A liquid chromatography detection method for related substances in pentoxifylline injection

Abstract

The invention discloses a liquid chromatography detection method for related substances in a pentoxifylline injection, comprising the following steps: liquid chromatography detection; liquid chromatography detection includes: injecting a reference solution and a test solution into a liquid chromatograph, and using a mobile phase gradient elution method for separation and detection; the chromatographic column used is a C8 column; the mobile phase gradient elution process is: at 0-10min, mobile phase A is 64%, and mobile phase B is 36%; at 10-15min, mobile phase A is uniformly reduced to 34%, and mobile phase B is uniformly increased to 66%; at 15-30min, mobile phase A is 34%, and mobile phase B is 66%; at 30-40min, mobile phase A is uniformly increased to 64%, and mobile phase B is uniformly reduced to 36%; at 40-60min, mobile phase A is 64%, and mobile phase B is 36%. The liquid chromatography detection method provided by the present invention has good accuracy, high sensitivity and good reliability.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a liquid chromatography detection method for related substances in pentoxifylline injection. Background Art

[0002] Pentoxifylline is a vasodilator that can improve blood rheology by reducing blood viscosity. It can be used for cerebral and peripheral blood circulation disorders; it can be used to treat peripheral vascular diseases, such as vascular headache, retinopathy, intermittent claudication and vascular occlusive vasculitis. For patients with chronic peripheral arterial disease, it can increase blood flow, improve microcirculation, and increase tissue oxygen supply, thereby alleviating the symptoms of the corresponding disease.

[0003] Related substances refer to impurities in drugs other than the main components. These impurities may come from raw material synthesis, production process, storage and transportation, etc. The content and nature of impurities directly affect the purity and safety of drugs. Therefore, the research and control of related substances of pentoxifylline is an important link in ensuring the quality of drugs.

[0004] High performance liquid chromatography has a high separation ability and can effectively separate the components in complex samples. It has high sensitivity: combined with appropriate detectors, liquid chromatography can detect trace components. Summary of the invention

[0005] In order to improve the accuracy and sensitivity of a liquid chromatography detection method for related substances in pentoxifylline injection, the present invention provides a liquid chromatography detection method for related substances in pentoxifylline injection.

[0006] According to an embodiment of the present invention, the present invention provides a liquid chromatography detection method for related substances in pentoxifylline injection, which adopts the following technical scheme:

[0007] A liquid chromatography detection method for related substances in pentoxifylline injection comprises the following steps: preparing a reference solution, preparing a test solution, and liquid chromatography detection; wherein the liquid chromatography detection comprises: injecting the reference solution and the test solution into a liquid chromatograph, and performing separation and detection by a mobile phase gradient elution method; the chromatographic column used is a C8 column; the mobile phase gradient elution process is: at 0-10min, the mobile phase A is 64%, the mobile phase B is 36%; at 10-15min, the mobile phase A is uniformly distributed in the liquid chromatograph; the mobile phase A is uniformly distributed in the liquid chromatograph; the mobile phase B is uniformly distributed in the liquid chromatograph; ... B is uniformly distributed in the liquid chromatograph; the mobile phase A is uniformly distributed in the liquid chromatograph; the mobile phase B is uniformly distributed in the liquid chromatograph; the mobile phase A is uniformly distributed in the liquid chromatograph; the mobile phase B is uniformly distributed in the liquid chromatograph; the mobile phase A is uniformly distributed in The mobile phase A decreases to 34% at a constant speed, and the mobile phase B increases to 66% at a constant speed; from 15 to 30 minutes, the mobile phase A is 34% and the mobile phase B is 66%; from 30 to 40 minutes, the mobile phase A increases to 64% at a constant speed, and the mobile phase B decreases to 36% at a constant speed; from 40 to 60 minutes, the mobile phase A is 64% and the mobile phase B is 36%; both the mobile phase A and the mobile phase B are measured in volume percentage; the mobile phase A is a potassium dihydrogen phosphate aqueous solution with a mass percentage concentration of 0.5% to 0.6%, and the mobile phase B is methanol.

[0008] The detection method according to the specific embodiment of the present invention has good detection accuracy, high sensitivity and good reliability.

[0009] According to some embodiments of the present invention, the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.544%.

[0010] According to some embodiments of the present invention, in the liquid chromatography detection, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0011] According to some embodiments of the present invention, in the liquid chromatography detection, the column temperature is 30-40°C.

[0012] According to some embodiments of the present invention, in the liquid chromatography detection, the detection wavelength is 272 nm.

[0013] According to some embodiments of the present invention, in the liquid chromatography detection, the chromatographic column is YMC-Triart C8, 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance, using octylsilane bonded silica gel as a filler.

