CN111595964A

CN111595964A - Fluoxetine hydrochloride raw material and analysis method of related substances in preparation thereof

Info

Publication number: CN111595964A
Application number: CN202010382957.2A
Authority: CN
Inventors: 徐小平; 王晓维; 罗国军
Original assignee: Shanghai Xiuxin Chenpon Pharmaceutical Technology Co ltd
Current assignee: Shanghai Xiuxin Chenpon Pharmaceutical Technology Co ltd
Priority date: 2020-05-08
Filing date: 2020-05-08
Publication date: 2020-08-28

Abstract

The invention discloses a method for analyzing related substances in a fluoxetine hydrochloride raw material and a preparation thereof, wherein the impurities in the fluoxetine hydrochloride raw material and the preparation thereof comprise: n-methyl-3-hydroxy-3-phenylpropylamine (impurity I), 4-trifluoromethylphenol (impurity II), N-methyl-3-phenylpropylamine (impurity III), and N-methyl-3- (3-trifluoromethylphenoxy) propylamine (impurity IV), characterized by comprising the steps of: s1, preparing 4 impurity reference substance solutions, separation degree solutions and test substance solutions; and S2, performing high performance liquid chromatography measurement on the 4 impurity reference substance solutions, the separation degree solution and the test sample solution. The invention adopts the high performance liquid chromatography technology, has high detection precision, good specificity and durability and simple and convenient operation.

Description

Fluoxetine hydrochloride raw material and analysis method of related substances in preparation thereof

Technical Field

The invention relates to the field of drug analysis and detection, in particular to a method for analyzing fluoxetine hydrochloride raw material and related substances in a preparation thereof.

Background

Fluoxetine hydrochloride is an antidepressant, belongs to a 5-HT reuptake inhibitor, and is listed as an antidepressant 'Wuduodatura flower', and can also be used for treating anxiety accompanied by depression and treating obsessive compulsive disorder and overeating (bulimia nervosa). Fluoxetine was discovered by the li-shi company in 1972 and was put to medical use in 1986. The medicine belongs to basic medicines of the world health organization and is one of the most important medicines required by a basic health system.

When the fluoxetine hydrochloride bulk drug is synthesized and prepared, impurities such as N-methyl-3-hydroxy-3-phenylpropylamine (impurity I), 4-trifluoromethylphenol (impurity II), N-methyl-3-phenylpropylamine (impurity III), N-methyl-3- (3-trifluoromethylphenoxy) propylamine (impurity IV) and the like can be generated, particularly the N-methyl-3-hydroxy-3-phenylpropylamine (impurity I) has larger polarity and is easy to flow out together with a solvent peak, and the N-methyl-3- (3-trifluoromethyl phenoxy) propylamine has a structure very similar to that of fluoxetine, if they are not separated well, the impurities cannot be detected or accurately quantified, thereby affecting the quality of the product.

In the traditional regulation methods, some impurities cannot be effectively separated (for example, in a related substance analysis method of a fluoxetine hydrochloride capsule in the United states Pharmacopeia, British Pharmacopeia and European Pharmacopeia, a peak-to-valley ratio is used to replace the separation condition of a main peak and a separation degree is regulated, the base line separation of the impurity IV and the main peak cannot be ensured, although the Chinese Pharmacopeia regulates that the separation degree of the two is not less than 1.5, in an experiment, only a new column can meet the requirement, the separation degree cannot meet the requirement after the column is used for a period of time, and in the limit level (0.15%) of the impurity IV and below, the method in each Pharmacopeia cannot meet the separation degree requirement), some impurities cannot be separated, but the impurities are not retained, are easily mixed with a solvent peak, and the separation degree of some impurities is not enough (for example, the related substance analysis method of the, the impurity IV and the fluoxetine peak can not realize baseline separation, the impurity I and a solvent peak flow out together in the fluoxetine hydrochloride and preparation related substance analysis method of Chinese pharmacopoeia), so that the quantification of the impurity is not accurate enough, and a large amount of tetrahydrofuran is added in a mobile phase of the method, so that a chromatographic analysis system is easily damaged, the health of workers is harmed, and the environment is polluted.

