CN113495101A - Method for determining genotoxic impurity meta-trifluoromethylphenylpropionaldehyde dimer in cinacalcet hydrochloride tablet - Google Patents

Abstract

The invention discloses a method for determining a genotoxic impurity m-trifluoromethylphenylpropionaldehyde dimer in cinacalcet hydrochloride tablets. The determination method comprises the following steps: respectively detecting the contents of the trifluoromethyl phenylpropyl aldehyde dimers by adopting a gas chromatography under the following detection conditions: a detector: gas chromatograph-electron capture detector; the chromatographic column is made of capillary, and the stationary phase is polysiloxane; the carrier gas is nitrogen or argon; eluting at column temperature by adopting a temperature programming mode; in the detection process, the temperature of the detector is 280-300 ℃; the sample introduction volume is 0.5-1.5 mul, the temperature of the sample introduction port is 270-290 ℃, and the flow rate of the purge gas of the detector is 20-50 ml/min in the detection process; the concentration of m-trifluoromethylphenylpropionaldehyde dimer was calculated by peak area according to an external standard method. The method has the advantages of high sensitivity, good specificity, strong durability and high accuracy, and ensures the safety and reliability of the cinacalcet hydrochloride tablet.

Description

Method for determining genotoxic impurity meta-trifluoromethylphenylpropionaldehyde dimer in cinacalcet hydrochloride tablet

Technical Field

The invention relates to a method for determining a genotoxic impurity, namely a trifluoromethyl phenylpropionaldehyde dimer in cinacalcet hydrochloride tablets, and belongs to the technical field of pharmaceutical analysis.

Background

Cinacalcet hydrochloride is a calcium mimetic agent developed by NPS Pharmaceuticals in the united states, is a specific drug for controlling hyperparathyroidism and inhibiting parathyroid hormone PTH, has become a first-line drug in most of domestic areas at present, and is listed in the medical insurance list in China, and more patients are used.

The level of genotoxic impurities is one of the important factors influencing the safety and the effectiveness of the medicine, so that how to accurately determine the content of the trifluoromethylphenylpropionaldehyde dimer among the genotoxic impurities in the cinacalcet hydrochloride becomes a problem to be solved urgently.

The m-trifluoromethyl phenylpropyl aldehyde dimer is a dimer generated by aldol condensation reaction of m-trifluoromethyl phenylpropyl aldehyde (an intermediate 3 in the cinacalcet hydrochloride raw material drug synthesis process) under an alkaline condition, and is a potential mutation-causing impurity with a warning structure.

At present, no relevant literature reports the determination method of m-trifluoromethylphenylpropionaldehyde dimer.

In order to effectively analyze the quality of the medicine and ensure the medication safety, according to the ICH M7 guiding principle, the M-trifluoromethylphenylpropyl aldehyde dimer is required to be controlled, the TTC limit is 15ppm, and the quantitative limit of the method meets the 30% TTC limit, namely 4.5 ppm. Therefore, a detection method with high sensitivity, good specificity, strong durability, convenience and effectiveness needs to be developed.

Disclosure of Invention

The invention aims to provide a method for measuring a genotoxic impurity, namely, a trifluoromethyl phenylpropyl aldehyde dimer in cinacalcet hydrochloride, aiming at the defects in the prior art.

The purpose of the invention can be realized by the following technical scheme:

a method for determining a genotoxic impurity, namely a trifluoromethyl phenylpropionaldehyde dimer in cinacalcet hydrochloride tablets comprises the following steps:

respectively detecting the content of the trifluoromethylphenylpropyl aldehyde dimer by adopting a gas chromatography, wherein the detection conditions of the gas chromatography are as follows:

a detector: gas chromatograph-electron capture detector;

the chromatographic column is made of capillary, and the stationary phase is polysiloxane;

the carrier gas is nitrogen or argon;

eluting at the column temperature by adopting a temperature programming mode: the initial temperature is maintained at 38-40 ℃ for 1-2 min, the temperature is increased to 230 ℃ at the rate of 20 ℃ per minute for 4-6 min, the temperature is increased to 280-300 ℃ at the rate of 48-50 ℃ per minute, and the temperature is maintained for 13-16 min;

in the detection process, the temperature of the detector is 280-300 ℃;

the sample injection volume is 0.5-1.5 mul, the temperature of the sample injection port is 270-290 ℃,

in the detection process, the flow rate of the purge gas of the detector is 20 ml/min-50 ml/min;

calculating the concentration of m-trifluoromethylphenylpropyl aldehyde dimer by peak area according to an external standard method;

wherein the structural formula of the m-trifluoromethyl phenylpropyl aldehyde dimer is as follows:

in a preferred embodiment of the measurement method of the present invention, the packing material of the chromatographic column is 100% dimethylpolysiloxane.

