CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution

Abstract

The invention discloses a CAD detection method for phosphoric acid and phosphorous acid in an alendronate sodium oral solution, and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: preparing a reference stock solution and a test solution; then setting high performance liquid chromatography detection conditions; a Shodex VN-504D chromatographic column is adopted, a CAD detector is adopted as a detector, a mixed solution of a mobile phase A and a mobile phase B is adopted as a mobile phase, the mobile phase A is ammonium formate, the mobile phase B is an acetonitrile solution, and gradient elution is performed by adopting the mobile phase; and respectively measuring the reference stock solution and the test solution, injecting into a liquid chromatograph, and recording a chromatogram. A CAD detector is adopted, the detection sensitivity is obviously improved, the quantitative concentration can reach 0.24 mu g / mL, and the detection concentration can reach 0.10 mu g / mL. The method is high in specificity, high in sensitivity, good in durability, rapid, simple, effective and reliable.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution. Background Technology

[0002] Alendronate sodium is a bisphosphonate sodium that acts as a specific inhibitor of osteoclast-mediated bone resorption. Bisphosphonates are synthetic analogs of pyrophosphates that bind to hydroxyapatite in bone. It is indicated for the treatment and prevention of osteoporosis in postmenopausal women and for increasing bone mass in men with osteoporosis.

[0003] Alendronate sodium is a third-generation bisphosphonate drug developed by Instituto Gentili in Italy and Merck in the United States. It was approved by the FDA in September 1995 for marketing in the United States, and is marketed as a tablet under the brand name [Brand Name Missing]. The specifications are available in 5mg, 10mg, 35mg, 40mg, and 70mg (based on C4H). 13 (NO7P2 calculation). Currently, tablets in 5mg, 10mg, 35mg, and 40mg strengths have been withdrawn from the US market. The oral solution formulation received FDA approval for marketing in the US in September 2003. The brand name is... The specification is EQ 70mg Base / 75mL, and it has been withdrawn from the market in the United States.

[0004] In the alendronate sodium raw material process, phosphoric acid and phosphorous acid are both listed in the standards for control in the Chinese Pharmacopoeia and the European Pharmacopoeia. Therefore, relevant studies were also conducted on alendronate sodium oral solution. The excipients contained in the oral solution are more complex and have greater interference, and phosphoric acid and phosphorous acid have no ultraviolet absorption, so the detection capabilities of multiple detectors such as ELSD and RID are insufficient. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution. This method has high specificity, high sensitivity, good durability, and is fast, simple, effective and reliable.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting phosphoric acid and phosphorous acid CAD in alendronate sodium oral solution, using high-performance liquid chromatography, includes the following steps:

[0008] 1) Prepare the reference standard stock solution and the test solution for later use;

[0009] 2) Set up high performance liquid chromatography detection conditions; use Shodex VN-50 4D column, CAD detector, and a mixed solution of mobile phase A and mobile phase B, wherein mobile phase A is ammonium formate and mobile phase B is formic acid acetonitrile solution, and gradient elution is performed using the mobile phase;

[0010] 3) Measure out the stock solution of the standard and the test solution separately, inject them into the liquid chromatograph, and record the chromatograms.

[0011] Further, in step 1), the preparation of the phosphoric acid stock solution in the reference stock solution is as follows: Weigh approximately 50 mg of phosphoric acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphoric acid stock solution; the preparation of the phosphorous acid stock solution is as follows: Weigh approximately 50 mg of phosphorous acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphorous acid stock solution.

[0012] Further, in step 1), the preparation of the test solution is as follows: accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, dilute to the mark with diluent, shake well, and use it as the test solution, which is then labeled as SPL-1 and SPL-2, containing 0.2 mg / mL of the main component.

[0013] Furthermore, in step 2), the Shodex VN-50 4D column has a specification of 4.6 × 150 mm and a packing particle size of 5 μm.

