CN113376297A

CN113376297A - Pretreatment method for tacrolimus ointment isomer determination

Info

Publication number: CN113376297A
Application number: CN202110624011.7A
Authority: CN
Inventors: 曹慧敏; 朱召贞; 王守彬; 吕敏; 刘宗银
Original assignee: Shandong Guanxiu Biopharmaceutical Co ltd
Current assignee: Shandong tingke Pharmaceutical Co.,Ltd.
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-09-10
Also published as: ZA202202829B; WO2022252731A1

Abstract

The invention discloses a pretreatment method for measuring tacrolimus ointment isomers. Then, the content of tacrolimus isomers was determined according to high performance liquid chromatography. The pretreatment method is simple to operate, so that the tacrolimus isomer is easy to extract from the tacrolimus ointment, the determination of the high performance liquid chromatography is further met, and the research is provided for the quality control of the tacrolimus ointment.

Description

Pretreatment method for tacrolimus ointment isomer determination

Technical Field

The invention belongs to the field of medical product detection, and particularly relates to a pretreatment method for tacrolimus ointment isomer determination.

Background

The quality control of tacrolimus ointment only controls the content of related substances of the tacrolimus ointment at present, and does not control the content of isomers of the tacrolimus ointment. Since the tacrolimus ointment contains oily viscous matrix and the instability of the tacrolimus isomers causes the extraction of the tacrolimus ointment isomers to be quite difficult, and further influences the effective determination of the subsequent tacrolimus isomers, the pretreatment in the determination of the tacrolimus ointment isomers is the key of the detection.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a pretreatment method for measuring a tacrolimus ointment isomer, which comprises the steps of adding n-hexane into the tacrolimus ointment for water bath dissolution according to the physical and chemical properties of the tacrolimus ointment, then adding acetonitrile or methanol, standing for layering, freezing, and taking an acetonitrile layer or a methanol layer as a sample solution. Then, the content of tacrolimus isomers was determined according to high performance liquid chromatography. The pretreatment method is simple to operate, so that the tacrolimus isomer is easy to extract from the tacrolimus ointment, the determination of the high performance liquid chromatography is further met, and the research is provided for the quality control of the tacrolimus ointment.

The technical scheme of the invention is realized as follows:

a pretreatment method for tacrolimus ointment isomer determination comprises the following steps: dissolving tacrolimus ointment in n-hexane water bath, adding acetonitrile or methanol, standing for layering, freezing, and taking the acetonitrile layer or the methanol layer as a sample solution.

The volume ratio of the n-hexane to the acetonitrile or methanol is 1: 1, preferably n-hexane with acetonitrile solvent system. In such a solvent system, the isomers of tacrolimus may be dissolved out from the tacrolimus ointment as completely as possible in the acetonitrile layer or the methanol layer, and the n-hexane layer may be completely separated from the acetonitrile layer or the methanol layer. The normal hexane, the acetonitrile and the methanol are all chromatographically pure.

The temperature of the water bath is 60-65 ℃, and 60 ℃ is preferred. If the water bath temperature is too low, the tacrolimus ointment is not sufficiently dissolved, the dissolution of subsequent isomers is influenced, and the detection of the content of the isomers is further influenced; if the temperature of the water bath is too high, the content of the tacrolimus isomer is increased, and the subsequent detection of the content of the isomer in the tacrolimus ointment is inaccurate.

The freezing condition is as follows: the freezing temperature is 18 ℃ below zero to 22 ℃ below zero, and the freezing time is 20min to 25 min. Preferably, the freezing temperature is minus 20 ℃ and the freezing time is 20min, so that the tacrolimus isomer component can be dissolved in the acetonitrile layer as completely as possible, and the matrix component in the tacrolimus ointment can be dissolved in the n-hexane layer.

Preferably, the pretreatment method for tacrolimus ointment isomer determination comprises the following steps: dissolving tacrolimus ointment in n-hexane in 60 ℃ water bath, adding acetonitrile, standing for layering, standing at-20 ℃ for 20min, and taking an acetonitrile layer as a test solution; the volume ratio of the n-hexane to the acetonitrile is 1: 1.

the test solution obtained by the pretreatment method of the invention is placed for 3 hours at room temperature, and has good stability. And (3) injecting the test solution into a liquid chromatograph, and detecting the content of the Tacrolimus isomer according to the chromatographic conditions in Tacrolimus USP 40.

In order to ensure the uniformity of the medicine quality, the control of the content of the tacrolimus isomer needs to be set in the quality standard of tacrolimus ointment, and the sum of the peak areas of the isomer I and the isomer II cannot exceed 8.0 percent of the area of the main peak.

The pretreatment method for measuring the tacrolimus ointment isomer is simple to operate, so that the tacrolimus isomer is easy to extract from the tacrolimus ointment, the measurement of the high performance liquid chromatography is further met, the content of the tacrolimus ointment isomer can be better monitored, and the research is provided for the quality control of the tacrolimus ointment.

Drawings

FIG. 1 is an HPLC chromatogram of an ointment isomer of tacrolimus as a reference formulation;

FIG. 2 is an HPLC chromatogram of an ointment isomer of tacrolimus described in example 1 of the present invention;

FIG. 3 is an HPLC chromatogram of the ointment isomers of tacrolimus described in example 2 of the present invention;

FIG. 4 is an HPLC chromatogram of the ointment isomers of tacrolimus described in example 3 of the present invention;

FIG. 5 is an HPLC chromatogram of tacrolimus ointment isomers when the test solution described in example 1 of the present invention is left for 0 h;

FIG. 6 is an HPLC chromatogram of an ointment isomer of tacrolimus when the test solution described in example 1 of the present invention is left for 1 hour;

FIG. 7 is an HPLC chromatogram of tacrolimus ointment isomers after the test solution described in example 1 of the present invention is left for 2 hours;

FIG. 8 is an HPLC chromatogram of the ointment isomers of tacrolimus when the test solution described in example 1 of the present invention was left for 3 hours.