In a preferable embodiment of the measuring method of the present invention, the chromatography column is a capillary; the length of the chromatographic column is 15 m-60 m, and preferably 30 m; the inner diameter of the chromatographic column is 0.25 mm-0.53 mm, and the more preferable diameter is 0.32 mm; the thickness of the liquid film of the column is 0.1 to 5 μm, preferably 1.5 μm

In a preferable embodiment of the measurement method of the present invention, the carrier gas is nitrogen.

In a preferable measurement method of the present invention, the temperature-increasing step is performed by gradient elution as follows: the initial temperature was maintained at 40 ℃ for 1min, 20 ℃ per minute to 230 ℃ for 4min, 50 ℃ per minute to 280 ℃ for 14 min.

As a preferable example of the measuring method of the present invention, the injection volume is 1.0. mu.l.

As a preferable mode of the measuring method of the present invention, the temperature of the injection port is 280 ℃ during the injection.

In a preferable measurement method of the present invention, the temperature of the detector is 290 ℃.

As a preferable mode of the measuring method of the present invention, the flow rate of the purge gas at the detector during the detection is 30 ml/min.

As a further preferable aspect of the measurement method of the present invention, the method comprises the steps of:

(1) weighing a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, and preparing a sensitivity solution;

(2) weighing a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, and preparing a reference solution;

(3) taking a proper amount of cinacalcet hydrochloride tablets, grinding the cinacalcet hydrochloride tablets into fine powder, weighing a proper amount of fine powder, preparing a solution, and filtering the solution by using a 0.22-0.45 mu m microporous membrane to prepare a test solution;

(4) detecting the contents of the trifluoromethylphenylpropyl aldehyde dimer in the sensitivity solution, the reference solution and the test solution respectively by using the gas chromatography method as set forth in any one of claims 1 to 9.

The method for determining the intermediate trifluoromethylphenylpropionaldehyde dimer of cinacalcet hydrochloride provided by the invention has the following beneficial effects:

(1) the invention adopts gas chromatography combined with an electron capture detector and utilizes a capillary chromatographic column taking polysiloxane as a filler to realize the detection of m-trifluoromethyl phenylpropyl aldehyde dimer for the first time.

(2) The detection limit of the method for detecting the intermediate trifluoromethylphenylpropionaldehyde dimer of the cinacalcet hydrochloride tablet is 1.5ppm (10% of the limit), the quantification limit is 3ppm (20% of the limit), and the signal-to-noise ratio is between 19.79 and 37.40 and is more than 10; the peak area RSD between 6 parallel samples is 5.5%, and the sensitivity can meet the requirement of impurity control. The method has good linearity in the concentration range of LOQ-150%. When the method is used for detection, the detected amount of the trifluoromethylphenylpropyl aldehyde dimer in 6 test sample solutions is 2.116 ppm-2.127 ppm, and the RSD is 0.3 percent and less than 10.0 percent. The results show that the method has good precision. The plum is added with 9 parts of standard test solution with the detection limit of 80%, 100% and 120% by the method, the recovery rate of m-trifluoromethylphenylpropyl aldehyde dimer is between 102.9% and 117.9%, the RSD is less than 10.0% and meets the requirement, and the method has the advantages of simple process, low cost, high safety and the like. The method has good accuracy.