[0014] According to some embodiments of the present invention, the related substances include: pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N,

[0015] Wherein, the structure of the impurity A is:

[0016] The structure of the impurity C is:

[0017] The structure of the impurity D is:

[0018] The structure of the impurity F is:

[0019] The structure of the impurity G is:

[0020] The structure of the impurity H is:

[0021] The structure of the impurity I is:

[0022] The structure of the impurity J is:

[0023] The structure of the impurity K is:

[0024] The structure of the impurity L is:

[0025] The structure of the impurity M is:

[0026] And the structure of the impurity N is:

[0027] According to some embodiments of the present invention, the limits of quantification of the pentoxifylline, the impurity A, the impurity C, the impurity D, the impurity F, the impurity G, the impurity H, the impurity I, the impurity J, the impurity K, the impurity L, the impurity M and the impurity N in the related substances are 0.0434 μg / mL, 0.0282 μg / mL, 0.0279 μg / mL, 0.0410 μg / mL, 0.0352 μg / mL, 0.1342 μg / mL, 0.0616 μg / mL, 0.0429 μg / mL, 0.0561 μg / mL, 0.0385 μg / mL, 0.1334 μg / mL, 0.3354 μg / mL, 0. The detection limits of the impurity A, the impurity C, the impurity D, the impurity F, the impurity G, the impurity H, the impurity I, the impurity J, the impurity K, the impurity L, the impurity M and the impurity N were 0.0174 μg / mL, 0.0113 μg / mL, 0.0112 μg / mL, 0.0164 μg / mL, 0.0141 μg / mL, 0.0537 μg / mL, 0.0246 μg / mL, 0.0171 μg / mL, 0.0225 μg / mL, 0.0154 μg / mL, 0.0533 μg / mL, 0.1342 μg / mL and 0.0283 μg / mL, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a chromatogram according to Example 1 of the present invention. DETAILED DESCRIPTION

[0029] The following is a liquid chromatography detection method for related substances in the pentoxifylline injection of the present invention described by specific examples.

[0030] The liquid chromatography detection method of related substances in pentoxifylline injection according to a specific embodiment of the present invention comprises the following steps:

[0031] Preparation of reference solution: Take 10 mg of pentoxifylline reference substance, weigh it accurately, and place it in a 10 ml volumetric bottle, then add 0.2 ml of each stock solution of impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, and impurity N, then add diluent to dissolve and dilute to scale, shake well, the preparation method of the above stock solution is as follows: Take about 2 mg of impurity A reference substance, weigh it accurately, place it in a 20 ml volumetric bottle, add diluent to dissolve and dilute to scale, shake well, and the stock solution of impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, and impurity N is the same as impurity A.

[0032] Preparation of test solution: Accurately measure 1 ml of pentoxifylline injection, place in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0033] Liquid chromatography detection includes: injecting the reference solution and the test solution into a liquid chromatograph, and using a mobile phase gradient elution method for separation and detection; the chromatographic column used is a C8 column, specifically, it can be YMC-Triart C8 with octylsilane bonded silica gel as a filler, 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance.

[0034] Specifically, the mobile phase gradient elution process is: from 0 to 10 minutes, the mobile phase A is 64% and the mobile phase B is 36%; during the first gradient elution, the mobile phase has a larger polarity, and the impurities M, impurity A, impurity C, impurity D and impurity F with larger polarity are eluted in turn, and there is a good separation between adjacent substances.

[0035] 10-15min, mobile phase A is uniformly reduced to 34%, and mobile phase B is uniformly increased to 66%; during the second gradient elution, the polarity of the mobile phase is uniformly reduced, and the flow rate and polarity change rate of the eluent are controlled to elute impurities L, impurity G, impurity K and pentoxifylline, and the separation degree between them and adjacent substances is good.

[0036] 15-30min, mobile phase A is 34%, mobile phase B is 66%; during the third gradient elution, the polarity of the mobile phase remains unchanged, impurity H, impurity I, impurity J, and impurity N are eluted and separated, and the separation degree of adjacent substances is good.

[0037] 30-40min, mobile phase A increases uniformly to 64%, and mobile phase B decreases uniformly to 36%; in the fourth gradient elution, the polarity of the mobile phase increases uniformly and rapidly, so that mobile phase A can be used to elute the chromatographic column.

[0038] At 40-60 minutes, mobile phase A is 64%, and mobile phase B is 36%. Both mobile phase A and mobile phase B are expressed in volume percentages. In the fifth gradient elution, mobile phase A is used as the elution for the chromatographic column to avoid residual substances on the chromatographic column.

[0039] Furthermore, the mobile phase A is a potassium dihydrogen phosphate aqueous solution with a mass percentage concentration of 0.5%-0.6%, and the mobile phase B is methanol.