Detailed Description

The content of the tacrolimus in the tacrolimus ointment is 0.1% w/w, and the matrix is propylene carbonate, white vaseline, mineral oil, white wax and paraffin. Tacrolimus, propylene carbonate, white vaseline, mineral oil, white wax and paraffin used in the self-made tacrolimus ointment can be purchased and obtained from manufacturers published on the official website of CDE. The reference formulation used was tacrolimus ointment manufactured by anstelan pharmaceuticals (china) limited.

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

Example 1

Determination of tacrolimus ointment isomer content:

(1) pretreatment of tacrolimus ointment

Taking about 2g of the product, placing the product in a 10mL glass tube with a plug, adding 4.0mL of n-hexane, dissolving in water bath at 60 ℃, precisely measuring 4.0mL of acetonitrile, placing the product in the same glass tube with the plug, standing for layering, placing the product in a freezing refrigerator at-20 ℃ for 20 minutes, and taking an acetonitrile layer as a sample solution;

(2) determination of tacrolimus ointment isomer content

Precisely measuring 20 μ L of sample solution, injecting into liquid chromatograph, and recording chromatogram, wherein the sum of peak areas of each isomer should not exceed 8.0% of main peak area.

The chromatographic conditions used were: octadecylsilane chemically bonded silica is used as a filling agent; taking 6mmol/L phosphoric acid as a solution A, acetonitrile-tert-butyl methyl ether (81: 19) as a solution B, taking the solution A-solution B (volume ratio of 4: 1) as a mobile phase A, and taking the solution A-solution B (volume ratio of 1: 4) as a mobile phase B; the detection wavelength is 220 nm; the flow rate was 1.5mL per minute; the column temperature is 60 ℃; gradient elution was performed as per table 1.

TABLE 1 gradient elution conditions

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	72	28
30	72	28
			53	15	85
54	72	28
			60	72	28

Example 2

Determination of tacrolimus ointment isomer content:

(1) pretreatment of tacrolimus ointment

Taking about 2g of the product, placing the product in a 10mL glass tube with a plug, adding 4.0mL of n-hexane, dissolving in 65 ℃ water bath, precisely measuring 4.0mL of methanol, placing the product in the same glass tube with the plug, standing for layering, placing the product in a freezing refrigerator at-18 ℃ for 25 minutes, and taking a methanol layer as a sample solution;

(2) determination of tacrolimus ointment isomer content

Example 3

Determination of tacrolimus ointment isomer content:

(1) pretreatment of tacrolimus ointment

Taking about 2g of the product, placing the product in a 10mL glass tube with a plug, adding 4.0mL of n-hexane, dissolving in 65 ℃ water bath, precisely measuring 4.0mL of acetonitrile, placing the product in the same glass tube with the plug, standing for layering, placing the product in a freezing refrigerator at-22 ℃ for 22 minutes, and taking an acetonitrile layer as a sample solution;

(2) determination of tacrolimus ointment isomer content

Stability testing of the test article solutions described in example 1:

respectively injecting samples according to the step (2) after the sample solution prepared in the step (1) in the example 1 is placed at room temperature for 0h, 1h, 2h and 3h, observing the stability of the sample solution, wherein the stability of the sample solution is shown in HPLC spectrograms in fig. 5-8, the observation result is shown in table 2, calculating the sum of the peak area and the main peak area, and the test result shows that the content measurement result of the isomer in the tacrolimus ointment is not changed greatly compared with 0 hour when the sample solution is placed at room temperature for 3 hours.

Table 2 results of stability test of the test solutions described in example 1

Specification (0.1%)	0h Peak area (V)	1h Peak area (V)	2h Peak area (V)	Peak area (V) for 3h
					Isomer II	73833	75201	87449	86659
Main peak	1886158	1920396	2213686	2211969
					Content%	3.91	3.92	3.95	3.92

Claims

1. A pretreatment method for tacrolimus ointment isomer determination is characterized in that: dissolving tacrolimus ointment in n-hexane water bath, adding acetonitrile or methanol, standing for layering, freezing, and taking the acetonitrile layer or the methanol layer as a sample solution.

2. The pretreatment method for tacrolimus ointment isomer measurement according to claim 1, characterized in that: the volume ratio of the n-hexane to the acetonitrile or methanol is 1: 1.

3. the pretreatment method for tacrolimus ointment isomer measurement according to claim 2, characterized in that: the temperature of the water bath is 60-65 ℃.

4. The pretreatment method for tacrolimus ointment isomer measurement according to claim 3, characterized in that: the freezing condition is as follows: the freezing temperature is 18 ℃ below zero to 22 ℃ below zero, and the freezing time is 20min to 25 min.

5. The pretreatment method for tacrolimus ointment isomer measurement according to claim 1, characterized in that: dissolving tacrolimus ointment in n-hexane in 60 ℃ water bath, adding acetonitrile, standing for layering, standing at-20 ℃ for 20min, and taking an acetonitrile layer as a test solution;

the volume ratio of the n-hexane to the acetonitrile is 1: 1.

6. the method for detecting a tacrolimus-related substance in a tacrolimus ointment according to claim 5, wherein the method comprises the following steps: the content of the tacrolimus in the tacrolimus ointment is 0.1% w/w, and the matrix is propylene carbonate, white vaseline, mineral oil, white wax and paraffin.