The invention comprehensively analyzes the source and the destination of the m-trifluoromethyl phenylpropyl aldehyde dimer according to the synthesis process of the cinacalcet hydrochloride raw material drug and the prescription process of the cinacalcet hydrochloride tablet. A gas chromatography, an electron capture detector, a capillary chromatographic column with polysiloxane as a filler and nitrogen as a carrier gas are adopted for elution, a pretreatment method of a cinacalcet hydrochloride tablet sample is optimized, and a determination method suitable for a trifluoromethyl phenylpropyl aldehyde dimer in the cinacalcet hydrochloride tablet is established. The method successfully realizes the control of the trifluoromethyl phenylpropyl aldehyde dimer among genotoxic impurities in the cinacalcet hydrochloride for the first time, has high sensitivity, good specificity, strong durability and high accuracy, ensures the safety and reliability of the cinacalcet hydrochloride and has practical significance.

Drawings

FIG. 1 is a chromatogram of a sensitive solution in an accuracy test according to the present invention

FIG. 2 is a chromatogram of a control solution in an accuracy test of the present invention

FIG. 3 is a chromatogram of a sample solution in an accuracy test according to the present invention

FIG. 4 is a chromatogram of the solution of the standard sample in the accuracy test of the present invention

Detailed Description

The invention is further illustrated by the following examples. It should be understood that: the embodiments of the present invention are given for illustration only, and not for limitation, and the simple modifications of the present invention based on the technical solutions of the present invention belong to the protection scope of the present invention.

The cinacalcet hydrochloride bulk drug, the cinacalcet hydrochloride tablet, the equipment, the m-trifluoromethyl phenylpropylaldehyde dimer reference substance, the ethyl acetate and the microporous filter membrane used in the specific embodiment of the invention are all known products, the cinacalcet hydrochloride tablet and the m-trifluoromethyl phenylpropylaldehyde dimer are prepared by the company, and the equipment, the ethyl acetate and the microporous filter membrane are obtained by purchasing commercially available products.

The main equipment sources are as follows:

7890 type a gas chromatograph available from agilent;

a muECD type electron capture detector, available from Agilent;

DB-1 column (30 m.times.0.32 mm.times.1.5 μm) from Agilent;

the main sources of the reagent, the test sample and the m-trifluoromethylphenylpropyl aldehyde dimer reference sample are as follows:

the batch number of the cinacalcet hydrochloride tablets is 181202, and the cinacalcet hydrochloride tablets are from Nanjing Hengsheng pharmaceutical Co.Ltd;

the m-trifluoromethyl phenylpropyl aldehyde dimer reference substance has a batch number of 191224, the content of 98.7 percent and is from Nanjing Biotech development Co., Ltd;

ethyl acetate lot 10942668812 from MERCK;

example 1 methodological study of the detection assay method of the invention

The following conditions were used for each test in this example:

the instrument comprises the following steps: 7890A gas chromatograph- μ ECD electron capture detector

A chromatographic column: agilent DB-1 with the specification of 30m multiplied by 0.32mm multiplied by 1.5 mu m

Flow rate of carrier gas: nitrogen, 1.0ml/min

Sample introduction volume: 1.0. mu.l

The split ratio is as follows: 5:1

Sample inlet temperature: 280 deg.C

Detector temperature: 290 deg.C

Flow rate of purge gas: 30ml/min

Column temperature: the initial temperature is maintained at 40 deg.C for 1min, the temperature is raised to 230 deg.C at a rate of 20 deg.C per minute for 4min, and the temperature is raised to 280 deg.C at a rate of 50 deg.C per minute for 14 min.

A detection step:

1. system applicability and system precision test

Blank solution: and taking a proper amount of diluent to a sample injection vial to obtain the product.

Sensitivity solution: taking a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, dissolving by using a diluent, and diluting to prepare a solution containing about 0.012 mu g of m-trifluoromethyl phenylpropyl aldehyde dimer in each 1 ml.

Control solution: taking a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, dissolving by using the diluent, and diluting to prepare a solution containing about 0.06 mu g of m-trifluoromethyl phenylpropyl aldehyde dimer in each 1 ml.

And (3) detection: the blank solution, the sensitivity solution and the control solution were measured accurately and measured at 1.0. mu.l each, and injected into a gas chromatograph, and the results are shown in tables 1 and 2.

