A method for determining the content of decanoic acid in daptomycin

Abstract

The present invention belongs to the field of pharmaceutical chemical analysis and relates to a method for determining the content of decanoic acid in daptomycin. The present invention adopts LC-MS method to determine the content of decanoic acid in daptomycin, uses liquid chromatography-mass spectrometry to detect decanoic acid, and extracts decanoic acid anion 171.1390 ​​[M-H] by extracting EIC mode. ‑ The method of the present invention is simple, rapid, highly specific, highly sensitive and reproducible. The linear relationship of decanoic acid is good in the range of 0.261 to 10.457 μg / mL, the average recovery rate is 98.76%, and the RSD is 1.98%.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemical analysis, and in particular relates to a method for determining the content of decanoic acid in daptomycin. Background Art

[0002] Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus. It is composed of 13 amino acids, 10 of which form an amino acid peptide ring and another 3 amino acids are arranged in a line. The terminal L-tryptophan of these 3 amino acids is followed by a decadecanoic acid chain. It is a calcium ion-dependent antibiotic.

[0003]

[0004] In the presence of calcium ions, daptomycin will bind to daptomycin binding proteins (DBPs) on the cell membrane in the form of non-covalent bonds. Daptomycin has a good bactericidal effect on Gram-positive bacteria, a high efficiency against highly pathogenic and resistant bacteria, good bactericidal effect, and low toxic side effects. It has been clinically used for (including but not limited to) infections caused by methicillin-resistant aureus and vancomycin-resistant enterococci.

[0005] Decanoic acid is a commonly used raw material in the process of synthesizing daptomycin. For example, U.S. Patent No. 4,885,243 describes an improved method for synthesizing daptomycin by supplying nutrients to the fermentation medium with decanoic acid or its lipid or salt; CN101880703B discloses a method for fermenting daptomycin with decanoate. Because decanoic acid itself has cytotoxic effects, the concentration of decanoic acid in the fermentation process of daptomycin needs to be strictly controlled. "Determination of decanoic acid in daptomycin fermentation broth by capillary gas chromatography" (China Journal of Pharmaceutical Industry, 2015, 46 (6): 626-627) reported the determination of decanoic acid content in daptomycin by gas chromatography, but because the temperature of the gas phase is high during the chromatographic separation process, the amide bond of daptomycin will be hydrolyzed at high temperature (column temperature is 210 ° C) to produce decanoic acid, which is not suitable for detecting the content of decanoic acid in daptomycin. Therefore, it is necessary to develop a new method for detecting decanoic acid content while avoiding the decomposition of daptomycin, which provides a basis for the quality control of daptomycin. Summary of the invention

[0006] The object of the present invention is to provide a method for determining the content of capric acid in daptomycin, which can detect the content of capric acid while avoiding the decomposition of daptomycin, and provide a basis for detecting the content of capric acid during the fermentation process of daptomycin and controlling the residual capric acid in the finished product.

[0007] To achieve the above object, the present invention develops a liquid mass spectrometry detection method for decanoic acid. Specifically, a method for determining the content of decanoic acid in daptomycin using LC-MS is developed, comprising the following steps:

[0008] a. Take a certain amount of the sample solution to be tested for LC-MS analysis;

[0009] b. Use EIC mode to extract m / z = 171.1390 ​​and calculate the content according to the formula:

[0010] Decanoic acid content in sample (ppm) = 10 6 × the peak area of ​​decanoic acid in the sample / (the peak area of ​​the reference substance / the concentration of the reference substance (mg / mL) × the concentration of the sample (mg / mL)).

[0011] Specifically, the LC described in step a is preferably ultra-high pressure liquid phase, and its chromatographic conditions are:

[0012] The chromatographic column was C18, 2.1 mm × 100 mm, 1.8 μm column;

[0013] The mobile phase was methanol and 10 mM ammonium formate in a volume ratio of 50:50;

[0014] Column temperature: 30°C;

[0015] Flow rate: 0.5 mL / min;

[0016] Injection volume: 10 μL.

[0017] The method for preparing the reference solution in the LC-MS determination is as follows: accurately weigh an appropriate amount of the capric acid reference, dissolve it in a 50% methanol aqueous solution, and quantitatively dilute it to prepare a solution with a concentration of 5 μg / mL.

[0018] The method for preparing the test solution in the ultra-high pressure liquid phase determination is as follows: accurately weigh an appropriate amount of decanoic acid raw material, add 50% methanol aqueous solution to dissolve it and quantitatively dilute it to prepare a solution with a concentration of 5 mg / mL.

[0019] The running time of the ultra-high pressure liquid phase is 6 minutes, and the data collection time is 6 minutes.

