Method for detecting oxidation impurities in phloroglucinol injection

Abstract

The invention discloses a method for detecting oxidative degradation impurities in phloroglucinol injection, and belongs to the technical field of medicine detection. According to the method, a high performance liquid chromatography is matched with a chromatographic column of octadecylsilane chemically bonded silica to detect various oxidative degradation impurities which easily cause color change of the phloroglucinol injection. A mixed solution prepared from the oxidized impurities is used for judging the separation degree of each impurity, and the separation degree is not lower than 1.5; taking a proper amount of phloroglucinol injection to prepare a test solution; taking a proper amount of a phloroglucinol impurity S reference substance solution, and preparing the impurity S reference substance solution; taking a proper amount of phloroglucinol reference substance, and preparing a phloroglucinol reference substance solution; then measuring the content of the oxidation impurities I and V by using an impurity S reference substance method added with correction factors; and measuring the content of the oxidation impurity III by adopting a phloroglucinol reference substance method with correction factors. According to the method, the content of the oxidative degradation impurities in the phloroglucinol injection can be accurately determined, so that the quality of a medicine is determined, and an effective and feasible method is provided for strictly controlling the content of the oxidative degradation impurities in the phloroglucinol injection.

Description

Technical Field

[0001] The present invention relates to the technical field of drug quality control, and in particular to a method for detecting oxidative impurities in related substances of phloroglucinol injection. Background Art

[0002] Phloroglucinol is a myotropic antispasmodic drug that can directly act on the smooth muscles of the gastrointestinal tract and urogenital tract. Compared with other types of smooth muscle antispasmodics, phloroglucinol does not have an anticholinergic effect. While exerting its antispasmodic effect, it does not produce a series of anticholinergic side effects, nor does it have adverse effects on cardiovascular function. It does not cause symptoms such as increased heart rate, hypotension, and arrhythmia, so it is widely used clinically. Because it has three phenolic hydroxyl groups, it is easily oxidized by oxygen to produce quinones or more complex oxidative degradation products, which causes the color of the injection to change, affecting the quality of the drug, reducing efficacy, and increasing toxicity and the risk of adverse reactions. The "Chinese Pharmacopoeia 2020 Edition" does not include the detection method for related substances of phloroglucinol injection. Although the "European Pharmacopoeia" 11.0 includes the detection method for related substances of phloroglucinol raw materials, it does not give the structure of the oxidized impurities and their specific detection methods.

[0003] After searching existing technologies at home and abroad, no literature reports on the detailed structure and detection methods of oxidative impurities in phloroglucinol injection have been found. Summary of the Invention

[0004] In view of the deficiencies of the prior art, in order to better detect oxidative impurities in phloroglucinol injection, the present invention provides a detection method for simultaneously detecting multiple oxidative impurities in phloroglucinol injection.

[0005] To achieve the above technical objectives, the inventors, based on years of experience in drug analysis and through extensive experimental research, have ultimately developed the following technical solution: a method for detecting multiple oxidative impurities in phloroglucinol injection, comprising the following steps:

[0006] (1) Prepare a mixed solution containing oxidized impurities I, III, and V, and examine the peak order and separation of impurities I, III, and V. Impurities I, III, and V should peak in sequence, and the separation between impurities I and III, and between impurities III and V should be no less than 1.5.

[0007] (2) Take an appropriate amount of phloroglucinol injection to prepare the test solution.

[0008] (3) Take an appropriate amount of impurity S reference substance to prepare an impurity S reference substance solution.

[0009] (4) Take an appropriate amount of phloroglucinol reference substance to prepare phloroglucinol reference substance solution.

[0010] (5) Obtain chromatograms of the test solution and the mixed reference solution under qualified separation conditions, and determine the content of each oxidized impurity in the phloroglucinol injection using the following formula.

[0011] Impurity I / Impurity V (%) = (A u / A s )×(C S / C U )×P s ×F s ×100

[0012] Where: A u is the peak area of ​​impurity I / impurity V in the test solution;

[0013] A s is the peak area of ​​impurity S in the reference solution;

[0014] C s is the concentration of impurity S reference solution (mg / ml);

[0015] C u is the concentration of the test solution (mg / ml);

[0016] P s is the purity of impurity S (%);

[0017] F s It is the correction factor of each impurity I / impurity V relative to impurity S.