TABLE 1 table of applicability test results of the system

TABLE 2 table of system precision test results

The results show that: the blank solution was not interfered with at the retention time of m-trifluoromethylphenylpropanal dimer; the peak signal-to-noise ratio of the middle trifluoro methyl phenylpropyl aldehyde dimer of the sensitivity solution is more than 19.95 and more than 10; the reference solution is continuously injected for 6 times, the retention time RSD of chromatographic peak is 0.02% < 1.0%, and the peak area RSD is 1.6% < 10.0%. The method has good system applicability.

2. Specificity test

Blank adjuvant solution: weighing 560mg of blank auxiliary materials, placing into a 20ml measuring flask, adding 10ml of diluent, performing ultrasonic treatment for 10min, cooling to room temperature, diluting the diluent to scale, shaking up, and filtering with 0.22 μm organic filter membrane.

Test solution: taking a proper amount of cinacalcet hydrochloride tablets after being ground into fine powder, dissolving and diluting the fine cinacalcet hydrochloride tablets by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride in each 1ml of the solution, and filtering the solution by using an organic filter membrane with the diameter of 0.22 mu m to obtain the cinacalcet hydrochloride tablet.

Adding standard 100% limit test solution: taking the cinacalcet hydrochloride tablets and a proper amount of m-trifluoromethyl phenylpropionaldehyde dimer after being ground into fine powder, dissolving and diluting the cinacalcet hydrochloride tablets and the m-trifluoromethyl phenylpropionaldehyde dimer by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride and 0.06 mu g of m-trifluoromethyl phenylpropionaldehyde dimer in each 1ml of the solution, and filtering the solution by using an organic filter membrane with the diameter of 0.22 mu m to obtain the cinacalcet hydrochloride.

Blank solution and control solution were as in item 1.

And (3) detection: precisely measuring blank solution, blank adjuvant solution, reference solution, sample solution, and 100% limit sample solution each 1.0 μ l, and injecting into gas chromatograph. The results of the specificity are shown in Table 3.

TABLE 3 specificity results

The results show that: the blank solution and the blank auxiliary materials have no interference to the sample test, and the retention time of the trifluoromethyl phenylpropyl aldehyde dimer peak in the middle of the reference solution, the test solution and the test solution added with the standard solution corresponds to each other.

3. Detection limit and quantification limit

Detection limiting solution: precisely measuring 2.0ml of reference substance solution, placing into a 20ml measuring flask containing about 10ml of diluent, diluting to scale, and shaking.

Quantitative limiting solution: precisely measuring 4.0ml of reference substance solution, placing into a 20ml measuring flask containing about 10ml of diluent, diluting to scale, and shaking. 6 portions of the limiting solution were diluted in parallel.

And (3) detection: precisely measuring blank solution, detection limit solution and quantification limit solution each 1.0 μ l, and injecting into gas chromatograph, wherein the detection limit result is shown in Table 4 and the quantification limit result is shown in Table 5.

TABLE 4 detection Limit results

TABLE 5 quantitative limit results

The results show that: the detection limit of m-trifluoromethylphenylpropionaldehyde dimer is 1.5ppm (10% of limit), the quantification limit is 3ppm (20% of limit), and the signal-to-noise ratio is between 19.79 and 37.40 and is more than 10; the peak area RSD between 6 parallel samples is 5.5%, and the sensitivity can meet the requirement of impurity control.

4. Linearity and range

The quantitative limiting solution is as under item 3;

linear solution: appropriate amount of m-trifluoromethylphenylpropyl aldehyde dimer is taken respectively, and dissolved and diluted by the diluent to prepare solutions containing about 0.024 μ g, 0.048 μ g, 0.06 μ g, 0.072 μ g and 0.09 μ g of m-trifluoromethylphenylpropyl aldehyde dimer in each 1ml as linear solutions with limit concentration of 40%, 80%, 100%, 120% and 150%, respectively.

And (3) detection: a blank solution, a quantitative limiting solution and a linear solution were precisely measured in an amount of 1.0. mu.l and injected into a gas chromatograph, and the results are shown in Table 6.

TABLE 6 results of the Linear test

The results show that: the method has good linearity in the concentration range of LOQ-150%.

5. Precision of the method

Test solution: taking a proper amount of cinacalcet hydrochloride tablets after being ground into fine powder, dissolving and diluting the fine cinacalcet hydrochloride tablets by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride in each 1ml of the solution, and filtering the solution by using a 0.22 mu m organic filter membrane to obtain 6 parts in parallel.