Disclosure of Invention

The invention aims to provide a method for analyzing fluoxetine hydrochloride raw material and related substances in a preparation thereof aiming at the defects in the prior art, and the method adopts a high performance liquid chromatography technology, has high detection precision, good specificity and durability and simple and convenient operation; in the method, all impurity components are reserved to a certain extent, the problem that the impurity I and a solvent peak flow out together in a regulation method is solved, the separation degrees among the main component, 4 impurities and 4 impurities are greater than 1.5, the sensitivity is high, and the qualitative and quantitative analysis can be well carried out, so that the quality of the fluoxetine hydrochloride bulk drug and the preparation can be effectively controlled.

In order to achieve the purpose, the invention adopts the technical scheme that:

provided is a method for analyzing related substances in a fluoxetine hydrochloride raw material and a preparation thereof, wherein impurities contained in the fluoxetine hydrochloride raw material and the preparation thereof comprise: n-methyl-3-hydroxy-3-phenylpropylamine (impurity I), 4-trifluoromethylphenol (impurity II), N-methyl-3-phenylpropylamine (impurity III), and N-methyl-3- (3-trifluoromethylphenoxy) propylamine (impurity IV), comprising the steps of:

s1, respectively dissolving N-methyl-3-hydroxy-3-phenylpropylamine reference substances, 4-trifluoromethylphenol reference substances, N-methyl-3-phenylpropylamine reference substances and N-methyl-3- (3-trifluoromethylphenoxy) propylamine reference substances to the concentration of 0.1-5 mu g/ml, and preparing 4 impurity reference substance solutions;

weighing a pure fluoxetine hydrochloride product, and respectively adding the pure fluoxetine hydrochloride product into the 4 impurity reference substance solutions to prepare separation degree solutions;

precisely weighing fluoxetine hydrochloride raw material medicine or a preparation thereof, and dissolving until the concentration of the fluoxetine hydrochloride is 0.1-5.0mg/ml to obtain a test solution;

s2, carrying out high performance liquid chromatography measurement on the 4 impurity reference substance solution, the separation degree solution and the test sample solution;

chromatographic conditions are as follows: the mobile phase A is water containing tetrabutyl ammonium hydroxide, sodium dihydrogen phosphate, n-butyl sodium sulfonate or sodium dodecyl sulfonate, and the mobile phase B is methanol; a C18 chromatography column or a pentafluorophenyl chromatography column; the column temperature is 20-50 ℃; the flow rate of the mobile phase is 0.5-1.5 ml/min; the sample amount is 1-100 mu L; gradient elution.

Preferably, the concentration of tetrabutylammonium hydroxide, sodium dihydrogen phosphate, sodium n-butyl sulfonate or sodium dodecyl sulfonate in the mobile phase A is 10-50 mmol/L.

Preferably, the mobile phase A is tetrabutylammonium hydroxide aqueous solution with the concentration of 12-45mmol/L, sodium dodecyl sulfate aqueous solution with the pH adjusted to 2.0-3.5 or 20-50mmol/L, and the pH adjusted to 2.0-3.5.

Preferably, in S3, the elution gradient is: and the volume fraction of the mobile phase A is reduced from 80% to 20% and the volume fraction of the methanol is increased from 20% to 80% in 0-30 min.

Preferably, in S3, the chromatographic conditions comprise: the column temperature is 30-40 ℃; the flow rate of the mobile phase is 1.0 ml/min; the sample size was 10. mu.L or 20. mu.L.

Preferably, in S1, the concentration of the test solution is 2.0mg/ml, and the concentration of the impurity control solution is 2 μ g/ml.

Preferably, in S2, the solvent for dissolving the sample solution is methanol or a mixture of methanol and water.

Preferably, in the mixed solution of methanol and water, the volume ratio of methanol to water is 80:20-20: 80.

Preferably, in S1, the solvent for preparing the 4 impurity control solutions is methanol or a mixture of methanol and water.