[0018] Impurity III (%) = (A u / A C )×(C C / C U )×P C ×F C ×100

[0019] Where: A u is the peak area of ​​impurity III in the test solution;

[0020] A C is the peak area of ​​pyrogallol in the reference solution;

[0021] C C is the concentration of phloroglucinol reference solution (mg / ml);

[0022] C u is the concentration of the test solution (mg / ml);

[0023] P C is the purity of phloroglucinol (%);

[0024] F Cis the correction factor of impurity III relative to phloroglucinol.

[0025] The separation conditions are as follows:

[0026] Chromatographic column: octadecylsilane bonded silica gel;

[0027] Column temperature: 35°C; injection temperature: room temperature;

[0028] Detection wavelength: 265nm;

[0029] Flow rate: 1.0 ml / min;

[0030] The first mobile phase was phosphate buffer at pH 3.0, and the second mobile phase was acetonitrile;

[0031] The first mobile phase was prepared as follows: 1.36 g of potassium dihydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH was adjusted to 3.0 with phosphoric acid)-acetonitrile (97:3)

[0032] The gradient elution program is as follows:

[0033]

[0034] The method for detecting oxidative impurities in phloroglucinol injection provided by the present invention can effectively measure the oxidative impurities in phloroglucinol injection, provide a basis for quality control of the drug, and reduce risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 : Chromatogram of the mixed solution of impurities I, III and V of the present invention.

[0036] Figure 2 : The separation spectrogram of the mixed test solution of the present invention.

[0037] Figure 3 : The separation spectrogram of the need testing solution of the present invention.

[0038] Figure 4 : Separation spectra of the detection limits of impurities I, III and V of the present invention.

[0039] Figure 5 : Separation spectra of the quantitative limits of impurities I, III and V of the present invention.

[0040] Figure 6 : Linear plot of impurity I of the present invention.

[0041] Figure 7 : Linear plot of impurity III of the present invention.

[0042] Figure 8 : Linear plot of impurity V of the present invention.

[0043] Figure 9 : Comparative Example of the present invention DETAILED DESCRIPTION

[0044] In order to have a further understanding of the technical solution and beneficial effects of the present invention, the technical solution and beneficial effects of the present invention are described in detail below with reference to the accompanying drawings.

[0045] Example 1: Detection of Various Oxidative Impurities in Phloroglucinol Injection

[0046] (1) Prepare the solvent: Dissolve 1.36 g of potassium dihydrogen phosphate in water and dilute to 1000 ml. Adjust the pH to 3.0 with phosphoric acid)-acetonitrile (90:10).

[0047] (2) Preparation of test solution: Accurately measure an appropriate amount of this product (equivalent to approximately 25 mg of phloroglucinol) into a 10 ml volumetric flask and quantitatively dilute with solvent to produce a solution containing approximately 2.5 mg of phloroglucinol per 1 ml.

[0048] (3) Preparation of impurity S reference solution: Take an appropriate amount of impurity S reference solution (equivalent to approximately 12.5 mg of impurity S), accurately weigh it, place it in a 100 ml volumetric flask, add solvent to dissolve it and dilute it to the scale, shake it well, accurately measure 2 ml, place it in a 25 ml volumetric flask, add solvent to dilute it to the scale, shake it well, and you will get a solution containing approximately 10 μg of impurity S per ml.

[0049] (4) Preparation of phloroglucinol reference solution: Take an appropriate amount of phloroglucinol reference solution (equivalent to approximately 12.5 mg of phloroglucinol), accurately weigh it, place it in a 50 ml volumetric flask, add solvent to dissolve it and dilute it to the scale, shake it well, accurately measure 1 ml, place it in a 50 ml volumetric flask, add solvent to dilute it to the scale, shake it well, and you will get a solution containing approximately 5 μg of phloroglucinol per ml.