And (3) detection: the results of the measurement performed according to the detection method of the present invention were calculated, and the results of the method precision are shown in Table 7.

TABLE 7 method precision results

From the above results, it was found that the amount of trifluoromethylphenylpropylaldehyde dimer detected in 6 test solutions was 2.116ppm to 2.127ppm, and RSD was 0.3% < 10.0%. The results show that the method has good precision.

6. Accuracy of

Accuracy solution 1 (spiked 80% limit): taking the cinacalcet hydrochloride tablets and a proper amount of m-trifluoromethyl phenylpropionaldehyde dimer after being ground into fine powder, dissolving and diluting the cinacalcet hydrochloride tablets and the m-trifluoromethyl phenylpropionaldehyde dimer by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride and 0.048 mu g of m-trifluoromethyl phenylpropionaldehyde dimer in each 1ml of the solution, and filtering the solution by using an organic filter membrane with the diameter of 0.22 mu m to obtain the cinacalcet hydrochloride. 3 parts are prepared in parallel.

Accuracy solution 2 (spiked 100% limit): taking the cinacalcet hydrochloride tablets and a proper amount of m-trifluoromethyl phenylpropionaldehyde dimer after being ground into fine powder, dissolving and diluting the cinacalcet hydrochloride tablets and the m-trifluoromethyl phenylpropionaldehyde dimer by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride and 0.06 mu g of m-trifluoromethyl phenylpropionaldehyde dimer in each 1ml of the solution, and filtering the solution by using an organic filter membrane with the diameter of 0.22 mu m to obtain the cinacalcet hydrochloride. 3 parts are prepared in parallel.

Accuracy solution 3 (spiked 120% limit): taking the cinacalcet hydrochloride tablets and a proper amount of m-trifluoromethyl phenylpropionaldehyde dimer after being ground into fine powder, dissolving and diluting the cinacalcet hydrochloride tablets and the m-trifluoromethyl phenylpropionaldehyde dimer by using a diluent to prepare a solution containing about 4mg of cinacalcet hydrochloride and 0.072 mu g of m-trifluoromethyl phenylpropionaldehyde dimer in each 1ml of the solution, and filtering the solution by using a 0.22 mu m organic filter membrane to obtain the cinacalcet hydrochloride tablet. 3 parts are prepared in parallel.

And (3) determination: sampling blank solution, reference solution, test solution and accuracy solution, recording chromatogram, calculating recovery rate and RSD of each limit test solution, and inspecting accuracy, wherein the accuracy result is shown in Table 8.

TABLE 8 accuracy results

FIG. 1 is a chromatogram of a sensitive solution in an accuracy test according to the present invention;

FIG. 2 is a chromatogram of a control solution in an accuracy test of the present invention;

FIG. 3 is a chromatogram of a sample solution in an accuracy test according to the present invention;

FIG. 4 is a chromatogram of a solution of a sample to be tested in an accuracy test according to the present invention;

the results show that: the recovery rate of the m-trifluoromethyl phenylpropyl aldehyde dimer is between 102.9% and 117.9%, and the RSD is less than 5.1% and less than 10.0%, which meets the requirement. The method has good accuracy.

7. Solution stability test solutions were as under item 5.

And (3) detection: the test method of the invention respectively tests the test solution for 0 hour, 12 hours, 1 day and 3 days, and the results are calculated, and the stability results of the test solution are shown in Table 9.

TABLE 9 test article solution stability results

Name (R)	Content (ppm)	AbsoluteDifference (ppm)
			Initial	2.121	N/A
Time interval12h	2.238	0.117
			Time interval1d	2.065	0.056
Time interval3d	2.167	0.046

Note: N/A indicates not applicable.

The results show that: the test solution is measured at room temperature on different days, the content of the trifluoromethylphenylpropyl aldehyde dimer is below 3ppm of the quantitative limit at different time points, and the test solution is stable after being placed at room temperature for 3 days.

