WO2022252730A1 - Method for detecting substances related to tacrolimus ointment - Google Patents

Abstract

A method for detecting substances related to tacrolimus ointment. A supercritical fluid is used in the pretreatment of a sample to extract substances related to tacrolimus, which solves the problems in the existing technology of incomplete extraction of substances related to tacrolimus ointment and steps being overly complicated.

Description

A method for detecting related substances in tacrolimus ointment field of invention

The invention belongs to the field of detection of pharmaceutical products, and in particular relates to a detection method for related substances of tacrolimus ointment.

Background technique

When detecting related substances in tacrolimus ointment, the extraction of related substances in tacrolimus is the key to detection. The pretreatment process of samples in the prior art is to obtain the test solution by solid phase extraction. The specific steps are as follows: The solid-phase extraction column is pretreated, and the sample is suspended by adding n-hexane-ethyl acetate, centrifuged, and the supernatant is passed through the pre-treated solid-phase extraction column; then, n-hexane-ethyl acetate suspension is added to the centrifuge tube, Centrifuge, and take the supernatant to pass through the pretreated solid phase extraction column; wash the solid phase extraction column with n-hexane-ethyl acetate 6 times to ensure that each wash is complete; then elute the solid phase extraction column twice with ethyl acetate , collected the eluate, and finally evaporated in a water bath to an oily residue, and added n-hexane-n-butyl chloride-acetonitrile mixed solution to dissolve to obtain the test solution. The above-mentioned processing is cumbersome and not efficient, and the operators may incompletely extract the relevant substances in the tacrolimus ointment due to lack of experience, and sometimes the effective extraction can last up to one day at a time, and the current method is difficult to carry out effective detection.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a method for detecting related substances of tacrolimus ointment, wherein the pretreatment of the sample adopts supercritical fluid to extract the related substances of tacrolimus, which mainly solves the problem of tacrolimus in the prior art. The problem of incomplete extraction of relevant substances in Si ointment and too cumbersome steps.

Technical scheme of the present invention is specifically realized like this:

A method for detecting related substances in tacrolimus ointment, including pretreatment of samples and detection of related substances in tacrolimus, wherein the pretreatment of samples uses supercritical fluid to extract related substances in tacrolimus;

The conditions of the supercritical fluid extraction: the supercritical fluid is carbon dioxide, the extraction temperature is 60°C-65°C, the extraction pressure is 20MPa-35MPa, the extraction time is 45min-60min, and the flow rate of the carbon dioxide is 0.1L/min- 0.8L/min.

Preferably, the conditions of the supercritical fluid extraction: the supercritical fluid is carbon dioxide, the extraction temperature is 60°C-65°C, the extraction pressure is 25MPa-30MPa, the extraction time is 60min, and the flow rate of the carbon dioxide is 0.6L/min -0.8L/min.

Wherein the specific steps of the pretreatment of the sample include:

Take a sample of tacrolimus ointment, dissolve it in a water bath at 60°C-65°C with n-hexane solvent, spray it into the extraction kettle at a pressure of 20MPa-35MPa, and inject carbon dioxide fluid for extraction at the same time, it is easily soluble in carbon dioxide and n-hexane The components in the extraction tank flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. The solvent is dissolved, that is: the test solution.

The content of tacrolimus in the tacrolimus ointment is 0.1% w/w, and the matrix is propylene carbonate, white petrolatum, mineral oil, white wax, paraffin. In the sample pretreatment process, the components that are not easily soluble in carbon dioxide and n-hexane include tacrolimus and propylene carbonate remaining in the extraction kettle, and the components that are easily soluble in carbon dioxide and n-hexane include white petrolatum, mineral oil, White wax and paraffin flow into the separation tank from the top of the extraction tank, so that the related substances of tacrolimus are completely separated.

The volume ratio of the n-hexane-n-butyl chloride-acetonitrile is 1:1:1.

The detection of related substances of tacrolimus adopts the determination method of related substances published in the registration standard of imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration.