[0040] Optionally, the flow rate is 0.8-1.2 ml per minute; the column temperature is 30-40° C.; the detection wavelength is 272 nm; and the injection volume is 20 μl.

[0041] According to the liquid chromatography detection method of related substances in pentoxifylline injection according to the specific embodiment of the present invention, the blank solution and the blank auxiliary material do not interfere with the inspection of the related substances of this product; in the chromatogram of the reference solution, the peak order is impurity A peak, impurity C peak, impurity F peak and pentoxifylline peak, and the peak separation of each component meets the requirements (the minimum value is 3.92); in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the peak height of the pentoxifylline chromatographic peak is 68.7, which is greater than 10; therefore, the method specificity of the related substances of this product is good. Under the conditions of column temperature (29°C to 31°C) and using different batches of chromatographic columns of the same brand, the blank solution and the blank auxiliary material do not interfere with the inspection of the related substances of this product; in the chromatogram of the reference solution, the peak order is impurity A peak, impurity C peak, impurity F peak and pentoxifylline peak, and the peak separation of each component meets the requirements (the minimum value is 3.81); in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the peak height of the main component chromatographic peak is greater than 10 (the minimum value is 50.5). The test results of the spiked solution of the test sample under each condition were calculated by the external standard method and the self-control method with correction factor, and the RSD of each impurity detection amount was less than 15% (the maximum value was 10.43%), and the test results were basically consistent. Therefore, the analysis method of related substances in the test sample of pentoxifylline injection has good durability.

[0042] The diluent is a methanol-0.544% potassium dihydrogen phosphate solution in a volume ratio of 1:1.

[0043] The blank solution was the diluent.

[0044] The preparation method of blank auxiliary material solution comprises the following steps: accurately measuring 1 ml of blank auxiliary material, placing it in a 20 ml volumetric bottle, diluting it to the scale with a diluent, and shaking it well.

[0045] The preparation method of pentoxifylline stock solution comprises the following steps: taking about 2 mg of pentoxifylline reference substance, accurately weighing it, placing it in a 20 ml volumetric bottle, adding a diluent to dissolve it and dilute it to the scale, and shaking it well.

[0046] Prepare impurity A, C, D, F, G, H, I, J, K, L, M, and N stock solutions in the same way.

[0047] The preparation method of the mixed reference solution comprises the following steps: taking about 10 mg of the pentoxifylline reference substance, accurately weighing it, placing it in a 10 ml volumetric flask, adding 0.2 ml of each of the stock solutions of impurities A, C, D, F, G, H, I, J, K, L, M, and N, adding a diluent to dissolve and dilute to the scale, and shaking well.

[0048] Test solution: Accurately measure 1 ml of pentoxifylline stock solution, place in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0049] According to some embodiments of the present invention, the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.544%. Such an arrangement can make the related substances separated in an orderly manner and can make the related substances have a better separation degree.

[0050] According to some embodiments of the present invention, in liquid chromatography detection, the flow rate of the mobile phase is 0.8 mL / min-1.2 mL / min. In this way, the flow rate of the mobile phase is suitable for the sequential desorption between the relevant substances and the chromatographic column, thereby achieving a separation effect.

[0051] According to some embodiments of the present invention, in the liquid chromatography detection, the column temperature is 30° C.-40° C. In this way, the column temperature is set at room temperature, which is convenient for separation operation.

[0052] According to some embodiments of the present invention, in the liquid chromatography detection, the detection wavelength is 272 nm. In this way, under the condition of 272 nm, the reaction sensitivity of the relevant substances is high, which is convenient for observation and detection.

[0053] According to some embodiments of the present invention, in liquid chromatography detection, the chromatographic column is a chromatographic column with octylsilane bonded silica as a filler YMC-Triart C8, 4.6mm×250mm, 5μm or equivalent performance. The chromatographic column with octylsilane bonded silica as a filler YMC-Triart C8, 4.6mm×250mm, 5μm or equivalent performance is used to separate related substances, and the adsorption and desorption effects of related substances are significantly distinguished. Combined with the gradient change of the eluent, the analysis and separation of related substances are achieved.

[0054] According to some embodiments of the present invention, the related substances include: pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N,

[0055] Among them, the structure of impurity A is:

[0056] The structure of impurity C is:

[0057] The structure of impurity D is:

[0058] The structure of impurity F is:

[0059] The structure of impurity G is:

[0060] The structure of impurity H is:

[0061] The structure of impurity I is:

[0062] The structure of impurity J is:

[0063] The structure of impurity K is:

[0064] The structure of impurity L is:

[0065] The structure of impurity M is:

[0066] And the structure of impurity N is: Among the related substances, pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N have similar structures and polarities and are not easy to separate. The specific technical method of the present invention can achieve better separation effect.