[0050] (5) Preparation of a mixed solution of impurities I, III, and V: Take appropriate amounts of impurities I, III, and V (about 1 mg each) and place them in a 100 ml volumetric flask. Add solvent to dissolve and dilute to the mark. Shake well to obtain a mixed solution of impurities I, III, and V.

[0051] (6) Chromatographic conditions: According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0512), octadecylsilane bonded silica gel was used as the filler (Sepax Sapphire C18 4.6 mm × 150 mm, 5 μm or a chromatographic column of equivalent performance), phosphate buffer (1.36 g of potassium dihydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH was adjusted to 3.0 with phosphoric acid)-acetonitrile (97:3) was used as the mobile phase A, and acetonitrile was used as the mobile phase B. Gradient elution was performed according to Table 2; the flow rate was 1.0 ml per minute; the column temperature was 35 °C; the detection wavelength was 265 nm; the injection volume was 20 μl. Accurately measure 20 μl of the mixed solution of impurities I, III, and V and inject it into the chromatogram. Impurities I, III, and V peaked in sequence, and the separation between impurities I and III, and impurities III and V should be no less than 1.5. Accurately measure 20 μl of the impurity S reference solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram. The chromatographic peaks in the test solution that coincide with the retention times of impurities I, III, and V should not exceed 0.2% of the labeled amount of phloroglucinol. The calculation formulas for impurities I, III, and V are as follows:

[0052] Impurity I / Impurity V (%) = (A u / A s )×(C S / C U )×P s ×F s ×100

[0053] Where: A u is the peak area of ​​impurity I / impurity V in the test solution;

[0054] A s is the peak area of ​​impurity S in the reference solution;

[0055] C s is the concentration of impurity S reference solution (mg / ml);

[0056] C u is the concentration of the test solution (mg / ml);

[0057] P s is the purity of impurity S (%);

[0058] F s It is the correction factor of each impurity I / impurity V relative to impurity S.

[0059] Impurity III (%) = (A u / A C )×(C C / C U )×P C ×F C ×100

[0060] Where: A u is the peak area of ​​impurity III in the test solution;

[0061] A C is the peak area of ​​pyrogallol in the reference solution;

[0062] C C is the concentration of phloroglucinol reference solution (mg / ml);

[0063] C u is the concentration of the test solution (mg / ml);

[0064] P C is the purity of phloroglucinol (%);

[0065] F C is the correction factor of impurity III relative to phloroglucinol.

[0066] Table 1 Correction factors for various impurities

[0067] Impurity name Impurity I Impurity III Impurity V Correction factor relative to impurity S 0.45 — 1.28 Compared with phloroglucinol (main ingredient) — 0.80 —

[0068] Table 2 Gradient elution table

[0069]

[0070] Example 2: Investigation of method specificity

[0071] Because phloroglucinol injection contains excipients other than phloroglucinol and a certain amount of trimethyl phloroglucinol, we need to investigate whether the blank excipients of phloroglucinol injection, trimethyl phloroglucinol and other impurities in phloroglucinol will interfere with the detection of oxidized impurities in phloroglucinol.

[0072] (1) Impurity S positioning solution: Take 12.5 mg of the impurity S reference substance and place it in a 100 ml volumetric flask. Add solvent to dissolve and dilute to the mark. Shake well to obtain the impurity S stock solution. Accurately measure 2 ml of the impurity S stock solution and place it in a 50 ml volumetric flask. Add solvent to dilute to the mark. Shake well to obtain the impurity S stock solution.

[0073] (2) Impurity I positioning solution: Take 12.5 mg of Impurity I reference substance, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well to obtain Impurity I stock solution, accurately measure 1 ml of Impurity I stock solution, place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well to obtain Impurity I stock solution.

[0074] (3) Impurity III positioning solution: Take 12.5 mg of the impurity III reference substance and place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well to obtain the impurity III stock solution, accurately measure 1 ml of the impurity III stock solution and place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0075] (4) Impurity V positioning solution: Take 12.5 mg of the impurity V reference substance, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well to obtain the impurity V stock solution, accurately measure 1 ml of the impurity V stock solution, place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0076] (5) Phloroglucinol localization solution: Take 12.5 mg of phloroglucinol reference substance and place it in a 50 ml volumetric flask. Add solvent to dissolve and dilute to the scale. Shake well and accurately measure 1 ml. Place it in a 50 ml volumetric flask. Add solvent to dilute to the scale and shake well.