[0014] Further, in step 2), the gradient elution conditions are as follows: 0–2 min, the volume content of mobile phase A is 17% and the volume content of mobile phase B is 83%; 2–19 min, the volume content of mobile phase A is 17–25% and the volume content of mobile phase B is 83–75%; 19–20 min, the volume content of mobile phase A is 25–35% and the volume content of mobile phase B is 75–65%; 20–25 min, the volume content of mobile phase A is 35% and the volume content of mobile phase B is 65%; 25–25.5 min, the volume content of mobile phase A is 35–17% and the volume content of mobile phase B is 65–83%; 25.5–30 min, the volume content of mobile phase A is 17% and the volume content of mobile phase B is 83%.

[0015] Furthermore, in step 2), the column temperature is 33–37°C.

[0016] Furthermore, in step 2), the flow rate is 0.7–0.9 mL / min.

[0017] Furthermore, in step 2), the injection volume is 10 μL.

[0018] Furthermore, in step 2), the temperature of the sample injection chamber is 5°C and the temperature of the evaporation chamber is 50°C.

[0019] Furthermore, in step 2), the filtering constant is 3.6s, the power function is 1.0, and the acquisition rate is 5Hz.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] This invention provides a CAD (Carbon Dioxide) detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution. Since neither phosphoric acid nor phosphorous acid absorbs ultraviolet light, conventional ultraviolet detectors are unsuitable. Detection methods derived from the European Pharmacopoeia and Chinese Pharmacopoeia have low RID (Residual Inhibitor Detection) sensitivity, and the high proportion of residual excipients in the oral solution significantly interferes with the detection of phosphoric acid and phosphorous acid, making development challenging. This invention employs high-performance liquid chromatography (HPLC) equipped with a CAD detector, effectively separating phosphoric acid, phosphorous acid, the main component, and all excipients in alendronate sodium oral solution. The use of a CAD detector significantly improves detection sensitivity, achieving a quantitative concentration of 0.24 μg / mL and a detection concentration of 0.10 μg / mL. This method is highly specific, sensitive, robust, rapid, simple, effective, and reliable. Attached Figure Description

[0022] Figure 1 This is the chromatogram of the detection method in this application;

[0023] Figure 2 This is the phosphoric acid standard curve for this application;

[0024] Figure 3 This is the standard curve of phosphorous acid in this application. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0026] Example 1

[0027] A method for detecting phosphoric acid and phosphorous acid CAD in alendronate sodium oral solution includes the following steps:

[0028] 1) Instrument and sample selection: The high performance liquid chromatograph was a Vanquish core model, equipped with a CAD detector; alendronate sodium oral solution, batch number AB9765A, was manufactured by Hikma Pharmaceuticals USA Inc.; phosphoric acid and phosphorous acid, and formic acid used in the solution preparation in the experiment were all provided by Aladdin; acetonitrile was provided by TEDIA.

[0029] 2) Setting up liquid chromatography detection conditions

[0030] The chromatographic column was a Shodex VN-50 4D (4.6 × 150 mm; 5 μm); mobile phase A was 20 mM ammonium formate; mobile phase B was acetonitrile solution; the diluent was mobile phase A-mobile phase B (50:50); the flow rate was 0.8 mL / min; the sample room temperature was 5 °C; the column temperature was 35 °C; the detector was a CAD detector; the evaporation chamber temperature was 50 °C; the filtration constant was 3.6 s; the power function was 1.0; the acquisition rate was 5 Hz; the injection volume was 10 μL; the elution program is shown in Table 1.

[0031] Table 1 Elution Procedure

[0032]

[0033]

[0034] 3) Sample solution preparation

[0035] Reference stock solution

[0036] Phosphoric acid stock solution: Weigh approximately 50 mg of phosphoric acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain phosphate stock solution (0.5 mg / mL).

[0037] Phosphorous acid stock solution: Weigh approximately 50 mg of phosphorous acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphorous acid stock solution (0.5 mg / mL).