[0067] According to some embodiments of the present invention, the quantitative limits of pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N in the related substances are 0.0434 μg / mL, 0.0282 μg / mL, 0.0279 μg / mL, 0.0410 μg / mL, 0.0352 μg / mL, 0.1342 μg / mL, 0.06 16μg / mL, 0.0429μg / mL, 0.0561μg / mL, 0.0385μg / mL, 0.1334μg / mL, 0.3354μg / mL, 0.0707μg / mL are set in this way, and the quantitative limits of pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N in the related substances are low, and the detection sensitivity is high.

[0068] According to other embodiments of the present invention, the detection limits of pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N are 0.0174 μg / mL, 0.0113 μg / mL, 0.0112 μg / mL, 0.0164 μg / mL, 0.0141 μg / mL, 0.0537 μg / mL, 0.0246 μg / mL, 0.0171 μg / mL, 0.0225 μg / mL, 0.0154 μg / mL, 0.0533 μg / mL, 0.1342 μg / mL and 0.0283 μg / mL, respectively. With such settings, the inspection limits of pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N in the related substances are low, and the detection sensitivity of the related substances is high at lower concentrations.

[0069] All reagents used in the present invention are commercially available products unless otherwise noted or described. Information on some of the products and instruments involved in the present invention is as follows: 1 Related substances

[0070] Table 1. Methodological validation of related substances - study impurities

[0071]

[0072]

[0073] Table 2. Methodological validation of related substances - sample information

[0074] name batch number Specification Manufacturer Pentoxifylline Injection 220701701 5ml: 100mg Huaxia Shengsheng Pharmaceutical (Beijing) Co., Ltd. Pentoxifylline Injection FCA14A 100mg / 5ml Sanofi Blank Accessories 22070701 / Preparation Room Pentoxifylline API YL-75220401 / CSPC Sinopharm Group Co., Ltd.

[0075] Table 3. Methodological validation of related substances - reference material information

[0076]

[0077]

[0078] Table 4. Methodological validation of related substances - information on main instruments and equipment

[0079]

[0080] 1.1 Exclusivity

[0081] The specificity of the method was examined, requiring that the blank solution and blank excipients must not interfere with the inspection of related substances of this product; in the chromatogram of the reference solution, the peak order is impurity A peak, impurity C peak, impurity F peak and pentoxifylline peak, and the separation between the peaks of each component should meet the requirements; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the pentoxifylline chromatographic peak height should be greater than 10.

[0082] Diluent: methanol-0.544% potassium dihydrogen phosphate solution (1:1).

[0083] Blank solution: diluent.

[0084] Blank excipient solution: Accurately measure 1 ml of blank excipient, place in a 20 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0085] Pentoxifylline stock solution: Take about 2 mg of pentoxifylline reference substance, weigh accurately, place in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0086] Prepare impurity A, C, D, F, G, H, I, J, K, L, M, and N stock solutions in the same way.

[0087] Mixed reference solution: Take about 10 mg of pentoxifylline reference substance, weigh accurately, place in a 10 ml volumetric flask, add 0.2 ml each of impurity A, C, D, F, G, H, I, J, K, L, M, N stock solutions, add diluent to dissolve and dilute to the scale, and shake well.

[0088] Pentoxifylline fixed solution: Accurately measure 2 ml of the pentoxifylline stock solution, place it in a 10 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0089] In the same way, prepare impurity A, C, D, F, G, H, I, J, K, L, M, and N location solutions.

[0090] Accurately measure the blank solution, blank excipient solution, mixed reference solution and each positioning solution, inject them into the liquid chromatograph, and record the chromatogram.

[0091] Table 5. Methodological validation of related substances - specificity

[0092]

[0093] Conclusion: Blank solution and blank excipients do not interfere with the inspection of related substances of this product; in the mixed reference solution, the separation between the impurity peaks meets the requirements (the minimum value is 1.55); therefore, the method specificity for related substances of this product is good.

[0094] 1.2 Instrument precision

[0095] Check whether the instrument precision meets the requirements, requiring retention time RSD ≤ 1.0% and peak area RSD ≤ 2.0%.

[0096] By injecting the reference solution continuously for 6 times, the retention time RSD of each impurity and main component was less than 1.0% (the maximum value was 0.09%), and the peak area RSD was less than 2.0% (the maximum value was 0.17%). Therefore, the instrument precision of the method for related substances of this product was good.

