CN111487334A - Detection method of low-limit canagliflozin impurities - Google Patents

Abstract

The invention discloses a method for detecting a low-limit canagliflozin impurity, wherein the low-limit impurity is a genotoxic impurity F. The method adopts a high performance liquid chromatography-mass spectrometry combined analysis method and uses [ M + NH ]₄ ⁺]Ammonium acetate is added as a parent ion to a diluent, and a volatile ammonium acetate solution and acetonitrile are used as mobile phases, so that the genotoxic impurity F can be separated and detected more efficiently. The method has the advantages of strong specificity, high sensitivity, accurate result and simple operation, and has important significance on quality control and medication safety of canagliflozin.

Description

A kind of detection method of low-limit canagliflozin impurities

technical field

The invention belongs to the technical field of medical analysis, and in particular relates to a detection method for low-limit canagliflozin impurities.

Background technique

Canagliflozin, the English name of Canagliflozin, is a potent, reversible and highly selective inhibitor of human renal sodium-glucose co-transporter (SGLT2) originally developed by Johnson & Johnson. fluid excretion, thereby lowering blood sugar. The chemical name of its hemihydrate is (1S)-1,5-anhydro-1-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl ]-D-glucitol hemihydrate, the molecular formula is C ₂₄ H ₂₅ FO ₅ S 1/2H ₂ O, the molecular weight is 453.5, and the structural formula is as follows:

On March 29, 2013, the U.S. Food and Drug Administration (FDA) approved canagliflozin tablets, the specifications are 0.1g, 0.3g, and the trade name is INVOKANA. On September 12, 2013, canagliflozin tablets were approved by the Therapeutic Goods Administration of Australia (TGA) and approved by the European Medicines Agency (EMA) on September 20 of the same year.

The genotoxic impurity F is the oxidative degradation impurity of canagliflozin, specifically, a hydroperoxy group (-O-OH) is generated by the oxidation of the adjacent methylene group of the thienyl group, and this group is genotoxic. Therefore, the residue of impurity F must be strictly controlled.

However, domestic and foreign pharmacopoeia have not yet included the quality standard of this impurity. According to the "Guidelines for the Limits of Genotoxic Impurities" issued by the European Pharmacopoeia Agency (EMEA), the maximum intake limit for genotoxic impurities is 1.5μg/day. Based on the maximum dose of canagliflozin of 300 mg/day, the control limit for this impurity is 5 ppm. Conventional gas-phase and liquid-phase detection methods cannot meet this detection limit requirement.

SUMMARY OF THE INVENTION

The object of the present invention is to overcome the inability to detect the genotoxic impurity F in the prior art, and provide a detection method of canagliflozin genotoxic impurity.

The specific technical solutions adopted to achieve the above objectives are:

A detection method for low-limit canagliflozin impurities, comprising the following steps: after a canagliflozin sample is diluted with a diluent, a high-performance liquid chromatography-mass spectrometer is used to elute with acetonitrile and an aqueous ammonium acetate solution as mobile phases ;

The low-limit canagliflozin impurity is genotoxic impurity F, and its chemical formula is:

Wherein, the ion mode adopted by the mass spectrometer is a positive ion mode; the ion pairs used are: m/z 494.2>380.9, m/z 494.2>222.9.

The mobile phase needs to contain ammonium acetate or another solvent that produces [M+NH ₄ ⁺ ]. The solvent such as ammonium acetate can generate ion pairs of 494.1>380.9 and 494.2>222.9, or other ion pairs with better responsiveness.

Wherein, the volume ratio of the ammonium acetate aqueous solution in the mobile phase is 30-70%.

Preferably, the ratio of acetonitrile and ammonium acetate aqueous solution in the mobile phase is specifically:

When the proportion of ammonium acetate aqueous solution in the mobile phase is 30-70%, the separation efficiency can be ensured, and the tailing and separation time can be reduced. The mobile phase can be isocratic or gradient. The ratio of the ammonium acetate aqueous solution may be 30%, 45%, 50%, 55%, 70%, and may be changed in the above ratio. A gradient mobile phase from the above table was used in a specific example. In the specific implementation process, a certain degree of adjustment can be made according to the concentration of the impurity F and the temperature and other conditions.

The concentration of the ammonium acetate aqueous solution is 1-10 mM, preferably 2-6 mM, and more preferably 5 mM.

The concentration of the ammonium acetate aqueous solution may be any value from 1 to 10 mM, and may be further limited to 2, 3, 4, 5, and 6 mM.

Wherein, the elution rate of the mobile phase is 0.5-2 mL/min, preferably 1 mL/min.

Wherein, the chromatographic column is: Agilent Eclipse XDB-C18.

Wherein, the column temperature of the chromatographic column during elution is 25-35°C, preferably 30°C.