Preferably, in the mixed solution of methanol and water, the volume ratio of methanol to water is 20:80-50: 50.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention adopts the high performance liquid chromatography technology to detect the related substances of the fluoxetine hydrochloride, and adopts a relatively cheap and relatively low-toxicity reagent to effectively retain 4 related substances on a chromatographic column by screening chromatographic conditions, thereby avoiding the interference of a solvent peak, solving the problem that the impurity I and the solvent peak co-flow out in the methods of Chinese pharmacopoeia and American pharmacopoeia, and ensuring that the high-polarity impurities can be accurately identified and quantitatively determined. Meanwhile, the problem of insufficient separation degree of the impurity IV and the fluoxetine peak in pharmacopoeia methods of various countries is solved, and particularly, when the separation degree of the impurity IV is lower than the limit level of the impurity IV, the separation degree of the impurity IV and the main peak can reach more than 1.5, so that the quantitative accuracy of the impurity IV is improved. And under the limit concentration level of the impurities, each impurity can be well separated from the main peak and each impurity, so that each impurity can be effectively detected and accurately quantified, and the quality control of the fluoxetine hydrochloride bulk drug and the preparation can be more conveniently and effectively carried out.

Drawings

FIG. 1 is a chromatogram of fluoxetine hydrochloride and 4 impurities in a resolution solution;

FIG. 2 is a chromatogram of 4 impurities in a limiting solution;

FIG. 3 is a chromatogram of 4 impurities in a detection limit solution;

FIG. 4 is a chromatogram of a sample solution to be tested;

FIG. 5 is a chromatogram of a sample spiking solution;

FIG. 6 is a linear relationship chart of impurity I (0.3-4.0. mu.g/ml);

FIG. 7 is a linear relationship chart of impurity II (0.5-4.0. mu.g/ml);

FIG. 8 is a linear relationship chart of impurity III (0.5-4.0. mu.g/ml);

FIG. 9 is a linear relationship chart of impurity IV (0.5-4.0. mu.g/ml).

Detailed Description

The invention is further described with reference to the following drawings and specific examples, which are not intended to be limiting.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

1. Instrument for measuring the position of a moving object

Thermo U3000 high performance liquid chromatograph, C18(4.6 x 250mm, 5 μm) column, C18(4.6 x 150mm, 3.5 μm) column, pentafluorophenyl column (4.6 x 150mm, 3 μm) column, pentafluorophenyl column (2.1 x 100mm, 1.7 μm) column.

2. Reagent

N-methyl-3-hydroxy-3-phenylpropylamine reference substance, 4-trifluoromethylphenol reference substance, N-methyl-3-phenylpropylamine reference substance, N-methyl-3- (3-trifluoromethylphenoxy) propylamine reference substance, fluoxetine hydrochloride pure product, fluoxetine hydrochloride bulk drug, methanol, sodium dodecyl sulfonate, tetrabutylammonium hydroxide and phosphoric acid.

3. Examination of Experimental conditions

3.1 examination of pretreatment method

Fluoxetine hydrochloride is easily soluble in methanol and ethanol and slightly soluble in water, and N-methyl-3-hydroxy-3-phenylpropylamine (impurity I), 4-trifluoromethylphenol (impurity II), N-methyl-3-phenylpropylamine (impurity III) and N-methyl-3- (3-trifluoromethylphenoxy) propylamine (impurity IV) contain the same parent nucleus structure, so in the invention, the solvent is selected from the mixed solution of methanol and water or methanol. Preferably, the solvent is selected from a mixed solution of methanol and water, and more preferably, the volume ratio of the methanol to the water is 20:80-50: 50.

When the test solution is prepared, if the concentration of fluoxetine hydrochloride is 0.4mg/ml, the response of each impurity is lower than the limit of quantitation after the impurity control with low concentration (e.g. 0.1%) is added. If the concentration of fluoxetine hydrochloride is 50mg/ml, the sample consumption is large, and the sample is easily overloaded on a chromatographic column. Therefore, in the present invention, it is preferable that the concentration of fluoxetine hydrochloride in the sample solution is 1.0 to 5.0 mg/ml.

3.2 examination of chromatographic conditions

Respectively dissolving N-methyl-3-hydroxy-3-phenylpropylamine reference substances, 4-trifluoromethylphenol reference substances, N-methyl-3-phenylpropylamine reference substances and N-methyl-3- (3-trifluoromethylphenoxy) propylamine reference substances, adding a mixed solution of methanol and water in a volume ratio of 20:80, and diluting until the concentration of an impurity I reference substance is 2 mu g/ml, the concentration of an impurity II reference substance is 2 mu g/ml, the concentration of an impurity III reference substance is 2 mu g/ml and the concentration of an impurity IV reference substance is 2 mu g/ml to prepare 4 impurity reference substance solutions.

