CN111679010B - High performance liquid chromatography detection method of remdesivir intermediate GS-441524 - Google Patents

Abstract

The invention discloses a high performance liquid chromatography detection method of a Rudexilwei intermediate GS-441524, which comprises the following steps: a. preparing a standard solution; b. detecting the standard solution by adopting reverse high performance liquid chromatography; c. the content of GS-441524 in the sample was calculated as peak area by external standard method. The detection method can lead GS-441524, raw materials and other impurities to be well separated on a high performance liquid chromatograph; within 1.0-20 mu g/mL, the linear relation is good, R²=0.9997, the average recovery rate is 97.79% -99.90%, and the precision RSD value of 9 parallel tests is 2.65%.

Description

High performance liquid chromatography detection method of remdesivir intermediate GS-441524

technical field

The invention relates to the technical field of analytical chemistry, in particular to a high-performance liquid chromatography detection method for remdesivir intermediate GS-441524.

Background technique

Remdesivir is a nucleoside analog, a new experimental broad-spectrum antiviral drug developed by Gilead Sciences, which can inhibit RNA-dependent RNA synthase (RdRp), GS-441524 It is an important intermediate for the synthesis of Remdesivir. The CAS registration number is 1191237-69-0, the molecular formula is C12H13N5O4, the molecular weight is 291.267, and the English name is: (2R, 3R, 4S, 5R)-2-(4-aminopyrrolo [2,1-f][1,2,4]triazin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-carbonitrile, its chemical structure is shown in Figure 1, GS-441524 It is both the precursor compound of Remdesivir and the active metabolite of Remdesivir.

In general, impurity compounds inevitably exist in GS-441524. The detection of impurity compounds is of great significance for the quality control of GS-441524 and Remdesivir. High-performance liquid chromatography can be classified into reversed-phase and normal-phase. It is determined by the relative polarity of the stationary phase and the mobile phase; among them, reversed-phase high-performance liquid chromatography: using a relatively weak stationary phase and a more polar solvent as the mobile phase, it is often used to separate and detect non-polar and polar It is widely used for compounds with weaker properties; normal-phase high performance liquid chromatography: using a polar stationary phase and a relatively weak solvent as the mobile phase, it is often used to separate and detect compounds with relatively strong polarity.

Whether as an intermediate or metabolite of Remdesivir, or as a drug itself, in the field of analytical chemistry and drug analysis, the content of GS-441524 is required to be accurately determined. At present, there is no GS-441524 in domestic and foreign literature. Therefore, there is an urgent need to develop a method for detecting the compounds shown in Figure 1 with simple operation and high resolution.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a high-performance liquid chromatography detection method of Remdesivir intermediate GS-441524.

The high-performance liquid chromatography detection method of Remdesivir intermediate GS-441524 provided by the invention comprises the following steps:

Step a. Preparation of standard solution: Precisely weigh 0.050g of GS-441524 reference substance, dissolve it in methanol/water (1:9, v/v) solution, and prepare a solution with a concentration of 0.2-20μg/mL, using 0.22μm Filtration with microporous membrane to obtain standard solution;

Step b, adopting reversed-phase high performance liquid chromatography to detect the standard solution, and the detection conditions are:

The stationary phase of the chromatographic column: silica gel with non-polar octadecyl functional groups (ODS) bonded on the surface as filler;

Mobile phase: methanol and 0.2% formic acid aqueous solution, fixed ratio 1:9 (v/v);

Detection wavelength: 230～320nm;

Step c. Calculate the content of GS-441524 in the sample by the peak area according to the external standard method.

Further, in step a, the concentration of the standard solution is 0.2-20 μg/mL.

Further, in step b, the inner diameter is 2.1 mm, the length is 50-150 mm, and the particle size of the filler is 1.7-1.8 μm.

Further, in step b, the chromatographic column is a C18 alkylsilane-bonded silica gel packed chromatographic column.

Further, in step b, two mobile phases, methanol and 0.2% formic acid aqueous solution, are used as eluents, and the volume ratio thereof is 1:9.

Further, in step b, the detector is a diode array or an ultraviolet detector, and the detection wavelength range is 230-320 nm.

Further, in step b, the column temperature is 35°C; the flow rate is 0.2-0.3 mL/min.

Further, in step b, the injection volume is 1-5 μL.

Further, in step b, the analysis time is 5-10 min.

Due to the adoption of the above technical solution, the advantages and positive effects of the present invention are as follows: the detection method of the present invention can achieve complete separation of GS-441524 and impurity compounds without gradient elution, the minimum separation degree is above 2.0, and the theoretical plate The detection method of the invention has the advantages of simple operation, accurate and reliable detection results, short analysis time and the like.