The flow rate of the mobile phase can be 0.5, 1, 1.5 or 2 mL/min. When the column capacity of the chromatographic column is large, the flow rate of the mobile phase can be appropriately increased; when the column capacity is small, the flow rate of the mobile phase should also be appropriately reduced to prevent the separation effect from being reduced due to the excessively fast flow rate.

Preferably, the diluent is a mixed solution of acetonitrile and a 5mM ammonium acetate aqueous solution in a volume ratio of 1:1, wherein the concentration of the ammonium acetate aqueous solution is 5mM.

The diluent must contain ammonium acetate, or other solvent that can generate [M+NH ₄ ⁺ ]. However, ammonium formate aqueous solution, formic acid, etc. are not included.

Wherein, the ionization technology used in the mass spectrometer is spray voltage ionization technology, the spray voltage is 5.5kV, and the ionization temperature is 550°C.

The spray voltage may not be limited to 5.5kV, but is preferably selected from 2-8kV, preferably 5.5kV. The ionization temperature can also be selected from 300 to 800°C, preferably 550°C

The invention has extremely low detection limit and can meet the detection of genotoxic impurities of canagliflozin. The present invention uses [M+NH ₄ ⁺ ] as the parent ion, and adds ammonium acetate into the diluent, and obtains the ion pair with high selectivity and high response through screening: m/z 494.2>380.9, m/z 494.2> 222.9, trace amount of impurity F can be detected using this ion pair, the detection limit is as low as 0.05ng/ml, and the quantification limit is as low as 0.125ng/ml. The method has strong specificity, high sensitivity, accurate results and simple operation, which is of great significance for the quality control and drug safety of canagliflozin

Detailed ways

The present invention will be further described below in conjunction with specific implementations, but these embodiments do not constitute any limitation to the present invention.

1. Instrument:

High performance liquid chromatograph model: Agilent 1260Infinity;

Mass spectrometer model: AB SCIEX 4500;

Chromatographic column: Agilent Eclipse XDB-C18, 4.6×250mm, 5um;

C18, 4.6×150mm, 2.7um, with octadecyl bonded silica gel as filler;

2. Reagents: acetonitrile (Merck, chromatographically pure), formic acid (Fisher, chromatographically pure), ammonium formate (Fisher, chromatographically pure), ammonium acetate (Fisher, chromatographically pure), ultrapure water (Milli-Q purification);

Diluent 1 is a mixed solution of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 55:45.

Diluent 2 is a solution of formic acid with a volume fraction of 0.1% (1 ml of formic acid is adjusted to 1000 ml with water).

Diluent 3 is 5 mM aqueous ammonium acetate-acetonitrile with a volume ratio of 1:1.

3. Impurity F reference substance

①Lot number: 178599C-HB-08(STD)

Impurity F acetonitrile solution with a concentration of 83.3 μg/ml was used as reference stock solution 1.

②Lot number: 20170301

Accurately weigh an appropriate amount of impurity reference substance F, add acetonitrile: water (45:55) mixed solution to dilute to make 7.52 μg/ml, and use as reference substance stock solution 2.

Example 1: Selection of Ion Pairs

(1) The reference substance solution preparation of impurity F:

Reference solution 1 (833ng/ml): Take 100μL of reference solution 1 in a 10ml volumetric flask, add diluent 1 to the volume;

Reference solution 2 (833ng/ml): Take 100μL of reference solution 1 in a 10ml volumetric flask, add diluent 2 to the volume;

Reference solution 3 (416ng/ml): Take 5ml of reference solution 1 in a 10ml volumetric flask, add 10mM ammonium formate buffer to the volume.

(2) Screening transitions:

The precursor ions were determined by Q1 Scan, and in positive ion mode, the response peaks of reference solution 1, 2, and 3 at 50-800 Da were scanned respectively. The precursor ion MS2 is then scanned to screen for highly responsive product ions.

The test results are shown in Table 1:

Table 1

There is a high-response parent ion in each reference solution, and three pairs of ion pairs are obtained by further screening. Among them, the response of [M+Na ⁺ ] in the determination was continuously reduced, so the reference solution 2 and 3 were selected for further verification.

(3) Verification and results

The chromatographic mass spectrometry conditions used were:

Flow rate: 1.0ml/min

Column temperature: 30℃

Injection volume: 10 μL

Mobile phase: acetonitrile: 0.1% aqueous formic acid = 70:30;

The mass spectrometry conditions are shown in Table 2: MRM mode

Table 2

Inject 10 μL of reference solution 2 and 3, respectively, and record the spectrum. And constantly dilute the reference solution 2 and 3 to verify the signal-to-noise ratio of the respective high-response ion pairs at low concentrations.