[0077] (6) Trimethylphloroglucinol positioning solution: Take 12.5 mg of trimethylphloroglucinol reference substance and place it in a 50 ml volumetric flask. Add solvent to dissolve and dilute to the scale. Shake well and accurately measure 1 ml. Place it in a 50 ml volumetric flask. Add solvent to dilute to the scale and shake well.

[0078] (7) Blank excipient solution: Accurately measure 2.5 ml of blank injection solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0079] (8) Specific mixed impurity stock solution: Accurately measure 2.5 ml of impurity I stock solution, 2.5 ml of impurity III stock solution, and 2.5 ml of impurity III stock solution, place them in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. (The concentration of impurities I, III, and V is 25 μg / ml)

[0080] (9) Mixed test solution: Accurately measure 2.5 ml of phloroglucinol injection, 2 ml of specific mixed impurity stock solution, and 0.5 ml of impurity S stock solution, place them in the same 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0081] Take the prepared blank excipient solution, impurity S, impurity I, impurity III, impurity V, phloroglucinol and trimethylphloroglucinol positioning solution, mix them with the test solution, and inject them for determination.

[0082] Table 3 Results of specificity investigation on oxidative degradation impurities in phloroglucinol injection

[0083]

[0084] According to the results of specific sampling, it can be seen that the blank excipient has no effect on the detection of impurities I, impurity III and impurity V, the peak areas of impurities I, impurity III and impurity V in the impurity positioning solution and the mixed test solution are equivalent, and the other components in the phloroglucinol injection have no effect on the detection of impurities I, impurity III and impurity V. The specificity of this method is good.

[0085] Example 3: Investigation of method sensitivity

[0086] The positioning solutions of impurity I, impurity III, and impurity V in Example 2 were diluted step by step, and the samples were injected for measurement. The concentration when the signal-to-noise ratio was about 10 was the quantitative limit concentration, and the concentration when the signal-to-noise ratio was about 3 was the detection limit concentration.

[0087] Table 4 Results of investigation on detection limits of impurities I, III and V

[0088] name Detection limit concentration μg / ml Signal-to-noise ratio Relative test sample concentration% Impurity I 0.001269 3.91、5.40 0.000050 Impurity III 0.06515 6.90、7.30 0.0026 Impurity V 0.006173 6.52、7.69 0.00025

[0089] Table 5 Results of investigation on impurity I, impurity III and impurity quantification limit

[0090] name Limit of quantification concentration μg / ml Signal-to-noise ratio Relative test sample concentration% Impurity I 0.003627 12.60—16.03 0.00015 Impurity III 0.1861 19.71—27.54 0.0074 Impurity V 0.01764 20.43—27.90 0.00071

[0091] According to the results of the limit of quantification and detection limit determination, the detection limits of impurities I, III, and V were all lower than 0.003% of the phloroglucinol sample, and the limits of quantification were all lower than 0.008% of the phloroglucinol sample. This method is highly sensitive for the detection of oxidative degradation impurities in phloroglucinol injection and meets the detection requirements.

[0092] Example 4: Investigation of method linearity and correction factor

[0093] Preparation of a linear series of solutions of impurity S: Take the impurity S stock solution prepared in Example 2, add solvent and dilute it step by step to prepare solutions containing 0.5, 1, 2.5, 5, 10, 25, 50, 75 and 100 μg of impurity S per ml.

[0094] Preparation of a linear series of solutions of impurity I: Take the stock solution of impurity I prepared in Example 2 and dilute it stepwise with solvent to prepare solutions containing 0.5, 1, 2.5, 5, 7.5, 10, 12.5 and 25 μg of impurity I per ml.

[0095] Preparation of a linear series of solutions of impurity III: Take the impurity III stock solution prepared in Example 2 and dilute it stepwise with solvent to prepare solutions containing 0.5, 1, 2.5, 5, 7.5, 10, 12.5 and 25 μg of impurity III per ml.

