A method for detecting medium-chain triglycerides in alfacalcidol soft capsules

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting medium-chain triglycerides in alfacalcidol soft capsules by high performance liquid chromatography. A charged aerosol detector (CAD detector) is used, a Fastcore Super C18 150mm×4.6mm, 2.6μm reverse phase chromatographic column is adopted, water-methanol is used as mobile phase A, acetonitrile is used as mobile phase B, flow rate, column temperature, injection volume, power function, filtration constant and evaporator temperature are set, and gradient elution is performed. The present invention controls the content of medium-chain triglycerides in alfacalcidol soft capsules for the first time. The method has simple mobile phase preparation, strong specificity, high separation, high sensitivity, good accuracy, and improves the efficiency of separation and detection.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting medium-chain triglycerides in alfacalcidol soft capsules by using high performance liquid chromatography. Background Art

[0002] Alfacalcidol regulates the balance of calcium and phosphorus in the human body, increases the absorption of calcium and phosphorus in the intestine, reduces the level of parathyroid hormone in plasma, and improves osteoporosis caused by menopause and hormone drugs in women. It is suitable for osteoporosis and rickets and osteomalacia caused by various reasons.

[0003] The soft capsule is composed of contents and a capsule shell. Adding medium-chain triglycerides as a capsule shell lubricant to the contents of the alfacalcidol soft capsule and combining it with a certain amount of antioxidant can effectively protect alfacalcidol and greatly improve the stability of the alfacalcidol soft capsule, thereby ensuring the effectiveness of the patient's medication and improving its bioavailability.

[0004] There are few liquid phase methods for detecting medium chain triglycerides reported in the literature. Patent CN109655536B discloses a method for detecting the content of caprylic acid diglyceride and capric acid diglyceride in medium chain triglycerides. Referring to the method in the patent, the inventor found that the baseline of the spectrum was poor during the experiment and the content of medium chain triglycerides could not be detected.

[0005] In order to ensure the quality of drugs and the safety and effectiveness of clinical use, the medium-chain triglycerides in alfacalcidol soft capsules must be controlled. Summary of the invention

[0006] The purpose of the present invention is to solve the problem of the lack of determination of the content of medium-chain triglycerides in alfacalcidol soft capsules in the prior art, and to provide a simple and easy-to-operate analysis method for simultaneously detecting the content of medium-chain triglycerides.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The invention discloses a method for detecting medium-chain triglycerides in alfacalcidol soft capsules. According to an embodiment of the invention, a high performance liquid chromatography method is used, a charged aerosol detector (CAD detector) is used, a C18 reverse phase chromatographic column with core-shell technology is adopted, water-methanol is used as a mobile phase A, an organic phase is used as a mobile phase B, a flow rate, a column temperature, an injection volume, a power function, a filtration constant and an evaporator temperature are set, and washing is performed according to the following gradient program in terms of volume ratio:

[0009] Elution started with 20% mobile phase A and 80% mobile phase B, and the proportion of mobile phase A was gradually reduced and the proportion of mobile phase B was increased. After 30 minutes, the proportion of mobile phase A was adjusted to 5%, and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20%, and the mobile phase B was adjusted to 80%. The elution was continued to maintain this mobile phase ratio for 51-60 minutes. This detection method can simultaneously detect the content of medium-chain triglycerides in alfacalcidol soft capsules.

[0010] The results of mass spectrometry analysis are shown in the table below.

[0011] Table 1 Mass spectrometry results

[0012]

[0013] The [M+Na] of this product was measured by mass spectrometry (ESI+) + The mass-to-charge ratio is 493.39, and the molecular weight of this product is 470.67, which is consistent with the molecular weight of tricaprylin. The above results are consistent with the structure of tricaprylin, and it is judged that the first chromatographic peak in medium-chain triglycerides is tricaprylin.

[0014] The [M+Na] of this product was measured by mass spectrometry (ESI+) + The mass-to-charge ratio is 521.49, and the molecular weight of this product is 498.73, which is consistent with the molecular weight of dicaprylic monocapric triglyceride. The above results are consistent with the structure of dicaprylic monocapric triglyceride, and it is judged that the second chromatographic peak in medium-chain triglycerides is dicaprylic monocapric triglyceride.