Description of drawings

The present invention has the following 7 accompanying drawings:

Figure 1 is the chemical structure of GS-441524;

Fig. 2 is the test result of embodiment 1 detection wavelength;

Fig. 3 is a linear relationship diagram of Test Example 1;

Fig. 4 is the detection result of embodiment 1 test sample solution;

Fig. 5 is the precision test result;

Fig. 6 is the test result of recovery rate;

Figure 7 is the sample stability test results.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The embodiments of the present invention, and all other embodiments obtained by those of ordinary skill in the art without creative work, fall within the protection scope of the present invention.

In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can do so without departing from the connotation of the present invention. Similar promotion, therefore, the present invention is not limited by the specific embodiments disclosed below.

The GS-441524 reference substance used in the examples was obtained from a commercially available product, and the purity was required to be higher than 99.0%, and the content of other single impurities should not exceed 0.5%.

Example 1

1. Instruments and Reagents

High performance liquid chromatograph (Agilent-1290 equipped with DAD detector, USA); Analytical balance (SartoriusBSA224S, Sartorius Scientific Instruments (Beijing) Co., Ltd.); Ultrasonic cleaner (KQ-100DE type, Kunshan Ultrasonic Instrument Co., Ltd.) ; GS-441524 reference substance (commercially available, content > 99%); methanol is chromatographically pure, pure water is prepared by a water purifier (Milli-QDirect8, Millipore, France), and other reagents are analytical grade reagents.

2. Content detection of GS-441524 in samples

A. Preparation of test samples

Accurately weigh 50 mg of the powdered test sample, dissolve it with about 200 mL of methanol/water (1:9, v/v) solution by ultrasonic, and then dilute the volume to a 250 mL volumetric flask to obtain the initial sample solution. The initial sample solution is methanol/water ( 1:9, v/v) solution was diluted 20 times to prepare the test sample solution. The test sample solution was filtered through a 0.22 μm microporous membrane to be tested.

B, the preparation of standard solutions

Accurately weigh 50 mg of GS-441524 reference substance, dissolve it with methanol/water (1:9, v/v) solution ultrasonically, and dilute the volume to 250 mL to obtain a mother liquor, which uses methanol/water (1:9, v/v) The solution was gradually diluted to obtain a series of standard solutions with concentrations of 0.2 μg/mL, 0.5 μg/mL, 1.0 μg/mL, 5.0 μg/mL, 10 μg/mL, 15 μg/mL, and 20 μg/mL. filter, to be tested.

3. Chromatographic conditions

The chromatographic column is ACQUITYUPLCBEHC18 (2.1×50mm, 1.7μm) (Waters, USA) from Waters Company; the volume ratio of mobile phase is methanol:water (0.2% formic acid)=1:9 (V/V), and an ultrasonic instrument is used before use. Ultrasonic degassing was performed for 30 minutes to remove air bubbles in the mobile phase; the column temperature was 35 °C, the detection wavelength of the UV detector was 240 nm, the flow rate was 0.2 mL/min, and the injection volume was 2 μL.

Determine the detection wavelength test:

The sample was scanned by UV spectrophotometer at the full wavelength of 190-400nm, and the UV absorption wavelength of GS-441524 was detected. The test results are shown in Figure 2.

The test results show that the wavelengths of 240nm and 300nm are the strong absorption peaks of GS-441524; among them, at the wavelength of 300nm, the absorption of other impurities is weak and cannot all be detected. All impurities are scanned out; the detection wavelength is in the range of 235nm to 245nm, which is suitable for the high performance liquid chromatography detection method of the present invention.

The type selection of mobile phase and the ratio of two phases were tested; when methanol was used as the organic phase, 0.2% formic acid water was used as the inorganic phase, and the ratio of organic and inorganic two phases was 1:9 (V/V), the chromatographic separation process was Completed within 8min, in the obtained spectrum, the peaks are symmetrical and the resolution is high. The retention time (RT) of GS-441524 is about 1.46min, and the test results are satisfactory.

4. Linear range inspection

Take 1.0 μg/mL, 5.0 μg/mL, 10 μg/mL, 15 μg/mL, and 20 μg/mL series of standard solutions, and analyze them in sequence. For each standard solution, take 2 μL and inject it into a liquid chromatograph, and measure according to the method of the present invention. Record the chromatogram, take the standard solution concentration C (μg/mL) as the abscissa and the peak area A as the ordinate to draw the standard curve, as shown in Figure 3, after calculating the standard curve data, the linear regression equation y=28.619x is obtained -3.5235, linear correlation coefficient R2=0.9997, indicating that GS-441524 has a good linear relationship in the range of 1.0μg/mL~20μg/mL.