[0096] Preparation of a linear series of solutions of impurity V: Take the stock solution of impurity V prepared in Example 2, add solvent and dilute it step by step to prepare solutions containing 0.5, 1, 2.5, 5, 7.5, 10, 12.5 and 25 μg of impurity V per ml.

[0097] Preparation of a linear series of phloroglucinol solutions: The phloroglucinol stock solution prepared in Example 2 was diluted with solvent stepwise to prepare solutions containing 0.5, 1, 2.5, 5, 7.5, 10, 12.5 and 25 μg of phloroglucinol per ml.

[0098] Take a linear series of solutions of impurity S, impurity I, impurity III, impurity V and phloroglucinol, inject them in order from low to high concentration, and record the peak area.

[0099] Table 6 Linearity test results of impurity S, impurity I, impurity III, impurity V and phloroglucinol (with concentration as the vertical axis)

[0100] name Linear equations Correlation coefficient Phloroglucinol y=0.05251011x-0.02540807 1.0000 Impurity S y=0.00423172x-0.57205693 0.9998 Impurity I y=0.00191662x-0.01031981 1.0000 Impurity III y=0.04197602x+0.01029007 0.9998 Impurity V y=0.00540012x-0.02245015 0.9999

[0101] The correction factors of impurities I, III and V relative to phloroglucinol are calculated by dividing the slopes of the linear equations of impurities I, III and V by the slope of the linear equation of phloroglucinol; the correction factors of impurities I, III and V relative to impurity S are calculated by dividing the slopes of the linear equations of impurities I, III and V by the slope of the linear equation of impurity S.

[0102] Table 7 Results of investigation on correction factors of impurities I, III and V

[0103] name Correction factor relative to phloroglucinol Correction factor relative to impurity S Impurity I 0.04 0.45 Impurity III 0.80 9.92 Impurity V 0.10 1.28

[0104] Due to the structural differences between oxidative degradation impurities I, III, and V and phloroglucinol, at a wavelength of 265 nm, the responses of impurities I and V differ significantly from those of phloroglucinol, but are relatively close to those of impurity S. The response of impurity III is similar to that of phloroglucinol. Considering the high cost, difficulty in obtaining, and poor stability of impurities I, III, and V, and the high availability and relatively good stability of phloroglucinol and impurity S, the amounts of impurities I and V were calculated using a correction factor relative to impurity S, while the amount of impurity III was calculated using a correction factor relative to that of phloroglucinol.

[0105] Example 5: Investigation of method accuracy

[0106] According to Examples 2 to 4, it can be seen that the method has strong specificity and high detection sensitivity, and reliable correction factors for impurities I, III and V are also obtained. Therefore, its accuracy is examined below.

[0107] (1) Mixed impurity accuracy stock solution ①: Take 5 ml of the impurity I stock solution, 5 ml of the impurity III stock solution, and 5 ml of the impurity V stock solution in Example 2, place them in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution. (Contains approximately 25 μg / ml each of impurity I, impurity III, and impurity V)

[0108] (2) Mixed impurity accuracy stock solution ②: Accurately measure 5 ml of mixed impurity accuracy stock solution ①, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. (Contains approximately 5 μg / ml each of impurity I, impurity III, and impurity V)

[0109] (3) Unspiked test sample: Accurately measure 2.5 ml of phloroglucinol injection into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well. Prepare 3 portions in the same manner.

[0110] (4) Spiked test sample (concentrations of impurities I, III, and V are 0.02%): Accurately measure 2.5 ml of phloroglucinol injection into a 10 ml volumetric flask. Accurately add 1 ml of mixed impurity preparation stock solution ②, dilute to the mark with solvent, and shake well. Prepare three parallel batches.

[0111] (5) Spiked test sample (concentrations of impurities I, III, and V are 0.2%): Accurately measure 2.5 ml of phloroglucinol injection into a 10 ml volumetric flask. Accurately add 2 ml of mixed impurity preparation stock solution ①, dilute to the mark with solvent, and shake well. Prepare three parallel batches.