The results are described in Table 3:

table 3

Example 2:

(1) The reference substance solution preparation of impurity F:

Reference substance solution 4 (376ng/ml): take 0.5ml of impurity reference substance stock solution 2 in a 10ml volumetric flask, add diluent 3 to the volume;

Reference substance solution 5 (37.6ng/ml): 1ml of reference substance solution 4 is placed in a 10ml volumetric flask, and diluent 3 is added to the volume.

(2) Screening transitions:

Determine the parent ion by Q1 Scan, and scan the response peak of reference solution 4 at 50-800 Da in positive ion mode. The precursor ion MS2 is then scanned to screen for highly responsive product ions.

The test results are shown in Table 4:

Table 4

(3) Verification and results

The chromatographic mass spectrometry conditions used were:

Flow rate: 1.0ml/min

Column temperature: 30℃

Injection volume: 5 μL

The mobile phase is shown in Table 5:

table 5

Mass spectrometry conditions are shown in Table 6: MRM mode

Table 6

Take 5 μL of reference solution 5 for injection, record the spectrum, repeat 6 times.

Results: The ion pair 494.2>380.9 had high responsiveness. When tested with reference solution 5 with a concentration of 37.6 ng/ml, its signal-to-noise ratio S/N was as high as 2457.9, and it was repeated 6 times, and the responsiveness was stable.

Example 3: Method Validation

(1) The reference substance solution preparation of impurity F:

Reference substance solution 6 (62.67ng/ml): Take 0.5ml of reference substance stock solution 2, add 2.5ml of diluent 3 and mix well, accurately measure 2.5ml of this solution into a 50ml measuring bottle, and use diluent 3 to make up to the mark , shake well.

Reference solution 7 (2.5ng/ml): Take 2ml of reference solution 6 (62.67ng/ml) in a 50ml volumetric flask, dilute to the mark with diluent 3, and shake well.

(2) Method conditions:

Chromatographic conditions:

Column temperature: 30℃

Flow rate: 1.0ml/min

Injection volume: 5 μL

The mobile phase is shown in Table 7:

Table 7

The mass spectrometry conditions are shown in Table 8: MRM mode

Table 8

(3) Methodological verification

a. Specificity test

Diluent 3 was injected and the chromatogram was recorded. The baseline was stable, indicating the specificity of the method.

b. System suitability

Take the reference solution 7 (2.5ng/ml) as the system suitability solution, inject 6 needles continuously, record the chromatogram, and calculate the RSD of the impurity peak area. The RSD results obtained are all less than or equal to 5%, which meets the requirements.

c. Limit of quantification detection limit

The reference solution 6 was diluted and injected step by step, and the signal-to-noise ratio of the impurity peak was detected. The Pharmacopoeia specifies a signal-to-noise ratio (S/N) of 3 for the limit of detection and a signal-to-noise ratio (S/N) of 10 for the limit of quantification. The experimental results show that the detection limit concentration of this method is 0.05ng/ml, and the quantitation limit concentration is 0.125ng/ml, as shown in Table 9.

Table 9

d. Linearity and range

Precisely pipette the reference solution 6, and dilute it to the concentration of 0.125ng/ml, 0.25ng/ml, 0.50ng/ml, 1.25ng/ml, 2.51ng/ml, 3.76ng/ml, 5.01ng/ml respectively. solution. 5 μL of each concentration solution was injected, and the chromatogram was recorded. The results are shown in Table 10.

Table 10

Take the concentration of impurity F as the x-axis and the peak area as the y-axis, and perform a linear regression. The regression equation of concentration and peak area is y=0.5772x-0.005, and the correlation coefficient R is 0.9999. It shows that the genotoxic impurity F has a good linearity in the concentration range of 0.125ng/ml～5.013ng/ml.

e. Recovery rate

Sample solution: Weigh about 25 mg of canagliflozin, place it in a 50 ml volumetric flask, and use diluent 3 to dilute to volume.

Recovery solution: Precisely weigh about 25mg of canagliflozin, place it in a 50mL volumetric flask, and precisely pipette 1.0ml, 2.0ml, and 3.0ml of reference solution 6, respectively, and dilute to volume with diluent 3. The solutions of 50%, 100%, and 150% impurities, in triplicate, were taken as the recovery solution.

Precisely measure 5 μl of the reference solution 7, the sample solution and the recovery solution, and inject the samples, and record the data. The measured value of the above-mentioned recovery rate solution is the measured total amount. The sample solution measurement is the background amount. Reference solution 7 was used as an external standard to calculate the total amount of measurement and the background amount.

According to the following formulas, the genotoxic impurity content and recovery rate in the samples were calculated respectively. The results are shown in Table 11.

formula:

Total measurement = external standard concentration / external standard peak area * measured peak area

Background amount = external standard concentration / external standard peak area * background peak area

Measured addition amount = measured total amount – background amount, recovery = measured addition amount/actual addition amount.