[0015] The [M+Na] of this product was measured by mass spectrometry (ESI+) + The mass-to-charge ratio is 549.44, and the molecular weight of this product is 526.79, which is consistent with the molecular weight of dicaprylic acid monocaprylic acid triglyceride. The above results are consistent with the structure of dicaprylic acid monocaprylic acid triglyceride, and it is judged that the third chromatographic peak in the medium-chain triglycerides is dicaprylic acid monocaprylic acid triglyceride.

[0016] The [M+Na] of this product was measured by mass spectrometry (ESI+) + The mass-to-charge ratio is 577.43, and the molecular weight of this product is 554.85, which is consistent with the molecular weight of capric triglyceride. The above results are consistent with the structure of capric triglyceride.

[0017] The inventors found in their research that the impurities in the alfacalcidol soft capsules could not be completely separated from the medium-chain triglycerides using C18 chromatographic columns such as Phenom Titank C18 150×4.6mm, 3μm, Phenom SuperLuC18-AQ, 250×4.6mm, 5μm. The use of a C18 reverse phase chromatographic column Phenom Fastcore Super C18 150mm×4.6mm, 2.6μm with added core-shell technology can completely separate the impurities in the alfacalcidol soft capsules from the medium-chain triglycerides. The analysis method of the present invention was determined by optimizing and screening the mobile phase composition, gradient, column temperature, power function, filtration constant and evaporator temperature. At the same time, according to the general rules of the four parts of the 2020 edition of the Chinese Pharmacopoeia <0512> (High Performance Liquid Chromatography) and General Rules <9101> The specificity validation was carried out according to the definition and validation method of (Guidelines for Validation of Analytical Methods for Pharmaceutical Quality Standards). The results showed that this method can simultaneously separate the four components of medium-chain triglycerides in alfacalcidol soft capsules, and effectively control the content of each impurity through the external standard method. The separation degree between each impurity peak is greater than 1.5.

[0018] According to an embodiment of the present invention, the reverse phase chromatography column in the high performance liquid chromatography method used in the present invention is selected from Pheromones Fastcore Super C18, with specifications of 150 mm×4.6 mm and 2.6 μm.

[0019] According to an embodiment of the present invention, the volume ratio of water to methanol in mobile phase A is selected from 2:8.

[0020] According to an embodiment of the present invention, the mobile phase B is an organic phase, selected from one or more of alcohol and acetonitrile.

[0021] Preferably, mobile phase B is selected from acetonitrile.

[0022] According to an embodiment of the present invention, in the high performance liquid chromatography disclosed in the present invention, when the flow rate is selected from 0.9~1.1ml / min, the column temperature is selected from 18~22°C, the injection volume is selected from 50μL, the evaporator temperature is selected from 35~50°C, the power function is 1.5, and the filtration constant is 5.0, the content of medium-chain triglycerides in alfacalcidol soft capsules can be detected, thereby ensuring that the quality of medium-chain triglycerides in alfacalcidol soft capsules is controllable.

[0023] Preferably, the flow rate is selected from 1.0 ml / min, the column temperature is selected from 20°C, and the evaporator temperature is selected from 35°C.

[0024] According to the embodiment of the present invention, the method of the present invention can effectively detect the content of medium-chain triglycerides in alfacalcidol soft capsules, and the separation degree between each impurity peak is greater than 1.5. The analysis process is shown in the example, and the results are shown in Table 2.

[0025] Table 2 Chromatographic separation parameters of known components

[0026]

[0027] The beneficial effects of the present invention are as follows: the present invention controls the content of medium-chain triglycerides in alfacalcidol soft capsules for the first time, the method has simple mobile phase preparation, strong specificity, high separation degree, high sensitivity and good accuracy, and improves the efficiency of separation and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shown is a high performance liquid chromatogram of medium chain triglycerides analyzed by mass spectrometry;

[0029] Figure 2 Shown are the mass spectra of tricaprylin and dicaprylic-capric triglyceride;

[0030] Figure 3 Shown are the mass spectra of dicaprylic monocaprylic triglyceride and capric triglyceride;

[0031] Figure 4 Shown is the HPLC chromatogram of alfacalcidol soft capsule prepared by the patent method according to Example 1;

[0032] Figure 5 The figure shows the HPLC chromatogram of the alfacalcidol soft capsule of Example 2 using the Pheromon Titank C18 150×4.6 mm, 3 μm chromatographic column;