[0112] (6) Spiked test sample (concentrations of impurities I, III, and V are 0.3%): Accurately measure 2.5 ml of phloroglucinol injection into a 10 ml volumetric flask, accurately add 3 ml of mixed impurity preparation stock solution ①, dilute to the mark with solvent, and shake well. Prepare three parallel batches.

[0113] (7) Impurity S reference solution: Take the impurity S stock solution in Example 2, accurately measure 2 ml, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. (Contains about 10 μg / ml of impurity S)

[0114] (8) Phloroglucinol reference solution: Take the phloroglucinol stock solution in Example 2, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well. (Contains approximately 5 μg / ml of phloroglucinol)

[0115] Take the impurity S reference solution, phloroglucinol reference solution, unspiked test sample, and spiked test samples of various concentrations for sampling and determination.

[0116] Table 8 Impurity I Accuracy Investigation Results

[0117]

[0118] Table 9 Impurity III accuracy investigation results

[0119]

[0120] Table 10 Impurity V Accuracy Investigation Results

[0121]

[0122] The recoveries of impurity I, impurity III, and impurity V were all in the range of 80%-120%, demonstrating the good accuracy of the method.

[0123] Example 6: Investigation of method repeatability

[0124] (1) Impurity S reference solution: Take the impurity S stock solution in Example 2, accurately measure 2 ml, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. (Contains about 10 μg / ml of impurity S)

[0125] (2) Phloroglucinol reference solution: Take the phloroglucinol stock solution in Example 2, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution (containing approximately 5 μg / ml of phloroglucinol).

[0126] (3) Mixed impurity stock solution: Take 5 ml of the impurity I stock solution, 5 ml of the impurity III stock solution, and 5 ml of the impurity V stock solution from Example 2, place them in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the mixed impurity solution (containing approximately 25 μg / ml each of impurity I, impurity III, and impurity V).

[0127] (4) Mixed test solution: Accurately measure 2.5 ml of phloroglucinol injection into a 10 ml volumetric flask, accurately add 2 ml of mixed impurity stock solution, dilute to the mark with solvent, and shake well. Prepare 6 parallel portions.

[0128] Take the impurity S reference solution, phloroglucinol reference solution and mixed test solution, and perform sampling and determination.

[0129] Table 11 Repeatability test results

[0130]

[0131] Six samples were repeatedly measured, and the results of impurity I, impurity III, and impurity V were consistent, with RSDs of 0, demonstrating that the method had good repeatability.

[0132] Example 7: Investigation of method robustness

[0133] (1) Impurity S reference solution: Take the impurity S stock solution in Example 2, accurately measure 2 ml, place it in a 25 ml volumetric flask, dilute to the mark with solvent, and shake well. (Contains about 10 μg / ml of impurity S)

[0134] (2) Phloroglucinol reference solution: Take the phloroglucinol stock solution in Example 2, accurately measure 1 ml, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution (containing approximately 5 μg / ml of phloroglucinol).

[0135] (3) Mixed impurity stock solution: Take 5 ml of the impurity I stock solution, 5 ml of the impurity III stock solution, and 5 ml of the impurity V stock solution from Example 2, place them in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the mixed impurity solution (containing approximately 25 μg / ml each of impurity I, impurity III, and impurity V).

[0136] (4) Mixed test solution: Accurately measure 2.5 ml of phloroglucinol injection and place it in a 10 ml volumetric flask. Accurately add 2 ml of mixed impurity stock solution, dilute to the mark with solvent, and shake well.

[0137] (4) Mixed solution of impurities I, III and V: Accurately measure 2 ml of the mixed impurity stock solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0138] The single-factor investigation method was used, and the column temperature was set at 33°C and 37°C, the flow rate was 0.9 ml / min and 1.1 ml / min, the detection wavelength was 264 nm and 266 nm, the mobile phase pH was 2.9 and 3.1, the ratio of mobile phase A was phosphate buffer (pH 3.0)-acetonitrile (475:15), phosphate buffer (pH 3.0)-acetonitrile (495:15), and the buffer salt concentration of mobile phase A was 1.224 g / L and 1.496 g / L. Under each condition, the impurity S reference solution, phloroglucinol reference solution, impurity I, III and V mixed solution, and mixed test solution were taken for sampling and determination.