Table 11

The results showed that the recovery rate of genotoxic impurity F in canagliflozin and the overall average recovery rate were between 100.8 and 102.2%, and the RSD and total RSD were both less than 1.9%. All indicators met the requirements. The accuracy of this method good.

f. Precision

Repeatability

Accurately weigh about 25 mg of canagliflozin, place it in a 50 mL volumetric flask, accurately pipette 2.0 ml of reference solution 6 and add it, use diluent 3 to the mark, and make six parallels as a 100% recovery solution. Take 5μl injection respectively and record the chromatogram. According to the above recovery calculation formula, the results are shown in Table 12. The recoveries of the genotoxic impurity F were 98.2%-104.8%, and the relative standard deviation was 2.6%, indicating that the method was reproducible.

Intermediate precision

Six 100% recovery solutions were prepared according to the above steps on different days, and the chromatograms were recorded. The calculation results are shown in Table 12. The relative standard deviation of the recovery rates of 12 solutions on different days was 2.0%, which met the requirements, indicating that the method had good intermediate precision.

Table 12

g. Durability

Changes in HPLC flow rate

Change the flow rate to 0.9ml/min and 1.1ml/min, other mass spectrometry and chromatographic conditions remain unchanged, take the reference solution 8 and inject 6 consecutive injections, calculate the impurity peak area RSD, the results are shown in Table 13.

Table 13

Changes in HPLC column temperature

Change the column temperature to 25°C and 35°C, and keep other mass spectrometry and chromatographic conditions unchanged, take the reference solution 8 and inject 6 consecutive injections, and calculate the RSD of the impurity peak area. The results are shown in Table 14.

Table 14

Conclusion: After appropriately changing the flow rate and column temperature, the applicability of the system meets the requirements, indicating that the method has good durability.

It has been verified that the method has good linearity, recovery rate, precision and robustness, is simple and effective, and can be used for the determination of genotoxic impurities in canagliflozin.

Example 4: Sample determination

Referring to the mass spectrometry and chromatographic conditions in Example 3, 5 μl of each of the above-mentioned sample solution and reference solution 8 were injected into the samples, the chromatograms were recorded, and the content of genotoxic impurities in the samples was calculated by the external standard method.

Table 15

batch number Impurity content C031-51701001 not detected C031-51701002 not detected C031-51701003 not detected

The results are shown in Table 15. No impurities were detected in each batch of samples, and all batches of samples were qualified.

The above examples have described the present invention in detail by using specific embodiments and tests, but on the basis of the present invention, some modifications or improvements can be made without departing from the gist of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made on the basis of not departing from the gist of the present invention all belong to the protection scope of the present invention.

Claims (10)

1. A method for detecting low-limit canagliflozin impurities is characterized by comprising the following steps: diluting a canagliflozin sample by using a diluent, and eluting by using an acetonitrile and ammonium acetate aqueous solution as a mobile phase by using a high performance liquid chromatography-mass spectrometer;

the low-limit canagliflozin impurity is a genotoxic impurity F, and the chemical formula of the low-limit canagliflozin impurity is as follows:

2. the detection method according to claim 1, wherein the ion mode adopted by the mass spectrometry is a positive ion mode; the ion pairs used were: m/z 494.2>380.9, m/z 494.2> 222.9.

3. The detection method according to claim 1, wherein the volume ratio of the ammonium acetate aqueous solution in the mobile phase is 30-70%.

4. The detection method according to claim 3, wherein the ratio of acetonitrile to the aqueous ammonium acetate solution in the mobile phase is in particular:

。

5. the detection method according to any one of claims 1 to 4, wherein the concentration of the ammonium acetate aqueous solution is 1 to 10mM, preferably 2 to 6mM, and more preferably 5 mM.

6. The detection method according to any one of claims 1 to 4, wherein the elution rate of the mobile phase is 0.5 to 2m L/min, preferably 1m L/min.

7. The detection method according to any one of claims 1 to 4, wherein the chromatographic column is: agilenteclipse XDB-C18.

8. The detection method according to claim 7, wherein the column temperature of the chromatographic column at the time of elution is 25 to 35 ℃, preferably 30 ℃.

9. The detection method according to any one of claims 1 to 4, wherein the diluent is a mixed solution of acetonitrile and an aqueous solution of ammonium acetate at a volume ratio of 1:1, wherein the concentration of the aqueous solution of ammonium acetate is 5 mM.

10. The detection method according to any one of claims 1 to 4, wherein the ionization technique used in the mass spectrometry is a spray voltage ionization technique, the spray voltage is 5.5kV, and the ionization temperature is 550 ℃.