The method for detecting related substances of tacrolimus ointment of the present invention, wherein the pretreatment of the sample adopts supercritical fluid to extract the related substances of tacrolimus, the extraction is complete, the steps are simple, and the related substances of tacrolimus can be effectively extracted, further Accurate monitoring of the content of related substances in tacrolimus ointment plays a major role in the quality research of tacrolimus ointment.

Description of drawings

Fig. 1 is the HPLC spectrogram of described tacrolimus crude drug;

Fig. 2 is the HPLC spectrogram of the blank excipient of described tacrolimus ointment;

Fig. 3 is the HPLC spectrogram of the related substance of tacrolimus ointment described in embodiment 1;

Fig. 4 is the HPLC spectrogram of the related substance of tacrolimus ointment described in embodiment 2;

Fig. 5 is the HPLC spectrogram of the related substance of tacrolimus ointment described in embodiment 3;

Fig. 6 is the HPLC spectrogram of the related substance of tacrolimus ointment described in embodiment 4;

Fig. 7 is the mass spectrogram of the substance XV related to the high-temperature destruction of the tacrolimus raw material drug;

Fig. 8 is the HPLC spectrogram of tacrolimus ointment related substance when need testing solution described in embodiment 1 is placed 0h;

Fig. 9 is the HPLC spectrogram of the related substances of tacrolimus ointment when the test solution described in Example 1 is placed for 1h;

Fig. 10 is the HPLC spectrogram of tacrolimus ointment related substance when need testing solution described in embodiment 1 is placed 3h;

Fig. 11 is the HPLC spectrogram of tacrolimus ointment related substance when the need testing solution described in embodiment 1 is placed 6h;

Fig. 12 is the HPLC spectrogram of tacrolimus ointment related substance when the need testing solution described in embodiment 1 is placed 9h;

Figure 13 is the HPLC spectrogram of the related substances of tacrolimus ointment when the test solution described in Example 1 was placed for 12 hours.

Detailed ways

The content of tacrolimus in the tacrolimus ointment of the present invention is 0.1% w/w, and the matrix is propylene carbonate, white petrolatum, mineral oil, white wax and paraffin. The tacrolimus, propylene carbonate, white petrolatum, mineral oil, white wax, and paraffin used in the homemade tacrolimus ointment can all be purchased from the manufacturers announced on the CDE official website. The reference preparation used was tacrolimus ointment produced by Astellas Pharmaceuticals (China) Co., Ltd.

The formula of the tacrolimus ointment: 0.1% w/w of tacrolimus, 0.4 g of propylene carbonate, 10 g of white petrolatum, 1.5 g of mineral oil, 1.5 g of white wax, and 1.5 g of paraffin.

Example 1

A method for detecting related substances in tacrolimus ointment, the specific steps comprising:

(1) Pretreatment of samples

Take 10g of tacrolimus ointment sample, dissolve it in a 60°C water bath with n-hexane solvent, spray it into the extraction kettle at a pressure of 25MPa, and at the same time inject a carbon dioxide fluid with a flow rate of 0.7L/min for extraction for 60min at an extraction temperature of 60°C. The components that are easily soluble in carbon dioxide and n-hexane flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) dissolved in solvent, that is: the test solution, the test solution can be stored at room temperature for 12 hours.

(2) Detection of related substances in tacrolimus ointment

The detection of the related substances adopts the determination method of the related substances announced in the registration standard of imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration.

Precisely measure 20 μL of the test solution and n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) solvent into the liquid chromatograph, and record the chromatogram to three times the retention time of the main component peak. Calculated by the area normalization method, if there is an impurity peak in the chromatogram of the test solution, the impurity peak with an impurity content less than 0.1% can be ignored, and the excipient peaks before RRT=0.25 and around RRT=0.36 are deducted. Isomer II and Isomer I are not counted as related substances. Among the known impurities, impurity XV (OD-1) must not exceed 2.5%, any single known impurity must not exceed 1.0%; any single unknown impurity must not exceed 0.6% %; total impurities shall not exceed 5.0%.