[0038] Standard curve solution

[0039] Linear stock solution L-Stock: Accurately weigh 1.0 mL each of phosphoric acid stock solution and phosphorous acid stock solution, place them in the same 100 mL volumetric flask, dilute to the mark with diluent, shake well, and use as linear stock solution, labeled as L-Stock (containing 5 μg / mL each of phosphoric acid and phosphorous acid).

[0040] Linear solutions L-LOQ~L-200%: Accurately transfer an appropriate amount of linear stock solution into different 5mL volumetric flasks, dilute to the mark with diluent, and shake well to obtain linear solutions of different concentrations. The preparation process is shown in Table 2.

[0041] Table 2 Linear solutions of different concentrations

[0042] name Volume (mL) of L-Stock transferred Volumetric flask (mL) Theoretical concentration (μg / mL) L-LOQ 0.25 5 0.25 L-50％ 0.5 5 0.5 L-80％ 0.8 5 0.8 L-100％ 1.0 5 1.0 L-150％ 1.5 5 1.5 L-200％ 2.0 5 2.0

[0043] Test solution: Accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, dilute to the mark with diluent, and shake well. This is the test solution, and it is labeled as SPL-1 and SPL-2 (containing 0.2 mg / L of the main component).

[0044] 4) Sample testing methods

[0045] Take the above-mentioned reference standard stock solution, standard curve solution, and test solution, respectively, and inject them into the liquid chromatograph according to the chromatographic conditions described in the method, and record the chromatograms. (Chromatograms are shown below.) Figure 1 As shown.

[0046] Depend on Figure 1 It can be seen that the diluent (Blank) and blank excipient solution do not interfere with the target peak. The retention time of the target peak in the phosphoric acid and phosphorous acid positioning solutions should be consistent with (±5%) the retention time of the target peak in the reference solution (L-100%) and the spiked test solution.

[0047] Example 2

[0048] The detection method disclosed in Example 1 was validated in terms of specificity, system suitability, limit of quantitation and limit of detection, accuracy, repeatability, intermediate precision, solution stability, and robustness, as detailed below:

[0049] 1. Specificity and System Applicability

[0050] Standard curve solution

[0051] Linear Stock Solution L-Stock: Accurately transfer 1.0 mL of phosphate reference stock solution and phosphorous acid reference stock solution into a 100 mL volumetric flask, dilute to the mark with diluent and shake well to obtain linear stock solution L-Stock (5 μg / mL).

[0052] Linear solutions L-LOQ~L-200%: Accurately transfer an appropriate amount of linear stock solution into different 5mL volumetric flasks, dilute to the mark with diluent, and shake well to obtain linear solutions of different concentrations. The preparation process is shown in Table 2.

[0053] Table 2 Linear solutions of different concentrations

[0054] name Volume (mL) of L-Stock transferred Volumetric flask (mL) Theoretical concentration (μg / mL) L-LOQ 0.25 5 0.25 L-50％ 0.5 5 0.5 L-80％ 0.8 5 0.8 L-100％ 1.0 5 1.0 L-150％ 1.5 5 1.5 L-200％ 2.0 5 2.0

[0055] Table 3. Results of the localization solution

[0056]

[0057]

[0058] As shown in Table 3, the retention times of the target peaks in the phosphoric acid and phosphorous acid positioning solutions were consistent with those in the spiked test sample solution (±5%).

[0059] Table 4 Results of the Phosphoric Acid Standard Curve

[0060]

[0061] From Table 4 and Figure 2 It can be seen that phosphoric acid exhibits a linear correlation within the concentration range of 0.2452 μg / mL to 1.9618 μg / mL, with a correlation coefficient r of 0.9980. The linear equation is y = 0.0812x² + 0.0436x + 0.0119. The results meet the requirements.

[0062] Table 5 Results of the standard curve for phosphorous acid

[0063]

[0064] From Table 5 and Figure 3 It can be seen that phosphorous acid exhibits a linear correlation within the concentration range of 0.2476 μg / mL to 1.9804 μg / mL, with a correlation coefficient r of 0.9993. The linear equation is y = 0.0115x² + 0.1369x - 0.0055. The results meet the requirements.