[0033] Figure 6 The HPLC chromatogram of the Thermoscientific ODS Hypersil 250×4mm, 5μm chromatographic column of Alfacalcidol Soft Capsules in Example 3 is shown;

[0034] Figure 7 Shown is the HPLC chromatogram of the blank solvent of Example 4;

[0035] Figure 8 Shown is the HPLC chromatogram of tricaprylin of Example 4;

[0036] Fig. 9 Shown is the HPLC chromatogram of capric triglyceride of Example 4;

[0037] Fig.10 Shown is the HPLC chromatogram of medium chain triglycerides in Example 4;

[0038] Fig.11 Shown is the HPLC chromatogram of Alfacalcidol Soft Capsules of Example 4;

[0039] Fig.12The HPLC chromatogram of alfacalcidol soft capsules in Example 5 at a column temperature of 18°C ​​is shown;

[0040] Fig.13 The HPLC chromatogram of alfacalcidol soft capsules of Example 6 at a column temperature of 22°C is shown;

[0041] Fig.14 Shown is the HPLC chromatogram of the alfacalcidol soft capsule of Example 7 with a flow rate of 0.9 ml / min;

[0042] Fig.15 Shown is the HPLC chromatogram of the alfacalcidol soft capsule of Example 8 at a flow rate of 1.1 ml / min;

[0043] Fig.16 Shown is the HPLC chromatogram of the alfacalcidol soft capsule in Example 9 with a mobile phase ratio of 18:82;

[0044] Fig.17 Shown is the HPLC chromatogram of the alfacalcidol soft capsule in Example 10 with a mobile phase ratio of 22:78;

[0045] Fig.18 Shown is the HPLC graph of the alfacalcidol soft capsule of Example 11 at an evaporator temperature of 50°C.

[0046] Note: "ZL-1" in the figure label represents the first component of medium chain triglycerides, tricaprylin, "ZL-2" represents the second component of medium chain triglycerides, dicaprylic monocaprylic triglyceride, "ZL-3" represents the third component of medium chain triglycerides, dicaprylic monocaprylic triglyceride, and "ZL-4" represents the fourth component of medium chain triglycerides, capric triglyceride. Specific implementation plan

[0047] The present invention discloses a method for detecting medium chain triglycerides in alfacalcidol soft capsules. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The detection method of the present invention has been described through preferred embodiments, and relevant personnel obviously modify or appropriately change and combine the method application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0048] Example 1

[0049] (1) Solvent: methanol;

[0050] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0051] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0052] (4) Detector: CAD;

[0053] (5) Chromatographic column: Pheromones SuperLu C18-AQ, 250×4.6 mm, 5 μm;

[0054] (6) Mobile phase A: water-acetonitrile (2:8), mobile phase B: acetonitrile-isopropanol (2:8), elution according to the following gradient:

[0055] Start elution with 60% mobile phase A and 40% mobile phase B, gradually reduce the proportion of mobile phase A and increase the proportion of mobile phase B. After 30 minutes, adjust the proportion of mobile phase A to 20% and the proportion of mobile phase B to 80%. Keep eluting for 40 minutes. After 1 minute, adjust the proportion of mobile phase A to 60% and the proportion of mobile phase B to 40%. Keep eluting at this proportion for 41-50 minutes.

[0056] (7) Flow rate is 1.0 ml per minute;

[0057] (8) Column temperature is 30°C;

[0058] (9) Injection volume: 50 μl;

[0059] (10) Evaporator temperature is 35°C;

[0060] (11) The power function is 1.0;

[0061] (12) The filtration constant is 3.6;

[0062] (13) Reference to patented methods, Figure 4 The medium-chain triglycerides in alfacalcidol soft capsules should be four chromatographic peaks, but the shape of the fourth peak is poor and the baseline of the chromatogram is poor, so the chromatographic column and mobile phase should be screened.

[0063] Example 2

[0064] (1) Solvent: methanol; (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0065] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0066] (4) Detector: CAD;

[0067] (5) Chromatographic column: Pheromones Titank C18 150×4.6 mm, 3 μm;

[0068] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0069] Start elution with 40% mobile phase A and 60% mobile phase B, gradually reduce the proportion of mobile phase A and increase the proportion of mobile phase B. After 30 minutes, adjust the proportion of mobile phase A to 10% and the proportion of mobile phase B to 90%. Keep eluting for 40 minutes. After 1 minute, adjust the mobile phase A to 40% and the mobile phase B to 60%. Keep this proportion of mobile phases and continue eluting for 41-50 minutes.