Example 2

A method for detecting related substances in tacrolimus ointment, the specific steps comprising:

(1) Pretreatment of samples

Take 10g of tacrolimus ointment sample, dissolve it in a 65°C water bath with n-hexane solvent, spray it into the extraction kettle at a pressure of 30MPa, and simultaneously inject a carbon dioxide fluid with a flow rate of 0.6L/min for extraction for 45min at an extraction temperature of 65°C. The components that are easily soluble in carbon dioxide and n-hexane flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) dissolved in solvent, that is: the test solution, the test solution can be stored at room temperature for 12 hours.

(2) Detection of related substances in tacrolimus ointment

The detection of the related substances adopts the determination method of the related substances published in the registration standard of the imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration.

Precisely measure 20 μL of the test solution and n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) solvent into the liquid chromatograph, and record the chromatogram to three times the retention time of the main component peak. Calculated by the area normalization method, if there is an impurity peak in the chromatogram of the test solution, the impurity peak with an impurity content less than 0.1% can be ignored, and the excipient peaks before RRT=0.25 and around RRT=0.36 are deducted. Isomer II and Isomer I are not counted as related substances. Among the known impurities, impurity XV (OD-1) must not exceed 2.5%, any single known impurity must not exceed 1.0%; any single unknown impurity must not exceed 0.6% %; total impurities shall not exceed 5.0%.

Example 3

A method for detecting related substances in tacrolimus ointment, the specific steps comprising:

(1) Pretreatment of samples

Take 10g of a tacrolimus ointment sample, dissolve it in a 62°C water bath with n-hexane solvent, spray it into the extraction kettle at a pressure of 20MPa, and at the same time inject a carbon dioxide fluid with a flow rate of 0.8L/min for extraction for 50min at an extraction temperature of 62°C. The components that are easily soluble in carbon dioxide and n-hexane flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) dissolved in solvent, that is: the test solution, the test solution can be stored at room temperature for 12 hours.

(2) Detection of related substances in tacrolimus ointment

The detection of the related substances adopts the determination method of the related substances published in the registration standard of the imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration.

Precisely measure 20 μL of the test solution and n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) solvent into the liquid chromatograph, and record the chromatogram to three times the retention time of the main component peak. Calculated by the area normalization method, if there is an impurity peak in the chromatogram of the test solution, the impurity peak with an impurity content less than 0.1% can be ignored, and the excipient peaks before RRT=0.25 and around RRT=0.36 are deducted. Isomer II and Isomer I are not counted as related substances. Among the known impurities, impurity XV (OD-1) must not exceed 2.5%, any single known impurity must not exceed 1.0%; any single unknown impurity must not exceed 0.6% %; total impurities shall not exceed 5.0%.

Example 4

A method for detecting related substances in tacrolimus ointment, the specific steps comprising:

(1) Pretreatment of samples

Take 10g of a tacrolimus ointment sample, dissolve it in a 60°C water bath with n-hexane solvent, spray it into the extraction kettle at a pressure of 20MPa, and simultaneously inject a carbon dioxide fluid with a flow rate of 0.8L/min for extraction for 60min at an extraction temperature of 60°C. The components that are easily soluble in carbon dioxide and n-hexane flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) dissolved in solvent, that is: the test solution, the test solution can be stored at room temperature for 12 hours.

(2) Detection of related substances in tacrolimus ointment

The detection of the related substances adopts the determination method of the related substances published in the registration standard of the imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration.

Precisely measure 20 μL of the test solution and n-hexane-n-butyl chloride-acetonitrile (volume ratio 1:1:1) solvent into the liquid chromatograph, and record the chromatogram to three times the retention time of the main component peak. Calculated by the area normalization method, if there is an impurity peak in the chromatogram of the test solution, the impurity peak with an impurity content less than 0.1% can be ignored, and the excipient peaks before RRT=0.25 and around RRT=0.36 are deducted. Isomer II and Isomer I are not counted as related substances. Among the known impurities, impurity XV (OD-1) must not exceed 2.5%, any single known impurity must not exceed 1.0%; any single unknown impurity must not exceed 0.6% %; total impurities shall not exceed 5.0%.