[0065] 2. Limit of Quantification, Limit of Detection

[0066] Accurately measure the reference solution, dilute the solution, and inject it according to the chromatographic conditions for content detection. Record the chromatogram. The limit of quantitation is defined as a signal-to-noise ratio (S / N) ≥ 10, and the limit of detection is defined as a signal-to-noise ratio (S / N) ≥ 3. The results are shown in Table 6-7.

[0067] Table 6 Results of Limit of Quantitation and Limit of Detection for Phosphate

[0068]

[0069] Table 7 Results of Limit of Quantitation and Limit of Detection for Phosphorous Acidity

[0070]

[0071]

[0072] As shown in Table 6-7, among the six solutions with the limit of quantitation, the RSD of the peak area of ​​phosphate was 9%, and the signal-to-noise ratio was between 17 and 24, while the signal-to-noise ratio of the limit of detection was 7; the RSD of the peak area of ​​phosphorous acid was 12%, and the signal-to-noise ratio was between 14 and 19, while the signal-to-noise ratio of the limit of detection was 8.

[0073] 3. Accuracy

[0074] Reference stock solution

[0075] Phosphoric acid stock solution: Weigh approximately 50 mg of phosphoric acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain phosphate stock solution (0.5 mg / mL).

[0076] Phosphorous acid stock solution: Weigh approximately 50 mg of phosphorous acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphorous acid stock solution (0.5 mg / mL).

[0077] Test solution: Accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, dilute to the mark with diluent, and shake well. This is the test solution, and it is labeled as SPL-1 and SPL-2 (containing 0.2 mg / L of the main component).

[0078] Accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, add an appropriate amount of linear stock solution L-Stock, and dilute to the mark with diluent to prepare spiked test solutions at different concentration levels. The preparation process is shown in Table 8. Inject the solution into the liquid chromatograph and record the chromatogram. The results are shown in Tables 9-10.

[0079] Table 8 Spiked test solutions at different concentration levels

[0080]

[0081] Table 9. Phosphoric acid accuracy results

[0082]

[0083] Table 10. Accuracy Results for Phosphorous Acidity

[0084]

[0085]

[0086] As shown in Table 9-10, the recoveries of the spiked test solutions of phosphoric acid at various concentration levels ranged from 92% to 120%, with an average recovery rate of 101% and an RSD of 12% (n=9). The recoveries of the spiked test solutions of phosphorous acid at various concentration levels ranged from 104% to 118%, with an average recovery rate of 107% and an RSD of 4% (n=9).

[0087] 4. Repeatability

[0088] Accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, add 1 mL of linear stock solution L-Stock, and dilute to the mark with diluent to prepare a medium-concentration spiked test solution. Prepare 6 parallel aliquots (the first 3 aliquots are prepared using the 100% concentration level spiked test solution from the recovery section) as shown in Table 11. Inject into the liquid chromatograph and record the chromatograms. The results are shown in Tables 12-13.

[0089] Table 11 shows the concentration levels of the spiked test solutions.

[0090]

[0091] Table 12 Repeatability Results of Phosphate

[0092]

[0093]

[0094] Table 13 Repeatability results for phosphorous acid

[0095]

[0096] As shown in Table 12-13, the average recovery rate of phosphoric acid in the six spiked test solutions was 101%, with an RSD of 5%; the average recovery rate of phosphorous acid was 107%, with an RSD of 2%.

[0097] 5. Intermediate precision

[0098] Related substances were tested on the same batch of samples by different operators, at different times, and using different instruments according to repeatability test methods. The results are shown in Tables 14-15 below.

[0099] Table 14 Comparison of intermediate precision and repeatability of phosphate

[0100]

[0101] Table 15 Comparison of intermediate precision and repeatability of phosphorous acid

[0102]

[0103]

[0104] As shown in Table 14-15, compared with repeatability, the RSD of the recovery rate of the target peak phosphoric acid in a total of 12 test solutions was 10% (n=12); the RSD of the recovery rate of the target peak phosphoric acid was 6% (n=12).