[0070] (7) Flow rate is 1.0 ml per minute;

[0071] (8) Column temperature is 30°C;

[0072] (9) Injection volume: 50 μl;

[0073] (10) Evaporator temperature is 35°C;

[0074] (11) The power function is 1.0;

[0075] (12) The filtration constant is 3.6;

[0076] (13) Figure 5 The peaks of medium-chain triglycerides have severe tailing, the separation degree between ZL-3 and the impurities behind is 0.86, and ZL-4 and the impurities in front are not separated.

[0077] Example 3

[0078] (1) Sample preparation: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and collect the supernatant;

[0079] (2) Reference solution: weigh about 10 mg of medium-chain triglycerides into a 50 ml volumetric flask, dissolve with solvent by vortexing, dilute to scale, and shake well;

[0080] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0081] (4) Detector: CAD;

[0082] (5) Chromatographic column: Thermoscientific ODS Hypersil 250×4mm, 5μm;

[0083] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0084] Start elution with 20% mobile phase A and 80% mobile phase B, gradually reduce the proportion of mobile phase A and increase the proportion of mobile phase B. After 30 minutes, adjust the proportion of mobile phase A to 10% and the proportion of mobile phase B to 90%. Keep eluting for 40 minutes. After 1 minute, adjust the mobile phase A to 20% and the mobile phase B to 80%. Keep this proportion of mobile phases and continue eluting for 41-50 minutes.

[0085] (7) Flow rate is 1.0 ml per minute;

[0086] (8) Column temperature is 30°C;

[0087] (9) Injection volume: 50 μl;

[0088] (10) Evaporator temperature is 35°C;

[0089] (11) The power function is 1.0;

[0090] (12) The filtration constant is 3.6;

[0091] (13) Figure 6 The peak shapes of medium-chain triglycerides were good. ZL-3 was not separated from the impurities behind it, and the separation degree of ZL-4 from the impurities in front was 0.91.

[0092] Example 4

[0093] (1) Solvent: methanol;

[0094] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0095] (3) Reference solution: weigh about 10 mg of medium-chain triglycerides into a 50 ml volumetric flask, dissolve with solvent by vortexing, dilute to scale, and shake well;

[0096] (4) Tricaprylin: Weigh about 2 mg of tricaprylin into a 20 ml volumetric flask, vortex with solvent to dissolve, dilute to scale, and shake well;

[0097] (5) Capric triglyceride: Weigh about 2 mg of capric triglyceride into a 20 ml volumetric flask, vortex to dissolve with solvent, dilute to scale, and shake well;

[0098] (6) Brand and model of HPLC: Thermo Fisher U3000;

[0099] (7) Detector: CAD;

[0100] (8) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0101] (9) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0102] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0103] (10) Flow rate is 1.0 ml per minute;

[0104] (11) Column temperature is 20°C;

[0105] (12) Injection volume: 50 μl;

[0106] (13) Evaporator temperature is 35°C;

[0107] (14) The power function is 1.5;

[0108] (15) The filtration constant is 5.0;

[0109] (16) Figure 7 The blank solvent does not interfere with the detection of alfacalcidol soft capsules. Figure 8 , Fig. 9 and Fig.10 The retention time of the peaks of caprylic triglyceride, capric triglyceride and medium-chain triglycerides is consistent, and the peak shapes of the components in medium-chain triglycerides are good. Fig.11 In the alfacalcidol soft capsules, the peaks are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0110] Example 5

[0111] (1) Solvent: methanol;

[0112] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0113] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0114] (4) Detector: CAD;

[0115] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0116] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0117] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0118] (7) Flow rate is 1.0 ml per minute;

[0119] (8) Column temperature is 18°C;

[0120] (9) Injection volume: 50 μl;

[0121] (10) Evaporator temperature is 35°C;

[0122] (11) The power function is 1.5;

[0123] (12) The filtration constant is 5.0;

[0124] (13) Fig.12 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0125] Example 6

[0126] (1) Solvent: methanol;