Table 1 Relative retention time table of related substances of tacrolimus

relative substance relative retention time Related substances VIII 0.61-0.65 Related Substance IX 0.69-0.74 Related Substance XV(OD-1) 0.75-0.79 Related substances XI 0.82-0.87 Unknown A 0.91-0.97 Tacrolimus 1.00 Isomer II 1.09-1.19 Isomer I 1.43-1.57 Related Substance VI 1.87-1.95

Table 2 Tacrolimus related substance HPLC spectrogram peak result of the present invention

Combined with accompanying drawings 1-6, deducting the peak of blank excipients, Table 2 combined with the "relative retention time of related substances of tacrolimus" published in the registration standard for imported drug tacrolimus ointment (standard number: JX20130245) issued by the State Drug Administration Table" (Table 1) shows that peak 1, peak 2, peak 3, and peak 4 are related substance XV (OD-1), unknown substance, tacrolimus, and isomer II, respectively.

From page 6 of the Japanese tacrolimus ointment 0.1% IF document (revised in July 2018 (19th edition) Japanese standard commodity classification number 872699), it is known that thermal decomposition of tacrolimus may produce related substances IX and related substances XI , Related substances XV. The tacrolimus raw material drug used in the tacrolimus ointment of the present invention was destroyed at high temperature, and the relevant mass spectrum was obtained in Figure 7, showing a molecular ion peak [M ⁺ NH ₄ ] 821.51580, which was consistent with the molecular weight of the related substance XV.

The stability test of need testing solution described in embodiment 1:

The need testing solution prepared by embodiment 1 step (1) is placed at room temperature for 0h, 1h, 3h, 6h, 9h, 12h, and then sample injection according to step (2) respectively, to investigate the stability of the need testing solution, see The HPLC spectrograms of Fig. 8-Fig. 13, the investigation results are shown in Table 3, and the sum of the peak area and the main peak area is calculated. The test results show that the test solution is placed at room temperature for 12 hours, and the content determination results of related substances in tacrolimus ointment Compared with 0 hours, there was little change, indicating that the stability of the test solution was good within 12 hours.

Table 3 embodiment 1 described need testing solution stability investigation result

Claims (5)

A method for detecting related substances in tacrolimus ointment, comprising sample pretreatment and detection of tacrolimus related substances, characterized in that: the sample pretreatment uses supercritical fluid to extract tacrolimus related substances; The conditions of the supercritical fluid extraction: the supercritical fluid is carbon dioxide, the extraction temperature is 60°C-65°C, the extraction pressure is 20MPa-35MPa, the extraction time is 45min-60min, and the flow rate of the carbon dioxide is 0.1L/min- 0.8L/min. The method for detecting related substances in tacrolimus ointment according to claim 1, characterized in that: the conditions for the supercritical fluid extraction: the supercritical fluid is carbon dioxide, the extraction temperature is 60°C-65°C, and the extraction pressure is 25MPa-30MPa, the extraction time is 60min, and the flow rate of the carbon dioxide is 0.6L/min-0.8L/min. According to the detection method of the related substance of tacrolimus ointment described in claim 1 or 2, it is characterized in that: the concrete steps of the pretreatment of described sample comprise: Take a sample of tacrolimus ointment, dissolve it in a water bath at 60°C-65°C with n-hexane solvent, spray it into the extraction kettle at a pressure of 20MPa-35MPa, and inject carbon dioxide fluid for extraction at the same time, it is easily soluble in carbon dioxide and n-hexane The components in the extraction tank flow into the separation tank from the top of the extraction tank, and the components that are not easily soluble in carbon dioxide and n-hexane remain in the extraction tank. The solvent is dissolved, that is: the test solution. The method for detecting related substances in tacrolimus ointment according to claim 3, characterized in that: the volume ratio of n-hexane-n-butyl chloride-acetonitrile is 1:1:1. The method for detecting related substances in tacrolimus ointment according to claim 4, characterized in that: the content of tacrolimus in the tacrolimus ointment is 0.1% w/w, and the matrix is propylene carbonate and white petrolatum , mineral oil, white wax, paraffin.

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