阅读说明：本技术 一种替诺福韦艾拉酚胺中溴乙烷的测定方法 (Method for determining ethyl bromide in tenofovir alafenamide ) 是由 沙薇 吴金平 李晨希 于 2018-09-12 设计创作，主要内容包括：本发明提供了一种替诺福韦艾拉酚胺中溴乙烷的测定方法,采用溶液直接进样法进行测定,将替诺福韦艾拉酚胺制成一定的溶液,用6%氰丙基苯基-94%二甲基聚硅氧烷为固定液的毛细管柱对微量的溴乙烷进行测定,用外标法计算出替诺福韦艾拉酚胺中溴乙烷含量。本发明方法只需用合适的溶剂将替诺福韦艾拉酚胺配制成溶液,前处理简单,易操作,且能更好测定替诺福韦艾拉酚胺中溴乙烷含量。(The invention provides a method for measuring ethyl bromide in tenofovir alafenamide, which adopts a solution direct injection method to measure, prepares tenofovir alafenamide into a certain solution, measures trace amount of ethyl bromide by using a capillary column taking 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid, and calculates the content of ethyl bromide in tenofovir alafenamide by an external standard method. The method only needs to use a proper solvent to prepare the tenofovir alafenamide into a solution, has simple pretreatment and easy operation, and can better determine the content of ethyl bromide in the tenofovir alafenamide.)

1. A method for determining ethyl bromide in tenofovir alafenamide is characterized by being carried out in the following mode:

chromatographic conditions are as follows: a capillary column using 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is heated to 220 ℃ at the initial temperature of 35-45 ℃ at the rate of 30 ℃/min per minute and is maintained for 5-8 minutes; the temperature of the detector is 250 ℃; the temperature of a sample inlet is 150-220 ℃, the split ratio is 2: 1-5: 1, the flow rate of a column is 2-4 ml/min, and the preparation of a sample solution: preparing a tenofovir alafenamide solution by using an organic solvent, wherein the selected solvent comprises methanol, ethanol, acetonitrile, acetone and dimethylformamide as a solvent, and measuring: and (3) directly injecting the solution into a gas chromatograph, recording a chromatogram, and analyzing, wherein the separation degree is more than 1.5, and the number of theoretical plates is not less than 5000.

2. The determination of the bromoethane content of tenofovir alafenamide by gas chromatography according to claim 1, characterized in that the carrier gas flow rate is 2 ml/min.

3. The determination of the bromoethane content of tenofovir alafenamide by gas chromatography according to claim 1, characterized by an onset temperature of 35 ℃.

4. The determination of the bromoethane content of tenofovir alafenamide by gas chromatography according to claim 1, characterized in that the sample solution is formulated to contain tenofovir alafenamide 0.1 g/ml.

5. The determination of the bromoethane content of tenofovir alafenamide by gas chromatography according to claim 1, characterized by a split stream of 3: 1.

6. The determination of the bromoethane content of tenofovir alafenamide by gas chromatography according to claim 1, characterized by equilibration at 35 ℃ for 5 minutes.

Technical Field

The invention belongs to the field of pharmaceutical analysis, and particularly designs a method for determining ethyl bromide in tenofovir alafenamide.

Background

The synthesis route of tenofovir alafenamide may generate a bromoethane by-product in the reaction process. 27/10/2017, published by international cancer research institute of world health organization, "precancerous list preliminary reference", bromoethane belongs to 3 kinds of carcinogenic substances. A plurality of literature reports exist in China for measuring the bromoethane, but the measurement of the bromoethane in tenofovir alafenamide is rarely reported in China at present, and the measurement is occasionally reported, the method is complex, and the sensitivity and the accuracy are not high.

Bromoethane is a colorless oily liquid with an ether-like odor and a burning odor. It turns yellow gradually when exposed to air or light, is easy to volatilize, can be mixed and dissolved with ethanol, diethyl ether, trichloromethane and most organic solvents, and has a boiling point of 38.4 ℃.

Disclosure of Invention

[1] The invention provides a method for determining ethyl bromide in tenofovir alafenamide, which has the advantages of simple operation, high sensitivity, high accuracy and the like.

[2] The specific technical scheme of the invention is as follows:

(1) the tenofovir alafenamide is formulated into a solution with a suitable solvent.

(2) Directly injecting sample, measuring by capillary gas chromatography, and calculating bromoethane content by external standard method, wherein the gas chromatography conditions are as follows: a capillary column using 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is heated to 220 ℃ at the initial temperature of 35-45 ℃ at the rate of 30 ℃/min per minute and is maintained for 5-8 minutes; the temperature of the detector is 250 ℃; the temperature of the sample inlet is 150-220 ℃, the split ratio is 2: 1-5: 1, and the flow rate of the column is 2-4 ml/min.