[0097] 1.3 Limit of quantification and limit of detection

[0098] The detection sensitivity of this method was investigated, and the signal-to-noise ratio was used as the evaluation index. The concentration corresponding to the signal-to-noise ratio of about 10 was required to be the quantitative limit, and the concentration corresponding to the signal-to-noise ratio of about 3 was required to be the detection limit.

[0099] Diluent: methanol-0.544% potassium dihydrogen phosphate solution (1:1).

[0100] Blank solution: diluent.

[0101] Pentoxifylline stock solution: Take about 2 mg of pentoxifylline reference substance, weigh accurately, place in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0102] Prepare impurity A, C, D, F, G, H, I, J, K, L, M, and N stock solutions in the same way.

[0103] Accurately measure an appropriate amount of the stock solution of impurities A, C, D, F, G, H, I, J, K, L, M, and N, and dilute them step by step until the S / N ratio is about 3. The corresponding concentration is the detection limit, and the corresponding concentration is about 10.

[0104] Table 6. Methodological validation of related substances - quantitation limits

[0105] name Concentration (μg / ml) Approximately equivalent to the test sample concentration (%) S / N Impurity M 0.3354 0.0335 11.8 Impurity D 0.0410 0.0041 13.9 Impurity G 0.1342 0.0134 16.9 Impurity K 0.0385 0.0038 14.3 Impurity H 0.0616 0.0062 16.8 Impurity I 0.0429 0.0043 11.1 Impurity N 0.0707 0.0071 15.2 Impurity A 0.0282 0.0028 14.9 Impurity C 0.0279 0.0028 12.6 Impurity F 0.0352 0.0035 14.4 Impurity L 0.1334 0.0133 17.7 principal component 0.0434 0.0043 12.1 Impurity J 0.0561 0.0056 12.3

[0106] Table 7. Methodological validation of related substances - detection limits

[0107] name Concentration (μg / ml) Approximately equivalent to the test sample concentration (%) S / N Impurity M 0.1342 0.0134 4.2 Impurity D 0.0164 0.0016 4.8 Impurity G 0.0537 0.0054 5.9 Impurity K 0.0154 0.0015 4.9 Impurity H 0.0246 0.0025 5.7 Impurity I 0.0171 0.0017 3.0 Impurity N 0.0283 0.0028 5.6 Impurity A 0.0113 0.0011 4.2 Impurity C 0.0112 0.0011 3.6 Impurity F 0.0141 0.0014 4.2 Impurity L 0.0533 0.0053 5.7 principal component 0.0174 0.0017 3.6 Impurity J 0.0225 0.0022 3.5

[0108] Conclusion: The limits of quantification and detection of each impurity and pentoxifylline are in compliance with the regulations; therefore, the detection method for related substances of this product has good sensitivity.

[0109] 1.4 Linear range and correction factor

[0110] The linearity of each impurity and pentoxifylline within the limit range of approximately LOQ to 200% was examined, requiring linear regression with concentration as the horizontal axis and peak area as the vertical axis, with a correlation coefficient of r≥0.998 and the Y-axis intercept within 25% of the 100% limit response value.

[0111] Linear stock solution: Accurately measure 2 ml each of impurity A, C, D, F, G, H, I, J, K, L, M, N stock solution and pentoxifylline stock solution; place in the same 50 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0112] Accurately measure the linear stock solutions respectively, prepare them according to the table below, and dilute them with diluent to make linear solution A.

[0113] Table 8. Linearity solutions

[0114]

[0115] Precisely measure the blank solution, each quantitative limit solution and each linear solution, inject them into the liquid chromatograph, record the chromatogram, and perform linear regression with concentration as the abscissa (X) and peak area as the ordinate (Y).

[0116] Table 9. Linear equations

[0117] name Concentration (equivalent to the percentage of test sample concentration) Linear equations Impurity M 0.03μg / ml~4.47μg / ml (LOQ~0.4%) y=4603.8650x+45.1243, r=0.9999 Impurity D 0.04μg / ml~4.10μg / ml (LOQ~0.4%) y=50854.6526x+495.7863,r=1.0000 Impurity G 0.13μg / ml~3.98μg / ml (LOQ~0.4%) y=28716.2191x+97.3264, r=0.9999 Impurity K 0.04μg / ml~3.85μg / ml (LOQ~0.4%) y=58407.3644x+319.4379, r=1.0000 Impurity H 0.06μg / ml~4.11μg / ml (LOQ~0.4%) y=32419.2325x+288.2004,r=1.0000 Impurity I 0.04μg / ml~4.29μg / ml (LOQ~0.4%) y=42108.6824x+417.4849, r=0.9999 Impurity N 0.07μg / ml~4.35μg / ml (LOQ~0.4%) y=35872.5307x+442.6662, r=1.0000 Impurity A 0.03μg / ml~4.52μg / ml (LOQ~0.4%) y=70181.9646x+717.9892,r=0.9999 Impurity C 0.03μg / ml~4.47μg / ml (LOQ~0.4%) y=69166.4609x+704.5831, r=0.9999 Impurity F 0.04μg / ml~4.03μg / ml (LOQ~0.4%) y=67400.3713x+645.2620, r=0.9999 Impurity L 0.13μg / ml~3.95μg / ml (LOQ~0.4%) y=30384.3550x+308.9025, r=0.9999 principal component 0.04μg / ml~4.34μg / ml (LOQ~0.4%) y=43900.1664x+385.4483, r=0.9999 Impurity J 0.06μg / ml~4.49μg / ml (LOQ~0.4%) y=39197.5921x+1103.8941, r=0.9998