5. Detection of GS-441524 content in samples

Take the test sample solution, inject 2.0 μL into the liquid chromatograph, develop according to the method of the present invention, record the chromatogram, obtain Figure 4, repeat 3 times, take the average value of the peak area of the corresponding chromatographic peak in the retention time RT=1.46min in the 3 experiments , and substituted into the linear regression equation to calculate the content of GS-441524 in the test sample.

The results show that the detection method of the present invention can enable GS-441524 to be well separated from raw materials and other impurities on high performance liquid chromatography, so that the content detection is accurate and reliable;

6. Precision inspection

Take the test sample solution, inject 9 times continuously according to the injection volume of 2 μL, measure according to the method of the present invention, record the peak area of the sample, and calculate the average value and relative standard deviation (RSD), the results are shown in Figure 5, indicating that this The method of the invention determines the content of GS-441524 with high precision, good reproducibility and small relative standard deviation.

7Recovery rate inspection

The test samples of low, medium and high concentrations were prepared with the reference substance. According to the method of the present invention, three parallel measurements were performed. The recovery rate, average recovery rate and RSD of the measurement results are shown in Figure 6.

8. Investigation of the stability of the solution

Take the prepared test sample and place it, take samples at 0, 2, 4, 6, 8, 12, and 24 hours, respectively, inject and measure according to the method described in the invention, record the chromatogram, and calculate the peak area of the substance according to GS-441524. RSD value, the results are shown in Figure 7, according to the RSD value, the solution of the test solution of the present invention has good stability when placed at room temperature for 24 hours.

9. Determination of detection limit and quantification limit

Taking the ratio of the peak height of the GS-441524 substance to the baseline noise (signal-to-noise ratio, S/N) in the chromatogram is greater than 3 as the minimum detection limit standard, the detection limit of the method of the present invention is known to be 0.5 μg/mL through experiments. The ratio (S/N) greater than 10 is the limit of quantification standard. Through experiments, it is known that the limit of quantification of the method of the present invention is 1.0 μg/mL.

The above 4-9 methodological investigation test results show that the method of the present invention has good specificity. The results show that GS-441524 exhibits a good linear relationship in the range of 1.0-20 μg/mL. The regression equation is y=28.619x-3.5235, and the correlation The coefficient R²=0.9997, the precision, stability and recovery are good, the average recovery rate is between 97.79% and 99.90%, the method is simple, the content determination is accurate and reliable, the test time is short, no interference peaks, resolution, tailing factor, The symmetry factors all meet the requirements of the Chinese Pharmacopoeia, and the test results are good, which can be used for the inspection and control of this content item.

Claims (2)

1. a high-performance liquid chromatography detection method of Remdesivir intermediate GS-441524, is characterized in that: the concrete steps of described method are as follows: a. Preparation of standard solution: Precisely weigh the GS-441524 reference substance, and dissolve the reference substance with a methanol/water mixed solution with a volume ratio of 1:9 to prepare concentrations of 0.2 μg/mL, 0.5 μg/mL, 1.0 A series of standard solutions of μg/mL, 5.0 μg/mL, 10 μg/mL, 15 μg/mL and 20 μg/mL were filtered with 0.22 μm microporous membrane; b. Prepare the test sample solution: accurately weigh the test sample, dissolve and dilute the test sample with a methanol/water mixed solution with a volume ratio of 1:9 to make the test sample solution; c. The standard solution and the test sample solution were detected by reversed-phase high performance liquid chromatography: octadecylsilane-bonded silica gel column was used as the detection method, and the column temperature was 35 ℃; the inner diameter of the column was 2.1 mm , the length is 50-150 mm, the particle size of the packing is 1.7-1.8 μm; the two phases are methanol and 0.2% formic acid aqueous solution as the mobile phase, the volume ratio is 1:9, and the column flow rate is 0.2-0.3 mL/min; The detector or diode array detector is the detector, the detection wavelength range is 230-320 nm; the single sample injection volume is 1-5 μL, and the analysis time is 5-10 min; d. Calculate the content of GS-441524 in the test sample by the peak area according to the external standard method. 2. The detection method according to claim 1, characterized in that: in step b, in terms of the weight of the test sample, the test sample solution concentration is 10.0 μg/mL.

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