.779 2080.565 11752 5 2.776 2059.899 11935 6 2.774 2053.525 11837 RSD 0.13％ 3.65％ ——

[0061] 3. Accuracy (Impurity Recovery Rate)

[0062] Take an appropriate amount of potassium di-tert-butyl phosphate, add an appropriate amount of potassium polyphosphate reference standard at 0.15% of the concentration of the test sample solution, prepare a spiking solution, and make 6 portions. Calculate the recovery rate for each portion.

[0063] Calculation formula:

[0064] Results: The results are shown in Table 2. The recovery rates ranged from 87.19% to 104.08%, with an RSD of 8.08%. The accuracy validation results met the acceptable criteria (acceptable criteria: the RSD of the recovery rate should not exceed 10%).

[0065] Table 2 Accuracy (Recovery Rate) Results

[0066]

[0067] 4. Durability

[0068] An appropriate amount of potassium di-tert-butyl phosphate was used to investigate the extent to which chromatographic parameters such as mobile phase ratio, flow rate, and column temperature were affected by minute changes in the determination results. Single-parameter adjustment was performed, using data from the same set under a single parameter condition as the evaluation object.

[0069] Measurement

[0070] ① Set the water-methanol ratio in the mobile phase to (87:13), (85:15), and (83:17), and inject samples for determination, calculating the RSD of the three impurity determination results. ② Set the flow rate to 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min, and inject samples for determination, calculating the RSD of the three impurity determination results. ③ Set the column temperature to 30℃, 35℃, and 40℃, and inject samples for determination, calculating the RSD of the three impurity determination results.

[0071] Results: The results are shown in Table 3. The RSD of impurity determination results in the same group of single-parameter data was 3.1%-3.6%, and the robustness verification results met the acceptable standard (acceptable standard: RSD should not exceed 5%).

[0072] Table 3 Durability Results

[0073]

[0074] 5. Testing of two batches of di-tert-butyl phosphate potassium salt

[0075] The method described in Example 1 was used to test two batches of potassium di-tert-butyl phosphate. The results are shown in Table 4. Figures 5-6 .

[0076] Table 4. Detection results of potassium di-tert-butyl phosphate

[0077]

[0078] Comparative Example 1

[0079] Octadecyl-bonded silica gel (YMC-Pack, ODS-AQ, 4.6 mm × 250 mm, 5 μm) was used as the packing material; water-methanol (80:20) was used as the mobile phase. Other conditions were the same as in Example 1. The potassium di-tert-butyl phosphate test solution was injected for analysis. The results showed that potassium polyphosphate eluted at 2.355 min, with an asymmetrical peak shape (tailing factor 0.63), and a theoretical plate number of only 2017. These conditions are not suitable for detecting the potassium polyphosphate content in potassium di-tert-butyl phosphate.

[0080] Comparative Example 2

[0081] Octyl-bonded silica gel (Zorbax Eclipse plus C8, 4.6 mm × 250 mm, 5 μm) was used as the packing material; water-methanol (80:20) was used as the mobile phase. Other conditions were the same as in Example 1. The potassium di-tert-butyl phosphate test solution was injected for analysis. The results showed that potassium polyphosphate eluted at 2.237 min, with an asymmetrical peak shape (tailing factor 0.78), and a theoretical plate number of only 2364. These conditions are not suitable for detecting the potassium polyphosphate content in potassium di-tert-butyl phosphate.

[0082] Comparative Example 3

[0083] Adamantyl-bonded silica gel (CAPCELL PAK ADME, 4.6 mm × 250 mm, 5 μm) was used as the packing material; a 0.02 mol / L dipotassium hydrogen phosphate solution-methanol (60:40) was used as the mobile phase. Other conditions were the same as in Example 1. Analysis of the potassium di-tert-butyl phosphate test solution did not reveal a potassium polyphosphate peak. These conditions are not suitable for detecting the potassium polyphosphate content in potassium di-tert-butyl phosphate.

Claims

1. A method for detecting potassium di-tert-butyl phosphate, characterized in that, It uses HPLC to detect potassium polyphosphate, an impurity in potassium di-tert-butyl phosphate. The chromatographic conditions are as follows: The chromatographic column is packed with adamantylsilane-bonded silica gel. The mobile phase consists of phase A (aqueous) and phase B (methanol), and isocratic elution is performed. The volume ratio of phase A to phase B is 80–90:20–10. The flow rate is 0.8–1.2 mL / min. The column temperature is 25–45 °C. The detector is a differential refractive index detector, an evaporative light scattering detector, or a conductivity detector. The temperature of the differential refractive index detector is 25–45 °C.

2. The method for detecting potassium di-tert-butyl phosphate according to claim 1, characterized in that: The chromatographic column is packed with OSAKA SODA CAPCELL PAK ADME, with dimensions of 4.6 mm × 250 mm and a particle size of 5 μm.

3. The method for detecting potassium di-tert-butyl phosphate according to claim 1 or 2, characterized in that: The volume ratio of phase A to phase B is 83–87:17–13; the flow rate is 0.8 mL / min, 1.0 mL / min, or 1.2 mL / min; the column temperature is 30–40 °C; and the differential refractive index detector temperature is 30–40 °C.

4. The method for detecting potassium di-tert-butyl phosphate according to claim 3, characterized in that: The volume ratio of phase A to phase B is 85:15; the column temperature is 30℃, 35℃ or 40℃.

5. The method for detecting potassium di-tert-butyl phosphate according to any one of claims 1-4, characterized in that, Includes the following steps: a. Prepare the potassium ditert-butyl phosphate solution and the potassium polyphosphate impurity reference solution for the test sample. b. Setting up liquid chromatography detection conditions: Setting up the chromatographic conditions as described in any one of claims 1-4; c. Accurately pipette the test solution and the impurity reference solution into the liquid chromatograph, start the detection and record the chromatogram; d. Calculate the content of impurity potassium polyphosphate in di-tert-butyl phosphate potassium salt using the external standard method based on peak area.

6. The method for detecting potassium di-tert-butyl phosphate according to claim 5, characterized in that, The preparation method of the test solution in step a is as follows: weigh the test sample potassium ditert-butyl phosphate, add diluent to dissolve and dilute to prepare a solution containing 20 mg of potassium ditert-butyl phosphate per 1 mL. The method for preparing the impurity reference solution is as follows: accurately weigh potassium polyphosphate reference standard, add diluent to dissolve and dilute to prepare a solution containing 40 μg of potassium polyphosphate per 1 mL.

7. The method for detecting potassium di-tert-butyl phosphate according to claim 6, characterized in that: The diluent is selected from water.

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