[0118] Conclusion: The linearity of pentoxifylline and impurities in this product is good within the range of LOQ~200% limit (about LOQ~0.4%).

[0119] 1.5 Accuracy

[0120] Accuracy tests were conducted on pentoxifylline injection with impurity limits of 50%, 100%, and 150%. The external standard method and the principal component external standard method with correction factors were used for calculation. At the limit concentrations of 50%, 100%, and 150%, the average recoveries should be between 80% and 120%, with RSD ≤ 10%.

[0121] Diluent: methanol-0.544% potassium dihydrogen phosphate solution (1:1).

[0122] Blank solution: diluent.

[0123] Spiked stock solution: Accurately measure 5 ml of impurity A, C, D, E, F, G, H, I, J, K, L, M, and N stock solutions, place them in the same 100 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0124] Reference solution: Accurately measure 8 ml of the spiked stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0125] Low concentration test solution (50% limit concentration): accurately measure 1 ml of the product, place it in a 20 ml volumetric flask, add 4 ml of the impurity spiked stock solution, dilute to the mark with diluent, and shake well. Prepare 3 copies in parallel.

[0126] Medium concentration test solution (100% limit concentration): accurately measure 1 ml of the product, place it in a 20 ml volumetric flask, add 8 ml of the impurity spiked stock solution, dilute to the mark with diluent, and shake well. Prepare 3 copies in parallel.

[0127] High concentration test solution (150% limit concentration): accurately measure 1 ml of the product, place it in a 20 ml volumetric flask, add 12 ml of the impurity spiked stock solution, dilute to the mark with diluent, and shake well. Prepare 3 copies in parallel.

[0128] Accurately measure the blank solution, blank excipient solution, sensitivity solution, each reference solution, each control solution and each test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0129] Table 10. Methodological validation of related substances - accuracy

[0130]

[0131]

[0132] Conclusion: The average recoveries of each impurity at the limits of about 50%, 100%, and 150% were all between 80% and 120%, and the RSDs were all less than 10% (the maximum value was 3.47%); therefore, the method accuracy for the related substances of this product was good.

[0133] The present invention is further described in detail below in conjunction with embodiments and performance testing experiments.

[0134] Example 1

[0135] A liquid chromatography detection method for related substances in pentoxifylline injection comprises the following steps:

[0136] Preparation of reference solution: Take 10 mg of pentoxifylline reference substance, weigh it accurately, and place it in a 10 ml volumetric bottle, then add 0.2 ml of each stock solution of impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity M, and impurity N, then add diluent to dissolve and dilute to scale, shake well, the preparation method of the above stock solution is as follows: Take about 2 mg of impurity A reference substance, weigh it accurately, place it in a 20 ml volumetric bottle, add diluent to dissolve and dilute to scale, shake well, and the stock solution of impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M, and impurity N is the same as impurity A.

[0137] To prepare the test solution, accurately measure 1 ml of pentoxifylline injection, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0138] Liquid chromatography detection: Inject the reference solution and the test solution into a liquid chromatograph, and use the mobile phase gradient elution method for separation and detection; the chromatographic column used is a C8 column, specifically, it can be YMC-Triart C8, 4.6mmriart, 5.6 or a chromatographic column with equivalent performance using octylsilane bonded silica gel as a filler.

[0139] The mobile phase gradient elution process is: from 0 to 10 minutes, the mobile phase A is 64% and the mobile phase B is 36%; in the first gradient elution, the mobile phase has a larger polarity, and the impurities M, impurity A, impurity C, impurity D and impurity F with larger polarity are eluted in turn, and there is a good separation degree between adjacent substances.

[0140] 10-15min, mobile phase A is uniformly reduced to 34%, and mobile phase B is uniformly increased to 66%; during the second gradient elution, the polarity of the mobile phase is uniformly reduced, and the flow rate and polarity change rate of the eluent are controlled to elute impurities L, impurity G, impurity K and pentoxifylline, and the separation degree between them and adjacent substances is good.