And precisely weighing a pure fluoxetine hydrochloride product, and respectively adding the pure fluoxetine hydrochloride product into the 4 impurity reference substance solutions to prepare separation degree solutions.

Precisely weighing fluoxetine hydrochloride raw material medicine or a preparation thereof, adding a mixed solution of methanol and water in a volume ratio of 20:80, and dissolving until the concentration of fluoxetine hydrochloride is 2mg/ml to obtain a test solution.

According to the chromatographic conditions shown in Table 1, sample introduction detection is carried out, the operation is carried out for 45min, a chromatogram is recorded, and the separation degree and the observation response value between the main component and each impurity are examined:

TABLE 1

Wherein, the elution conditions of example 1 are as follows: and (3) 0min-30min, the volume fraction of the mobile phase A is reduced from 80% to 20%, and the volume fraction of the methanol is increased from 20% to 80%.

The elution conditions for example 2 were: the volume fraction of the mobile phase A is reduced from 80% to 20% and the volume fraction of the methanol is increased from 20% to 80% within 0-25 min.

The elution conditions for example 3 were: and (3) 0min-30min, the volume fraction of the mobile phase A is reduced from 80% to 20%, and the volume fraction of the methanol is increased from 20% to 80%.

The elution conditions for example 4 were: and (3) 0min-15min, the volume fraction of the mobile phase A is reduced from 80% to 20%, and the volume fraction of the methanol is increased from 20% to 80%.

The results of examples 1-4 are as follows:

in example 1, after all the main components and the impurities are peaked within 30 minutes, the main components and the impurity baselines are separated, the chromatogram baselines are stable, and the peak response value is high.

In example 2, the peaks of the main component and the impurities are all completed within 25 minutes, the peak types of the components are good, the base lines of the main component and the impurities are separated, and the base lines of the chromatogram are stable.

In example 3, the peaks of the main component and impurities were all completed within 30 minutes, the peak patterns of the components were good, and the degrees of separation between impurities were good.

In example 4, the main component and the impurities all showed peaks within 15 minutes, and the separation degree between the impurities was good.

4. Determination of the composition

Accurately weighing an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving and diluting until the concentration of the impurity I reference substance is 2 mug/ml, the concentration of the impurity II reference substance is 2 mug/ml, the concentration of the impurity III reference substance is 2 mug/ml and the concentration of the impurity III reference substance is 2 mug/ml, thus obtaining 4 reference substance solutions.

Precisely weighing a fluoxetine hydrochloride pure product, an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving, and diluting until the concentration of the impurity I reference substance is 2 mug/ml, the concentration of the impurity II reference substance is 2 mug/ml, the concentration of the impurity III reference substance is 2 mug/ml, the concentration of the impurity IV reference substance is 2 mug/ml and the concentration of the fluoxetine hydrochloride is 2mg/ml, thus obtaining a separation solution.

Precisely weighing fluoxetine hydrochloride raw material medicine, adding a mixed solution of methanol and water in a volume ratio of 20:80, dissolving, and diluting until the fluoxetine hydrochloride concentration is 2mg/ml to obtain a test solution.

According to the chromatographic conditions of example 1 in Table 1, the 4 kinds of reference solution, separation degree solution, test solution and blank solution are subjected to sample injection detection, the sample injection detection is carried out for 45min, the chromatogram is recorded, and the retention time of each solution is shown in Table 2:

TABLE 2

The results showed that the impurities all peaked within 30 minutes, the separation degree of each component was good, the peak pattern of each component was good, and the separation degree between impurities reached 1.5 or more. The position of the target impurity peak in the blank solution is not interfered, and peaks near the target peak in the sample solution and the resolution solution are not interfered; as can be seen from the data in Table 2, the retention times of the target peaks of the impurities in the test solution, the separation solution and the impurity control solutions were all consistent. As can be seen from fig. 1, the chromatogram baseline of the resolution solution was stable, the sensitivity was high, and four impurities in the resolution solution were confirmed.

5. Determination of detection limits and quantification limits

And continuously diluting the concentration of each impurity reference substance, carrying out sample introduction test on the resolution solution with each concentration to obtain a chromatogram, and determining the detection limit and the quantification limit of 4 impurities according to the chromatogram of each concentration.