[0139] Table 12 Summary of separation of impurities I, III and V under various conditions

[0140]

[0141] Table 13 Detection results of impurities I, III and V under various conditions

[0142]

[0143] The chromatographic conditions were fine-tuned, and the separation degree of each impurity in the mixed solution of impurities I, III and V was greater than 1.5; the RSDs of the determination results of impurities I, III and V in the mixed test solution did not exceed 20%, demonstrating the good durability of this method.

[0144] Comparative Example

[0145] Since the European Pharmacopoeia contains a detection method for related substances of phloroglucinol raw materials, in order to test whether this condition can be used for the separation and quantitative detection of oxidized impurities I, III and V in phloroglucinol injection, we used this method to investigate the oxidized impurities I, III and V of phloroglucinol.

[0146] (1) Prepare the solvent: Dissolve 1.36 g of potassium dihydrogen phosphate in water and dilute to 1000 ml. Adjust the pH to 3.0 with phosphoric acid)-acetonitrile (90:10).

[0147] (2) Preparation of impurity I, III and V mixed solution: Take about 5 mg of impurities I, III and V respectively, place them in the same 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately take 1 ml, place it in a 50 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain

[0148] Chromatographic conditions: According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0512), octadecylsilane bonded silica gel was used as the filler [EC 250 / 4Nucleodur C18 Pyramid (250×4.0mm, 5μm) or a chromatographic column with equivalent performance], phosphate buffer (1.36g of potassium dihydrogen phosphate was dissolved in water and diluted to 1000ml, and the pH was adjusted to 3.0 with phosphoric acid) as the mobile phase A, acetonitrile as the mobile phase B, and gradient elution was performed according to Table 14; the flow rate was 1.0ml per minute; the column temperature was 35°C; the detection wavelength was 265nm; the injection volume was 20μl, and the mixed solution of impurities I, III and V was injected for determination.

[0149] Table 14 Comparative Example Gradient Elution Table

[0150] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 9 100 0 15 50 80 25 20 50 30 20 50 31 100 0 40 100 0

[0151] The chromatogram obtained from the mixed solution of impurities I, III and V detected by the European Pharmacopoeia method is shown in Figure 9 Under this condition, due to the drastic change in the gradient, a relatively obvious blank gradient peak appeared at 12-18 minutes, with a peak height of about 18 mAu, which seriously affected the detection of impurities. In addition, the known impurities concentrated their peaks at 16-17 minutes, and the separation degree of impurities III and V could not reach 1.5, resulting in difficulty in separating the impurities and inability to perform accurate quantitative detection.

[0152] Oxidation impurities I

[0153] Chemical structure:

[0154]

[0155] Chemical name: 4,7,9-trihydroxydibenzo[b,d]furan-1,3(2H,4H)-dione

[0156] Molecular formula: C 12 H8O6

[0157] Molecular weight: 248.19

[0158] Oxidation impurities III

[0159] Chemical structure:

[0160]

[0161] Chemical name: 5,7,9,13,15,17-hexahydroxy-2,20-dioxolane[11.7.0.0^{1,9}.0^{3,8}.0^{14,19}]icosa-3,5,7,14,16,18-hexaen-11-one

[0162] Molecular formula: C 18 H 14 O9

[0163] Molecular weight: 374.30

[0164] Oxidation impurities V

[0165] Chemical structure:

[0166]

[0167] Chemical name: 4,6-dihydroxy-2-(2-oxopropyl)benzofuran-3(2H)-one

[0168] Molecular formula: C 11 H 10 O5

[0169] Molecular weight: 222.20

[0170] Impurity S

[0171] Chemical structure:

[0172]

[0173] Chemical name: 2,4,6-trihydroxybenzoic acid

[0174] Molecular formula: C7H6O5

[0175] Molecular weight: 170.12

[0176] Although the present invention has been described using the above preferred embodiments, they are not intended to limit the scope of protection of the present invention. Any person skilled in the art may carry out various activities and modifications relative to the above embodiments without departing from the spirit and scope of the present invention. The scope of protection of the present invention shall be based on the definition of the claims.