8. Durability

The content of the middle trifluoromethylphenylpropyl aldehyde dimer in the test sample is determined by changing various chromatographic conditions, so as to evaluate the bearing degree of the system and the determination result which are not influenced when the determination conditions are changed to a certain extent, wherein various test conditions are shown in a table 10, and the determination result is shown in a table 11.

TABLE 10 durability test conditions

TABLE 11 durability test results

Note: N/A indicates not applicable.

The results show that: after the test conditions are changed, the measurement results of the test solution are not changed and are all less than the limit of quantitation of 3ppm, which indicates that the method has better durability.

In conclusion, the invention firstly creates a method suitable for measuring the genotoxic impurity-m-trifluoromethylphenylpropylaldehyde dimer in the cinacalcet hydrochloride, the method has good specificity and extremely high sensitivity, the detection limit can reach 0.006 mu g/ml, meanwhile, the method has better durability, precision and accuracy, provides a convenient detection method for monitoring the genotoxic impurity-m-trifluoromethylphenylpropylaldehyde dimer in the cinacalcet hydrochloride, and ensures the product quality and the medication safety of patients.

Claims (10)

1. A method for determining a genotoxic impurity, namely a trifluoromethyl phenylpropionaldehyde dimer in cinacalcet hydrochloride tablets is characterized by comprising the following steps of:

respectively detecting the content of the trifluoromethylphenylpropyl aldehyde dimer by adopting a gas chromatography, wherein the detection conditions of the gas chromatography are as follows:

a detector: gas chromatograph-electron capture detector;

the chromatographic column is made of capillary, and the stationary phase is polysiloxane;

the carrier gas is nitrogen or argon;

eluting at the column temperature by adopting a temperature programming mode: the initial temperature is maintained at 38-40 ℃ for 1-2 min, the temperature is increased to 230 ℃ at the rate of 20 ℃ per minute for 4-6 min, the temperature is increased to 280-300 ℃ at the rate of 48-50 ℃ per minute, and the temperature is maintained for 13-16 min;

in the detection process, the temperature of the detector is 280-300 ℃;

the sample injection volume is 0.5-1.5 mul, the temperature of the sample injection port is 270-290 ℃,

in the detection process, the flow rate of the purge gas of the detector is 20 ml/min-50 ml/min;

calculating the concentration of m-trifluoromethylphenylpropyl aldehyde dimer by peak area according to an external standard method;

wherein the structural formula of the m-trifluoromethyl phenylpropyl aldehyde dimer is as follows:

2. the assay of claim 1, wherein the chromatography column packing is 100% dimethylpolysiloxane.

3. The method according to claim 1, wherein the chromatography column is a capillary; the length of the chromatographic column is 15 m-60 m, preferably 30 m; the inner diameter of the chromatographic column is 0.25 mm-0.53 mm, preferably 0.32 mm; the thickness of the liquid film of the chromatographic column is 0.1-5 μm, preferably 1.5 μm.

4. The method according to claim 1, wherein the carrier gas is nitrogen.

5. The method according to claim 1, wherein the temperature-raising program is eluted with a gradient of: the initial temperature was maintained at 40 ℃ for 1min, 20 ℃ per minute to 230 ℃ for 4min, 50 ℃ per minute to 280 ℃ for 14 min.

6. The assay method according to claim 1, wherein the injection volume is 1.0 μ l.

7. The assay method according to claim 1, wherein the temperature of the injection port during the injection is 280 ℃.

8. The method of claim 1, wherein the temperature of the detector is 290 ℃.

9. The assay of claim 1, wherein the flow rate of the detector purge gas during detection is 30 ml/min.

10. The method according to any one of claims 1 to 9, comprising the steps of:

(1) weighing a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, and preparing a sensitivity solution;

(2) weighing a proper amount of m-trifluoromethyl phenylpropyl aldehyde dimer, and preparing a reference solution;

(3) taking a proper amount of cinacalcet hydrochloride tablets, grinding the cinacalcet hydrochloride tablets into fine powder, weighing a proper amount of fine powder, preparing a solution, and filtering the solution by using a 0.22-0.45 mu m microporous membrane to prepare a test solution;

(4) detecting the contents of the trifluoromethylphenylpropyl aldehyde dimer in the sensitivity solution, the reference solution and the test solution respectively by using the gas chromatography method as set forth in any one of claims 1 to 9.

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