Accurately weighing an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving and diluting until the concentration of the impurity I reference substance is 0.2 mu g/ml, the concentration of the impurity II reference substance is 0.3 mu g/ml, the concentration of the impurity III reference substance is 0.3 mu g/ml and the concentration of the impurity IV reference substance is 0.3 mu g/ml, thus obtaining the detection limit solution.

According to the chromatographic conditions of example 1 in Table 1, the detection limiting solution is injected and detected, the operation is carried out for 45min, and the chromatogram is recorded, as shown in figure 3.

Calculating the signal-to-noise ratio of the chromatogram of FIG. 3, requiring a signal-to-noise ratio greater than 3, and determining that 0.2. mu.g/ml can be used as the detection limit of impurity I, and 0.3. mu.g/ml can be used as the detection limit of 3 other impurities.

Accurately weighing an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving and diluting until the concentration of the impurity I reference substance is 0.3 mu g/ml, the concentration of the impurity II reference substance is 0.5 mu g/ml, the concentration of the impurity III reference substance is 0.5 mu g/ml and the concentration of the impurity IV reference substance is 0.5 mu g/ml, thus obtaining a quantitative limit solution.

According to the chromatographic conditions of example 1 in Table 1, the above quantitative limiting solution is subjected to sample injection detection, run for 45min, and record chromatogram, as shown in FIG. 2.

Calculating the signal-to-noise ratio of the chromatogram of FIG. 2, requiring a signal-to-noise ratio greater than 10, and determining that 0.3. mu.g/ml can be used as the limit of quantitation for impurity I, and 0.5. mu.g/ml can be used as the limit of quantitation for 3 other impurities.

6. Precision survey

(1) Accurately weighing an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving and diluting until the concentration of the impurity I reference substance is 0.3 mu g/ml, the concentration of the impurity II reference substance is 0.5 mu g/ml, the concentration of the impurity III reference substance is 0.5 mu g/ml and the concentration of the impurity IV reference substance is 0.5 mu g/ml, thus obtaining a quantitative limit solution.

According to the chromatographic conditions of example 1 in Table 1, the quantitative limiting solution is continuously injected into 6 needles, the operation is carried out for 45 minutes each time, the chromatogram is recorded, the separation degree among the impurities and the reproducibility of the peak area of each impurity are examined, and the result is shown in Table 3;

TABLE 3

As shown in fig. 2, the baseline of the spectrogram is stable, each component generates a peak within 30 minutes, the separation degree among impurities is good, and the repeatability of the peak area of each impurity is good.

(2) Precisely weighing a fluoxetine hydrochloride raw material drug, an impurity I reference substance, an impurity II reference substance, an impurity III reference substance and an impurity IV reference substance, adding a mixed solution of methanol and water with a volume ratio of 20:80, dissolving, and diluting until the concentration of the impurity I reference substance is 2 mug/ml, the concentration of the impurity II reference substance is 2 mug/ml, the concentration of the impurity III reference substance is 2 mug/ml, the concentration of the impurity IV reference substance is 2 mug/ml and the concentration of fluoxetine hydrochloride is 2mg/ml, thus obtaining a sample added standard solution.

According to the chromatographic conditions of example 1 in Table 1, adding a standard solution into a sample, continuously injecting the sample into the sample for 6 needles, operating for 45 minutes each time, recording a chromatogram, and inspecting the separation degree among various impurities and the peak area reproducibility of each impurity, wherein the results are shown in Table 4;

TABLE 4

Serial number	Peak area of impurity I	Peak area of impurity II	Peak area of impurity III	Area of impurity IV peak
					1	19.039	21.299	28.456	37.881
2	19.386	21.904	28.043	38.276
					3	20.211	21.314	27.332	38.521
4	19.509	21.282	26.609	38.726
					5	19.051	21.844	28.007	40.552
6	20.794	22.372	27.561	37.725
					Mean value of	19.665	21.669	27.500	37.614
RSD％	3.6	2.1	2.2	2.6

The result shows that the base line of the spectrogram is stable, all components generate peaks within 30 minutes, the separation degree among impurities is good, and the repeatability of the peak area of each impurity is good.