Claims

1. A method for detecting oxidative impurities in phloroglucinol injection, characterized in that include: Step S1: taking a mixed solution containing oxidized impurities I, III, and V, and testing the separation conditions using the mixed solution, wherein the separation degree of each impurity peak should be no less than 1.5; Step S2: Take an appropriate amount of phloroglucinol injection to prepare a test solution; Step S3: taking appropriate amounts of phloroglucinol and phloroglucinol impurity S reference substance to prepare phloroglucinol reference substance solution and phloroglucinol impurity S reference substance solution respectively; Step S4: Obtain chromatograms of the test solution and the reference solution under qualified separation conditions, and determine the content of oxidized impurities in the phloroglucinol injection using the following formula; Impurity I / Impurity V (%) = (A u / A s )×(C S / C U )×P s ×F s ×100 Where: A u is the peak area of ​​impurity I / impurity V in the test solution; A s is the peak area of ​​impurity S in the reference solution; C s is the concentration of impurity S reference solution (mg / ml); C u is the concentration of the test solution (mg / ml); P s is the purity of impurity S (%); F s It is the correction factor of each impurity I / impurity V relative to impurity S. Impurity III (%) = (A u / A C )×(C C / C U )×P C ×F C ×100 Where: A u is the peak area of ​​impurity III in the test solution; A C is the peak area of ​​pyrogallol in the reference solution; C C is the concentration of phloroglucinol reference solution (mg / ml); C u is the concentration of the test solution (mg / ml); P C is the purity of phloroglucinol (%); F C is the correction factor of impurity III relative to phloroglucinol.

2. The method for detecting oxidative impurities in phloroglucinol injection according to claim 1, wherein: The separation conditions are as follows: Filler: Octadecylsilane bonded silica gel Column temperature: 33℃~37℃ Detection wavelength: 264nm~266nm Flow rate: 0.9ml / min~1.1ml / min The first mobile phase is selected from a mixed solution of phosphate buffer and acetonitrile, and the second mobile phase is selected from acetonitrile.

3. The method for detecting oxidative impurities in phloroglucinol injection according to claim 2, wherein: The first mobile phase was prepared as follows: 1.36 g of potassium dihydrogen phosphate was weighed, dissolved in purified water and diluted to 1000 ml, the pH was adjusted to 3.0 with phosphoric acid, and then 970 ml was added with 30 ml of acetonitrile and mixed evenly.

4. The method for detecting oxidative impurities in phloroglucinol injection according to claim 2, wherein: The gradient elution program is as follows:

5. The method for detecting oxidative impurities in phloroglucinol injection according to claim 2, wherein: The system has good applicability, with the separation between each oxidized impurity greater than 1.5; good specificity (blank solvent, blank injection solution, impurities, etc. do not interfere with the detection of oxidized impurities); high detection sensitivity (the detection limit does not exceed 0.0026%; The limit of quantification did not exceed 0.0074%); The linear relationship is good (the correlation coefficient r is not less than 0.9998); the accuracy is high (the recovery rate of each impurity is within the range of 80% to 120%); the repeatability is good (RSD is 0); and the durability is good.

6. The method for detecting oxidative impurities in phloroglucinol injection as claimed in claim 2, characterized in that: The correction factor of impurity I compared to phloroglucinol impurity S is 0.45, the correction factor of impurity III compared to phloroglucinol is 0.80, and the correction factor of impurity V compared to phloroglucinol impurity S is 1.

28.

7. The method for detecting oxidative impurities in phloroglucinol injection as claimed in claim 2, characterized in that: The chemical structural formula of phloroglucinol impurity S is as follows:

8. The method for detecting oxidative impurities in phloroglucinol injection as claimed in claim 2, characterized in that: The chemical structure of the oxidized impurity I is as follows:

9. The method for detecting oxidative impurities in phloroglucinol injection as claimed in claim 2, characterized in that: The chemical structure of the oxidized impurity III is as follows:

10. The method for detecting oxidative impurities in phloroglucinol injection as claimed in claim 2, characterized in that: The chemical structure of the oxide impurity V is as follows:

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