[0141] 15-30min, mobile phase A is 34%, mobile phase B is 66%; during the third gradient elution, the polarity of the mobile phase remains unchanged, impurity H, impurity I, impurity J, and impurity N are eluted and separated, and the separation degree of adjacent substances is good.

[0142] 30-40min, mobile phase A increases uniformly to 64%, and mobile phase B decreases uniformly to 36%; in the fourth gradient elution, the polarity of the mobile phase increases uniformly and rapidly, so that mobile phase A can be used to elute the chromatographic column.

[0143] At 40-60 minutes, mobile phase A is 64%, and mobile phase B is 36%. Both mobile phase A and mobile phase B are expressed in volume percentages. In the fifth gradient elution, mobile phase A is used as the elution for the chromatographic column to avoid residual substances on the chromatographic column.

[0144] Furthermore, the mobile phase A is a potassium dihydrogen phosphate aqueous solution with a mass percentage concentration of 0.544%, and the mobile phase B is methanol.

[0145] Optionally, the flow rate is 1.0 ml per minute; the column temperature is 35° C.; the detection wavelength is 272 nm; and the injection volume is 20 μl.

[0146] The elution conditions are shown in Table 11. The obtained spectrum is shown in Figure 1 shown.

[0147] Table 11. Chromatographic conditions

[0148]

[0149] Example 2

[0150] Example 2 is substantially the same as Example 1, except that, in the liquid chromatography detection, the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.5%, the flow rate of the mobile phase is 0.8 mL / min, and the column temperature is 30°C.

[0151] Example 3

[0152] Example 3 is substantially the same as Example 1, except that, in the liquid chromatography detection, the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.6 g / L, the flow rate of the mobile phase is 1.2 mL / min, and the column temperature is 40°C.

[0153] Comparative Example 1

[0154] Pentoxifylline Injection ChP2020 Method Reproduction:

[0155] Chromatographic column: YMC Triart C8, 250mm×4.6mm, 5μm; mobile phase: methanol-0.544% potassium dihydrogen phosphate solution (3:7) as mobile phase A, methanol-0.544% potassium dihydrogen phosphate solution (7:3) as mobile phase B, elution according to Table 12 below; flow rate: 1.0ml per minute; column temperature: 30°C; detection wavelength: 272nm; injection volume: 20μl.

[0156] Table 12. Chromatographic conditions

[0157]

[0158]

[0159] Test results: In the related substance method of pentoxifylline injection ChP2020, the separation degree between the main component and impurity J in the chromatogram of the reference solution was low.

[0160] Comparative Example 2

[0161] Reproduce the method for related substances in the USP-NF2022 standard for pentoxifylline:

[0162] Chromatographic column: YMC Triart C18, 250mm×4.6mm, 5μm; mobile phase: methanol-tetrahydrofuran-acetonitrile-1g / L perchloric acid solution (2:2.5:15:80), flow rate: 0.7ml per minute; column temperature: 30°C; detection wavelength: 273nm; injection volume: 20μl, running time: 100min.

[0163] Conclusion: In the related substance method of pentoxifylline USP-NF2022 standard, the separation degree between impurity F and pentoxifylline is 24.22, which is greater than 10.0; however, impurities J and impurity N did not show peaks and could not be effectively detected. Therefore, this method is not suitable for the detection of related substances of this product.

[0164] Comparative Example 3

[0165] Comparative Example 3 is substantially the same as Example 1, except that the chromatographic column is filled with octadecylsilane bonded silica gel (CAPCELLPAK C18 MGⅢ column, 4.6 mm×250 mm, 5 μm).

[0166] Comparative Example 4

[0167] Comparative Example 4 is substantially the same as Example 1, except that the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.8%.

[0168] Comparative Example 5

[0169] Comparative Example 5 is substantially the same as Example 1, except that the mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.3%.

[0170] Comparative Example 6

[0171] Comparative Example 6 is substantially the same as Example 1, except that the flow rate of the mobile phase is 0.5 mL / min.

[0172] Comparative Example 7

[0173] Comparative Example 7 is substantially the same as Example 1, except that the flow rate of the mobile phase is 1.5 mL / min.

[0174] Accurately measure the test solution and the reference solution according to the method of Examples 1-3 and Comparative Examples 1-7, inject them into the liquid chromatograph, and record the chromatograms, as shown in Table 13 below.