[0020] Specifically, the MS used in step a is a quadrupole time-of-flight mass spectrometer, and the specific mass spectrometry detection conditions are as follows:

[0021] The mass spectrometer used -ESI scanning mode with a scanning range of 100 m / z-1700 m / z;

[0022] Mass spectrometer switching valve: 0-1min: waste, 1-6min: MS,

[0023] Drying gas temperature 320℃; Drying gas flow rate 8L / min;

[0024] Sheath gas temperature 350°C; sheath gas flow rate 11L / min;

[0025] Capillary voltage 4000V; nozzle voltage 0V; capillary outlet voltage 95V.

[0026] Furthermore, the mass spectrometry was performed using an extraction EIC mode, extracting m / z=171.1390 ​​for quantitative analysis.

[0027] Beneficial effects of the present invention:

[0028] The invention discloses a method system for determining the content of decanoic acid in daptomycin by liquid chromatography-mass spectrometry, wherein daptomycin and decanoic acid are separated by liquid chromatography, and decanoic acid is quantified by a mass spectrometer. The method of the invention is simple, rapid, highly specific, highly sensitive, and reproducible, and the linear relationship of decanoic acid is good within the range of 0.261 to 10.457 μg / mL, the average recovery rate is 98.76%, and the RSD is 1.98%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The gas chromatogram of daptomycin sample solution under the detection conditions of Example 1 is shown in FIG.

[0030] Figure 2 The gas chromatogram of the capric acid reference substance solution under the detection conditions of Example 1 is

[0031] Figure 3 Spectrum of blank solution under the detection conditions of Example 3

[0032] Figure 4 The spectrum of the reference solution under the detection conditions of Example 3

[0033] Figure 5 The spectrum of the test sample spiked solution under the detection conditions of Example 3 DETAILED DESCRIPTION

[0034] The present invention will be described in detail below in conjunction with specific implementations, but the scope of the present invention is not limited thereto.

[0035] In the following specific examples, unless otherwise specified, the reagents and instruments used are commonly used reagents and instruments in the field and can be obtained commercially; the methods used are conventional methods in the field, and those skilled in the art can know how to specifically implement the methods and achieve corresponding results based on the contents of the examples.

[0036] The high performance liquid chromatograph used in the following specific examples is Agilent 1290 (Agilent, USA).

[0037] The quadrupole time-of-flight mass spectrometer was Agilent QTOF 6545 (Agilent, USA);

[0038] Methanol and ammonium formate were both LCMS grade and commercially available; the decanoic acid reference substance was supplied by Sigma with a purity of 99.5%;

[0039] The experimental water was Milli-Q ultrapure water;

[0040] The daptomycin API sample was sourced from Hangzhou Sino-US Huadong Pharmaceutical Co., Ltd., batch number: 180601YTX. Unless otherwise specified, the daptomycin API / samples in the examples were sourced from this source.

[0041] Example 1

[0042] (1) Preparation of control solution and sample solution

[0043] Reference solution: Weigh 100 mg of capric acid reference substance into a 100 mL volumetric flask, dilute to the mark with methanol, shake well, transfer 5 mL of the solution into a 50 mL volumetric flask, dilute to the mark with methanol, shake well to obtain 100 μg / mL capric acid reference solution.

[0044] Sample solution: Weigh 0.1 g of daptomycin API into a 5 mL volumetric flask, dilute to the mark with methanol, and mix well.

[0045] (2) Detect the decanoic acid content in daptomycin under the following gas phase conditions

[0046] The gas phase detection method conditions are:

[0047] Chromatographic column: HP-INNOWAX (30m×0.32mm×0.5μm)

[0048] Injection volume: 1.0 μL

[0049] Inlet temperature: 280°C

[0050] Split ratio: 10:1

[0051] Column flow rate: 1mL / min

[0052] Heating program: 40℃ for 3 min, increase to 200℃ at 20℃ / min and hold for 12 min, increase to 240℃ at 20℃ / min and hold for 2 min

[0053] Detector: FID

[0054] Detector parameters: temperature: 280°C, air: 400 mL / min, hydrogen: 30 mL / min

[0055] The results showed that the decanoic acid content in the sample solution was relatively high, about 3000 ppm.

[0056] The gas chromatogram of daptomycin sample solution is attached. Figure 1, the gas chromatogram of the capric acid reference solution is attached Figure 2 .

[0057] Example 2

[0058] Weigh 0.2 g of daptomycin sample into a centrifuge tube, add 10 mL of water to dissolve, then add 10 mL of ethyl acetate, cover and seal, shake for 5 minutes, centrifuge at 10000 r / min for 15 minutes, take the upper ethyl acetate layer and detect it using the gas phase detection conditions of Example 1. The test results show that no decanoic acid is detected in the ethyl acetate layer.