[0127] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0128] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0129] (4) Detector: CAD;

[0130] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0131] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0132] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0133] (7) Flow rate is 1.0 ml per minute;

[0134] (8) Column temperature is 22°C;

[0135] (9) Injection volume: 50 μl;

[0136] (10) Evaporator temperature is 35°C;

[0137] (11) The power function is 1.5;

[0138] (12) The filtration constant is 5.0;

[0139] (13) Fig.13 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0140] Example 7

[0141] (1) Solvent: methanol;

[0142] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0143] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0144] (4) Detector: CAD;

[0145] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0146] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0147] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0148] (7) Flow rate is 0.9 ml per minute;

[0149] (8) Column temperature is 20°C;

[0150] (9) Injection volume: 50 μl;

[0151] (10) Evaporator temperature is 35°C;

[0152] (11) The power function is 1.5;

[0153] (12) The filtration constant is 5.0;

[0154] (13) Fig.14 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0155] Example 8

[0156] (1) Solvent: methanol;

[0157] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0158] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0159] (4) Detector: CAD;

[0160] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0161] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0162] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0163] (7) Flow rate is 1.1 ml per minute;

[0164] (8) Column temperature is 20°C;

[0165] (9) Injection volume: 50 μl;

[0166] (10) Evaporator temperature is 35°C;

[0167] (11) The power function is 1.5;

[0168] (12) The filtration constant is 5.0;

[0169] (13) Fig.15 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0170] Example 9

[0171] (1) Solvent: methanol;

[0172] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0173] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0174] (4) Detector: CAD;

[0175] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0176] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0177] Elution started with 18% mobile phase A and 82% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 18% and the mobile phase B was adjusted to 82%. The elution was continued at this mobile phase ratio for 51-60 minutes.

[0178] (7) Flow rate is 1.0 ml per minute;

[0179] (8) Column temperature is 20°C;

[0180] (9) Injection volume: 50 μl;

[0181] (10) Evaporator temperature is 35°C;

[0182] (11) The power function is 1.5;

[0183] (12) The filtration constant is 5.0;

[0184] (13) Fig.16 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0185] Example 10

[0186] (1) Solvent: methanol;

[0187] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0188] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0189] (4) Detector: CAD;

[0190] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0191] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0192] Elution started with 22% mobile phase A and 78% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 22% and the mobile phase B was adjusted to 78%. The elution was maintained at this proportion for 51-60 minutes.

[0193] (7) Flow rate is 1.0 ml per minute;

[0194] (8) Column temperature is 20°C;

[0195] (9) Injection volume: 50 μl;

[0196] (10) Evaporator temperature is 35°C;

[0197] (11) The power function is 1.5;

[0198] (12) The filtration constant is 5.0;

[0199] (13) Fig.17 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0200] Embodiment 11

[0201] (1) Solvent: methanol;

[0202] (2) Test solution: Take 5 Alfacalcidol soft capsules, weigh them, cut the capsule shell with scissors, place them in a 50 ml volumetric bottle, rinse the surface of the scissors with solvent, add an appropriate amount of solvent, vortex for 5 minutes, dilute to the scale with solvent, shake well, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant;

[0203] (3) Brand and model of HPLC: Thermo Fisher U3000;

[0204] (4) Detector: CAD;

[0205] (5) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0206] (6) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0207] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0208] (7) Flow rate is 1.0 ml per minute;

[0209] (8) Column temperature is 20°C;

[0210] (9) Injection volume: 50 μl;

[0211] (10) Evaporator temperature is 50°C;

[0212] (11) The power function is 1.5;

[0213] (12) The filtration constant is 5.0;

[0214] (13) Fig.18 The peaks of Alfacalcidol Soft Capsules are well separated and can achieve baseline separation, which meets the requirements of the Chinese Pharmacopoeia and can detect the content of medium-chain triglycerides in the sample.