[0175] Table 13. Chromatographic peak data

[0176] name Minimum separation between peaks of each substance Example 1 1.55 Example 2 1.54 Example 3 1.53 Comparative Example 1 1.37 Comparative Example 2 Impurity J and impurity N did not appear Comparative Example 3 1.26 Comparative Example 4 1.41 Comparative Example 5 1.11 Comparative Example 6 0.83 Comparative Example 7 0.47

[0177] Combining the test results of the embodiments, comparative examples and tables, it can be seen that when the detection method of the present invention is used for detection, the separation degree of each substance is good, the retention time deviation is small, and the detection result is reliable. If the detection conditions of the present invention are changed, the reliability of the detection will be significantly affected. Combining Comparative Examples 1 to 3, it can be seen that if the composition of the mobile phase is changed, taking Comparative Example 1 as an example, the minimum separation between the peaks is 1.37, which is less than 1.5; if the elution gradient is changed, taking Comparative Example 2 as an example, impurity J and impurity N do not peak under this chromatographic condition and cannot be effectively detected. If the chromatographic column is changed, taking Comparative Example 3 as an example, the minimum separation between the peaks is 1.26, which is less than 1.5; if the phosphate concentration is increased, taking Comparative Example 4 as an example, the minimum separation between the peaks is 1.41, which is less than 1.5; if the phosphate concentration is reduced, taking Comparative Example 5 as an example, the minimum separation between the peaks is 1.26, which is less than 1.5; if the flow rate is increased, taking Comparative Example 6 as an example, the minimum separation between the peaks is 0.83, which is less than 1.5; if the flow rate is reduced, taking Comparative Example 7 as an example, the minimum separation between the peaks is 0.47, which is less than 1.5.

Claims

1. A liquid chromatography detection method for related substances in pentoxifylline injection, characterized in that: The following steps are involved: Prepare reference solution, prepare test solution, and perform liquid chromatography detection; The liquid chromatography detection includes: injecting the reference solution and the test solution into a liquid chromatograph, and using a mobile phase gradient elution method for separation and detection; the chromatographic column used is a C8 column; The mobile phase gradient elution process is as follows: at 0-10 min, mobile phase A is 64% and mobile phase B is 36%; at 10-15 min, mobile phase A is uniformly reduced to 34% and mobile phase B is uniformly increased to 66%; at 15-30 min, mobile phase A is 34% and mobile phase B is 66%; at 30-40 min, mobile phase A is uniformly increased to 64% and mobile phase B is uniformly reduced to 36%; at 40-60 min, mobile phase A is 64% and mobile phase B is 36%; mobile phase A and mobile phase B are both expressed in volume percentages; The mobile phase A is a potassium dihydrogen phosphate aqueous solution with a mass percentage concentration of 0.5%-0.6%, and the mobile phase B is methanol.

2. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: The mass percentage concentration of the potassium dihydrogen phosphate aqueous solution is 0.544%.

3. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: In the liquid chromatography detection, the flow rate of the mobile phase is 0.8-1.2 mL / min.

4. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: In the liquid chromatography detection, the column temperature is 30-40°C.

5. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: In the liquid chromatography detection, the detection wavelength is 272 nm.

6. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: In the liquid chromatography detection, the chromatographic column is YMC-Triart C8, 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance, using octylsilane bonded silica gel as a filler.

7. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: The relevant substances include: pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N, Wherein, the structure of the impurity A is: , The structure of the impurity C is: , The structure of the impurity D is: , The structure of the impurity F is: , The structure of the impurity G is: , The structure of the impurity H is: , The structure of the impurity I is: , The structure of the impurity J is: , The structure of the impurity K is: , The structure of the impurity L is: , The structure of the impurity M is: , And the structure of the impurity N is: .

8. The liquid chromatography detection method for related substances in pentoxifylline injection according to claim 1, characterized in that: The limits of quantification of the pentoxifylline, impurity A, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M and impurity N in the related substances are 0.0434 μg / mL, 0.0282 μg / mL, 0.0279 μg / mL, 0.0410 μg / mL, 0.0352 μg / mL, 0.1342 μg / mL, 0.0616 μg / mL, 0.0429 μg / mL, 0.0561 μg / mL, 0.0385 μg / mL, 0.1334 μg / mL, 0.3354 μg / mL and 0.0707 μg / mL, respectively. The detection limits of the pentoxifylline, the impurity A, the impurity C, the impurity D, the impurity F, the impurity G, the impurity H, the impurity I, the impurity J, the impurity K, the impurity L, the impurity M and the impurity N were 0.0174 μg / mL, 0.0113 μg / mL, 0.0112 μg / mL, 0.0164 μg / mL, 0.0141 μg / mL, 0.0537 μg / mL, 0.0246 μg / mL, 0.0171 μg / mL, 0.0225 μg / mL, 0.0154 μg / mL, 0.0533 μg / mL, 0.1342 μg / mL and 0.0283 μg / mL, respectively.

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