[0059] By extraction with ethyl acetate, decanoic acid is extracted into the ethyl acetate layer, while daptomycin has good water solubility and is mainly in the water layer. The extraction experiment proves that the sample actually used does not contain decanoic acid or the decanoic acid content is very low. However, when directly using gas chromatography for detection, the decanoic acid content in the sample solution is measured to be relatively high (the detection result of Example 1 is about 3000 ppm). It can be seen that the direct use of gas chromatography is not conducive to the control of decanoic acid content in the actual fermentation process and the quality control of daptomycin, because this method cannot determine whether decanoic acid is contained in the sample or is produced by hydrolysis of the amide bond of daptomycin due to the high detection temperature.

[0060] Example 3

[0061] (1) Preparation of test solution

[0062] Dilution and blank solution: Mix equal volumes of water and methanol to obtain a 50% methanol-water solution.

[0063] Decanoic acid reference substance stock solution (50 μg / mL): Accurately weigh 10 mg of decanoic acid reference substance and dilute it with diluent to contain 50 μg of decanoic acid per 1 mL.

[0064] Decanoic acid reference solution (5 μg / mL, system suitability solution): Accurately pipette 1.0 mL of the decanoic acid reference stock solution into a 10 mL volumetric flask and dilute to the mark with diluent.

[0065] Test solution: Accurately weigh 100 mg of daptomycin raw material, dissolve it in a 20 mL volumetric flask, add diluent to dissolve and dilute to the scale, shake well to obtain a 5 mg / mL test solution.

[0066] Test sample spiked solution: Accurately weigh about 100 mg of daptomycin API, dissolve it in a 20 mL volumetric flask, add an appropriate amount of diluent, and accurately add 2.0 mL of capric acid reference stock solution, dilute to the scale with diluent, and shake well.

[0067] (2) The reference solution and the test solution were injected and tested under the following test conditions:

[0068] Liquid phase condition settings:

[0069] Chromatographic column: Agilent ZORBAX XDB-C18 (2.1 mm × 100 mm, 1.8 μm);

[0070] Column temperature: 30°C;

[0071] Flow rate: 0.5 mL / min;

[0072] Injection volume: 10 μL;

[0073] Mobile phase: V (acetonitrile): V (10 mM ammonium formate) = 50:50;

[0074] MS condition settings:

[0075] The mass spectrometer was scanned in ESI mode.

[0076] Scanning range: 100m / z-1700m / z,

[0077] Mass spectrometer switching valve: 0-1min: waste, 1-6min: MS,

[0078] Drying gas temperature: 320℃; Drying gas flow rate: 8L / min;

[0079] Sheath gas temperature: 350℃; sheath gas flow rate: 11L / min;

[0080] Capillary voltage: 4000V; Nozzle voltage: 0V; Capillary outlet voltage: 95V;

[0081] The quantitative analysis was performed using the extraction EIC mode and extracting m / z: 171.1390.

[0082] Results: Decanoic acid was not detected in the samples.

[0083] Example 4

[0084] System suitability test

[0085] A decanoic acid reference solution (5 μg / mL) was taken as the system suitability solution, and the system suitability solution was continuously injected 6 times according to the detection conditions set in Example 3, and the decanoic acid peak area RSD was measured to be 0.63%.

[0086] Take the blank solution, capric acid reference solution and test sample spiked solution in Example 3 and inject them under the above conditions, and record the spectra (see Appendix Figure 3-5 ), the results showed that the blank solvent had no interference; there were no other interfering peaks in the reference solution and the test solution, and the specificity was good.

[0087] Example 5

[0088] Linearity test

[0089] Accurately measure an appropriate amount of the reference stock solution, dilute it with the diluent, and prepare a series of decanoic acid solutions with concentrations of approximately 0.2, 0.5, 2.5, 5, 7.5 and 10 μg / mL, respectively. Shake well, and inject and measure them respectively according to the detection conditions set in Example 3.

[0090] The quantitative limit was 0.2 μg / mL (S / N greater than 10), and the sample was diluted step by step for detection. The detection limit was 0.05 μg / mL when the S / N was about 3. The linear regression of the decanoic acid peak area (Y) and the decanoic acid solution concentration (X) was performed, and the regression equation was Y=0.3923X-0.0421, R=0.9999.

[0091] The linear range of decanoic acid is 0.261μg / mL~10.457μg / mL, the correlation coefficient r is 0.9999, and the concentration and peak area show a good linear relationship.