[0215] Example 12

[0216] (1) Linear stock solution (400%): Weigh about 40 mg of medium-chain triglycerides into a 50 ml volumetric flask, vortex to dissolve with solvent, dilute to scale, and shake well;

[0217] 200%: Accurately measure 10 ml of the linear stock solution into a 20 ml volumetric flask and dilute to volume with methanol;

[0218] 100%: Accurately measure 5 ml of the linear stock solution into a 20 ml volumetric flask and dilute to volume with methanol;

[0219] 80%: Accurately measure 4 ml of the linear stock solution into a 20 ml volumetric flask and dilute to volume with methanol;

[0220] 50%: Accurately measure 2.5 ml of the linear stock solution into a 20 ml volumetric flask and dilute to volume with methanol;

[0221] 20%: Accurately measure 1 ml of the linear stock solution into a 20 ml volumetric flask and dilute to volume with methanol;

[0222] (2) Brand and model of HPLC: Thermo Fisher U3000;

[0223] (3) Detector: CAD;

[0224] (4) Chromatographic column: Pheromon Fastcore Super C18, 150 mm × 4.6 mm, 2.6 μm;

[0225] (5) Mobile phase A: water-methanol (2:8), mobile phase B: acetonitrile, elution according to the following gradient:

[0226] Elution started with 20% mobile phase A and 80% mobile phase B, gradually reducing the proportion of mobile phase A and increasing the proportion of mobile phase B. After 30 minutes, the proportion of mobile phase A was adjusted to 5% and the proportion of mobile phase B was adjusted to 95%. The elution was maintained for 50 minutes. After 1 minute, the mobile phase A was adjusted to 20% and the mobile phase B was adjusted to 80%. The elution was continued at this proportion for 51-60 minutes.

[0227] (6) Flow rate is 1.0 ml per minute;

[0228] (7) Column temperature is 20°C;

[0229] (8) Injection volume: 50 μl;

[0230] (9) Evaporator temperature is 35°C;

[0231] (10) The power function is 0.8~1.5;

[0232] (11) The filtration constant is 5.0;

[0233] (12) The higher the power function, the better the linearity. The optimal power function is 91.5. The results are shown in the table below.

[0234]

Claims

1. A method for detecting medium-chain triglycerides in alfacalcidol soft capsules, characterized in that: The invention adopts high performance liquid chromatography, uses a charged aerosol detector (CAD detector), adopts a C18 reverse phase chromatographic column with added core-shell technology, uses water-methanol as mobile phase A, uses acetonitrile as mobile phase B, sets flow rate, column temperature, injection volume, power function, filtration constant and evaporator temperature, and performs gradient elution. The gradient elution is as follows: in terms of volume ratio, elution starts with 18-22% of mobile phase A and 82-78% of mobile phase B, gradually reduces the proportion of mobile phase A, increases the proportion of mobile phase B, adjusts the proportion of mobile phase A to 5% and the proportion of mobile phase B to 95% after 30 minutes, maintains elution for 50 minutes, adjusts mobile phase A to 20% and mobile phase B to 80% after 1 minute, and maintains the proportion of mobile phases to continue elution for 51-60 minutes. The detection method can simultaneously detect the contents of four medium chain triglycerides in alfacalcidol soft capsules.

2. The method for detecting medium-chain triglycerides in alfacalcidol soft capsules according to claim 1, characterized in that: The reverse phase chromatographic column is selected from Phenom FastCore SuperC18, with specifications of 150mm×4.6mm and 2.6µm.

3. The method for detecting medium-chain triglycerides in alfacalcidol soft capsules according to claim 1, characterized in that: The volume ratio of water to methanol in the mobile phase A is selected from 2:

8.

4. The method for detecting medium chain triglycerides in alfacalcidol soft capsules according to claim 1, characterized in that: The mobile phase B is selected from one or more of alcohol and acetonitrile.

5. The method for detecting medium-chain triglycerides in alfacalcidol soft capsules according to claim 4, characterized in that: The B mobile phase is selected from acetonitrile.

6. The method for detecting medium chain triglycerides in alfacalcidol soft capsules according to claim 1, characterized in that: In the high performance liquid chromatography method, the flow rate is selected from 0.9 to 1.1 ml / min, the column temperature is selected from 18 to 22° C., the injection volume is selected from 50 μL, the evaporator temperature is selected from 35 to 50° C., the power function is 1.5, and the filtration constant is 5.

0.

7. The method for detecting medium chain triglycerides in alfacalcidol soft capsules according to claim 6, characterized in that: In the high performance liquid chromatography method, the flow rate is selected from 1.0 ml / min, the column temperature is selected from 20° C., and the evaporator temperature is selected from 35° C.

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