[3] Detailed description of the preferred embodiments

The following examples are provided to illustrate specific steps of the present invention, but are not intended to limit the present invention. The invention is described in further detail below with reference to specific examples and data, it being understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

(1) Selection of solvents

The tenofovir alafenamide is dissolved in organic solvents such as methanol, acetonitrile, acetone and dimethylformamide. Because the limit of ethyl bromide is lower, tenofovir alafenamide needs to be dissolved as much as possible, and methanol, acetonitrile, acetone and dimethylformamide can be selected as solvents according to the solubility information of the tenofovir alafenamide. Dimethylformamide has a boiling point of 152.8 ℃ and the difference between the boiling points of dimethylformamide and bromoethane is large, and dimethylformamide is preferably used as a solvent.

(2) Selection of chromatographic conditions

A capillary column using 6% cyanopropylphenyl-94% dimethylpolysiloxane as a stationary liquid is heated to 220 ℃ at the initial temperature of 35-50 ℃ at the rate of 30 ℃/min per minute and is maintained for 5-8 minutes; the temperature of the detector is 250 ℃; the temperature of a sample inlet is 150-220 ℃, the split ratio is 2: 1-5: 1, the flow rate of the column is 2-4 ml/min, and the flow rate of the carrier gas is 1.5-3 ml/min.

Influence of the onset temperature: the initial temperature is preferably 35 ℃ in consideration of the influence of the peak height, the degree of separation, the peak pattern and the number of theoretical plates due to the change in the column temperature.

Influence of Carrier gas flow: in order to obtain better separation determination effect, the method preferably has carrier gas flow of 2ml/min

Influence of split ratio: in order to better achieve the separation effect of each peak and ensure the detection sensitivity, the split ratio is preferably 3: 1.

Influence of equilibration time: the length of the equilibration time affects the diffusion rate of the molecules of the component being measured from the sample matrix to the gas phase. The samples are balanced for 5-8 minutes at 35-50 ℃, and the sample injection is inspected, so that the temperature is overhigh, the balance time is overlong, and the separation degree is good, therefore, the balance time is preferably 5 minutes at 35 ℃.

(3) The method of the invention is used for determining the content of ethyl bromide in tenofovir alafenamide

Standard curve test: an appropriate amount of bromoethane was weighed out precisely, dissolved in dimethylformamide and diluted to a solution containing about 10.62. mu.g per 1ml, and shaken up. Precisely measuring 0.2 ml, 0.5ml, 1.0 ml, 4.0 ml, 6.0 ml and 8.0ml of bromoethane stock solution respectively, placing the bromoethane stock solution into a 20ml measuring flask respectively, diluting the bromoethane stock solution to a scale by using dimethylformamide, and shaking up the bromoethane stock solution to obtain linear relation test solution; and precisely measuring 1 mu l of each linear relation test solution respectively, injecting the linear relation test solution into a gas chromatograph, and recording the chromatogram. Linear regression was performed with the concentration of bromoethane as abscissa x and the peak area of bromoethane as ordinate y to find the linear equation y =0.0329x +0.0053, R²=0.9954, the linear relationship was good, and the results are shown in table 1 and fig. 1.

TABLE 1 Linear relationship test

Precision analysis: dissolving and diluting dimethylformamide to prepare a reference substance solution containing 0.531 mug per 1ml, precisely measuring 1 mug, injecting into a gas chromatograph, recording a chromatogram, continuously injecting samples for 6 times, wherein the RSD of the peak area of the reference substance is 7.65%, the RSD of the retention time is 0.12%, the precision of the instrument is good, the separation degree of bromoethane in each reference substance solution is more than 1.5, the separation is complete, the number of theoretical plates is more than 10000, and the peak pattern is good. The results are shown in Table 2.

TABLE 2 precision test

Sample introduction sequence number	1	2	3	4	5	6	Average	RSD%
									Peak area	0.024	0.022	0.027	0.026	0.023	0.024	0.024	7.65
Degree of separation	2.47	2.3	2.31	2.49	2.27	2.17	/	/
									Number of theoretical plates	32854	26797	22604	26572	27030	25413	/	/

Recovery rate test: a spiking recovery test was used. : 1g of sample is taken and precisely weighed, the sample is placed into a 10ml measuring flask, 0.5ml of bromoethane stock solution is added, the solution is dissolved and diluted to the scale by dimethylformamide, and the mixture is shaken up to 6 parts in total. The measured amount of the bromoethane is calculated by the peak area according to an external standard method, the calculated recovery rate is 97.95-112.65%, the RSD is 5.30%, and the result is shown in Table 3, which shows that the method has high accuracy in measuring the bromoethane.

TABLE 3 recovery test