[0105] 6. Solution stability

[0106] The reference solution was prepared by taking L-100% linear solution, and the test solution was prepared by taking spiked test solution RM#1. The chromatograms were recorded. The results are shown in Tables 16-17.

[0107] Table 16 Stability results of reference solutions

[0108]

[0109] Table 17 Stability results of spiked test sample solutions

[0110]

[0111] As shown in Table 16-17, when the reference solution was stored at 5℃ for less than 22.0 h, the ratio of the peak area of ​​the phosphoric acid target to the peak area at 0 h was between 111% and 118% at each time point, and the ratio of the peak area of ​​the phosphorous acid target to the peak area at 0 h was between 107% and 115%, indicating that the reference solution was stable at 5℃ for less than 22.0 h. When the spiked test solution was stored at 5℃ for less than 16.0 h, the ratio of the peak area of ​​the phosphoric acid target to the peak area at 0 h was between 102% and 105% at each time point, and the ratio of the peak area of ​​the phosphorous acid target to the peak area at 0 h was between 117% and 119%, indicating that the spiked test solution was stable at 5℃ for less than 16.0 h.

[0112] 7. Durability

[0113] The effects of column temperature, initial organic phase ratio, and flow rate on the system were investigated. The results are shown in Table 18.

[0114] Table 18 Results of Durability System Applicability

[0115]

[0116] As shown in Table 18, the system applicability under each condition should meet the method requirements, and the durability is good.

[0117] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting phosphoric acid and phosphorous acid CAD in alendronate sodium oral solution, characterized in that: High-performance liquid chromatography (HPLC) is used, including the following steps: 1) Prepare the reference standard stock solution and the test solution for later use; 2) Set up high performance liquid chromatography detection conditions; use Shodex VN-50 4D column, CAD detector, and a mixed solution of mobile phase A and mobile phase B, wherein mobile phase A is ammonium formate and mobile phase B is acetonitrile solution, and gradient elution is performed using the mobile phase; 3) Measure out the stock solution of the standard and the test solution separately, inject them into the liquid chromatograph, and record the chromatograms.

2. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 1), the preparation of the phosphoric acid stock solution in the reference stock solution is as follows: Weigh approximately 50 mg of phosphoric acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphoric acid stock solution; the preparation of the phosphorous acid stock solution is as follows: Weigh approximately 50 mg of phosphorous acid accurately, place it in a 100 mL volumetric flask, dilute to the mark with purified water, and shake well to obtain the phosphorous acid stock solution.

3. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 1), the test solution is prepared as follows: accurately transfer 1.0 mL of alendronate sodium oral solution into a 5 mL volumetric flask, dilute to the mark with diluent, shake well, and use this as the test solution, labeled as SPL-1 and SPL-2 respectively, containing 0.2 mg / mL of the main component.

4. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the Shodex VN-50 4D column has a specification of 4.6 × 150 mm and a packing particle size of 5 μm.

5. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the gradient elution conditions are as follows: 0–2 min, mobile phase A volume content is 17% and mobile phase B volume content is 83%; 2–19 min, mobile phase A volume content is 17–25% and mobile phase B volume content is 83–75%; 19–20 min, mobile phase A volume content is 25–35% and mobile phase B volume content is 75–65%; 20–25 min, mobile phase A volume content is 35% and mobile phase B volume content is 65%; 25–25.5 min, mobile phase A volume content is 35–17% and mobile phase B volume content is 65–83%; 25.5–30 min, mobile phase A volume content is 17% and mobile phase B volume content is 83%.

6. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the column temperature is 33–37°C.

7. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the flow rate is 0.7–0.9 mL / min.

8. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the injection volume is 10 μL.

9. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the temperature of the sample injection chamber is 5°C and the temperature of the evaporation chamber is 50°C.

10. The CAD detection method for phosphoric acid and phosphorous acid in alendronate sodium oral solution according to claim 1, characterized in that: In step 2), the filtering constant is 3.6s, the power function is 1.0, and the acquisition rate is 5Hz.

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