7. Method for detecting fluoxetine hydrochloride related substances

Adding a mixed solution of methanol and water with the volume ratio of 20:80 into the impurity I reference substance, the impurity II reference substance, the impurity III reference substance and the impurity IV reference substance, and diluting impurity reference substance solutions with different concentration gradients after dissolving.

And adding a mixed solution of methanol and water in a volume ratio of 20:80 into the fluoxetine hydrochloride to be detected, dissolving, and diluting until the concentration of the fluoxetine hydrochloride to be detected is 2mg/ml to obtain a sample solution to be detected.

According to the chromatographic conditions of example 1 in Table 1, impurity reference solutions and sample solutions to be detected with different concentrations are subjected to sample injection detection, the operation is carried out for 45 minutes, and a chromatogram is recorded.

The chromatograms of the sample solutions to be detected are respectively shown in fig. 4, and as can be seen from fig. 4, the impurities i, ii, iii and iv in the fluoxetine hydrochloride bulk drug to be detected are all less than the detection limit of 0.3 μ g/ml.

And respectively adding 0.1% of impurities I, II, III and IV into the sample solution to be detected to obtain a sample labeling solution.

According to the chromatographic conditions of example 1 in Table 1, the sample adding standard solution is subjected to sample injection detection, the operation is carried out for 45 minutes, and chromatograms of impurities I, II, III and IV in the sample to be detected and added standard are recorded, as shown in figure 5.

Recording the corresponding impurity peak areas of the impurities I, II, III and IV reference substances with different concentration gradients according to the chromatograms of the reference substances with the impurities I, II, III and IV with different concentration gradients, and making a standard working curve according to the concentrations of the impurities and the peak areas of the corresponding impurities, as shown in figures 6-9.

While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

1. A method for analyzing related substances in a fluoxetine hydrochloride raw material and a preparation thereof, wherein impurities contained in the fluoxetine hydrochloride raw material and the preparation thereof comprise: n-methyl-3-hydroxy-3-phenylpropylamine (impurity I), 4-trifluoromethylphenol (impurity II), N-methyl-3-phenylpropylamine (impurity III), and N-methyl-3- (3-trifluoromethylphenoxy) propylamine (impurity IV), characterized by comprising the steps of:

2. The method for analyzing fluoxetine hydrochloride raw material and related substances in its preparation according to claim 1, wherein the concentration of tetrabutylammonium hydroxide, sodium dihydrogen phosphate, sodium n-butyl sulfonate or sodium dodecyl sulfonate in said mobile phase A is 10-50 mmol/L.

3. The method of claim 2, wherein the mobile phase A is tetrabutylammonium hydroxide solution with a concentration of 12-45mmol/L, sodium dodecyl sulfate solution with a pH of 2.0-3.5 or 20-50mmol/L, and pH of 2.0-3.5.

4. The method of analyzing fluoxetine hydrochloride starting material and related substances in its formulation according to claim 1, wherein in S3, said elution gradient is: and the volume fraction of the mobile phase A is reduced from 80% to 20% and the volume fraction of the methanol is increased from 20% to 80% in 0-30 min.

5. The method of analyzing fluoxetine hydrochloride starting material and related substances in its formulation according to claim 1, wherein in S3, said chromatographic conditions comprise: the column temperature is 30-40 ℃; the flow rate of the mobile phase is 1.0 ml/min; the sample size was 10. mu.L or 20. mu.L.

6. The method of claim 1, wherein in S1, the concentration of the test sample solution is 2.0mg/ml and the concentration of the impurity control solution is 2 μ g/ml.

7. The method of claim 1, wherein the solvent for dissolving the sample solution in S2 is methanol or a mixture of methanol and water.

8. The method of analyzing a fluoxetine hydrochloride starting material and related substances in its formulation according to claim 7, wherein the volume ratio of methanol to water in the mixed solution of methanol and water is 80:20-20: 80.

9. The method of claim 1, wherein the solvent used to prepare the 4 reference solutions of impurities is methanol or a mixture of methanol and water in S1.

10. The method of analyzing a fluoxetine hydrochloride starting material and related substances in its formulation according to claim 9, wherein the volume ratio of methanol to water in the mixed solution of methanol and water is 20:80 to 50: 50.