[0092] Example 6

[0093] Precision test

[0094] Repeatability test: Accurately weigh 100 mg of daptomycin raw material, place it in a 20 mL volumetric flask, add an appropriate amount of diluent, accurately add 2.0 mL of capric acid reference stock solution, dilute to the scale with diluent, shake well, prepare 6 copies in parallel, and measure according to the conditions and methods of Example 3. After deducting the background amount, the average recovery rate of capric acid in the 6 samples was calculated to be 100.91%, and the recovery rate RSD was 0.78%, with good repeatability.

[0095] Intermediate precision: At different times, different personnel used the same chromatographic column to conduct repeatability tests. The average recovery of 6 samples was calculated to be 99.37%, and the RSD of the recovery was 1.29%.

[0096] Comparing the data results obtained from the repeatability test and the intermediate precision test, the average recovery rate of decanoic acid in 12 test solutions was 100.14%, and the RSD of the recovery rate of decanoic acid in 12 test solutions was 1.29%, so the precision was good.

[0097] Example 7

[0098] Recovery test

[0099] The recovery test was designed at three concentrations of 2.5, 5 and 7.5 μg / mL: accurately weigh about 100 mg of daptomycin raw material, place it in a 20 mL volumetric flask, add an appropriate amount of diluent, and add an appropriate amount of 1 mL, 2 mL and 3 mL of capric acid reference stock solution respectively, dilute to the scale with diluent, shake well and prepare 3 copies for each concentration in parallel.

[0100] The reference solution, the test solution and the test solution of each concentration recovery rate were measured according to the method in Example 3, and the recovery rate of decanoic acid was calculated by the peak area according to the external standard method. The recovery rate of decanoic acid at each concentration point was between 96.14% and 102.22%, the average recovery rate was 98.76%, and the total recovery rate RSD was 1.98%, indicating that the method had good accuracy.

[0101] Example 8

[0102] Accuracy of Quantitative Limit 100 mg of daptomycin raw material was accurately weighed and placed in a 20 mL volumetric flask. An appropriate amount of diluent was added, and 1.0 mL of capric acid reference solution was accurately added. The sample was diluted to the mark with the diluent, shaken, and 6 portions were prepared in parallel. The sample was determined according to the conditions of Example 3, and the background amount was deducted. The average recovery of capric acid in the 6 samples was calculated to be 95.31%, and the RSD of the recovery was 5.5%, indicating that the accuracy of the quantitative limit of the method was good.

Claims

1. A method for determining the content of capric acid in daptomycin, characterized in that: The content of decanoic acid in daptomycin was determined by LC-MS: The test solution was prepared as follows: an appropriate amount of daptomycin raw material was weighed, dissolved in 50% methanol aqueous solution and quantitatively diluted to make a solution with a concentration of 5 mg / mL; LC is ultra-high pressure liquid chromatography, and the ultra-high pressure liquid chromatography conditions are as follows: mobile phase: acetonitrile and 10 mM ammonium formate aqueous solution in a volume ratio of 50:50; column temperature 30°C; flow rate 0.5 mL / min; chromatographic column C18, 2.1 mm × 100 mm, 1.8 μm column; The mass spectrometer used the ESI scanning mode with a scanning range of 100 m / z-1700 m / z; The mass spectrometry was carried out in the extraction EIC mode, and m / z = 171.1390 ​​was extracted for quantitative analysis.

2. The method for determining the content of capric acid in daptomycin according to claim 1, characterized in that: The method for preparing the reference solution in the ultra-high pressure liquid chromatography determination is as follows: weigh an appropriate amount of the capric acid reference, add 50% methanol aqueous solution to dissolve it, and quantitatively dilute it to prepare a solution with a concentration of 5 μg / mL.

3. The method for determining the content of capric acid in daptomycin according to claim 1, characterized in that: The running time of the ultra-high pressure liquid chromatography is 6 minutes, and the data collection time is 6 minutes.

4. The method for determining the content of capric acid in daptomycin according to claim 1, characterized in that: The MS of the LC-MS method uses a quadrupole time-of-flight mass spectrometer.

5. The method for determining the content of capric acid in daptomycin according to claim 4, characterized in that: Mass spectrometer switching valve: 0-1min: waste, 1-6min: MS, Drying gas temperature 320℃; Drying gas flow rate 8L / min; Sheath gas temperature 350°C; sheath gas flow rate 11L / min; Capillary voltage 4000V; nozzle voltage 0V; capillary outlet voltage 95V.

6. The method for determining the content of capric acid in daptomycin according to claim 1, characterized in that: Under the established detection conditions, the determination steps are as follows: a. Take the sample solution to be tested for LC-MS analysis; b. Use EIC mode to extract m / z = 171.1390 ​​and calculate the content according to the formula: The unit of decanoic acid content is ppm, and the units of reference substance concentration and sample concentration are both mg / mL.

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