Separation and detection method of tedizolid phosphate and enantiomer

Abstract

The invention provides a tedizolid phosphate and enantiomer separation and detection method, and belongs to the field of pharmaceutical analysis. The tedizolid phosphate and the enantiomer can be effectively separated and accurately measured by adopting phenyl-hexyl bonded silica gel as a filling agent and taking a reversed-phase mixed solvent as a flowing phase. The method disclosed by the invention is simple, convenient, high in sensitivity, good in separation degree and reproducibility and accurate and reliable in result, and can be used for detecting tedizolid phosphate enantiomers.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a high performance liquid chromatography separation and detection method for tedizolid phosphate and its enantiomers. Background Art

[0002] Tedizolid phosphate is a prodrug of oxazolidinone phosphate. The antibacterial activity of tetazole is mediated by binding to the 50S subunit of the bacterial ribosome, thereby inhibiting protein synthesis. Tedizolid phosphate is mainly active against Gram-positive bacteria. Tedizolid phosphate has antibacterial effects on enterococci, staphylococci and streptococci in vitro. The chemical name is: (5R)-(3-{3-fluoro-4-[6-(2-methyl-2H-tetrazol-5-yl)pyridin-3-yl]phenyl}-2-oxooxazolidin-5-yl)methyl dihydrogen phosphate, and its chemical structure is as follows:

[0003]

[0004] There is a chiral center in tedizolid phosphate. According to the synthetic process route of tedizolid phosphate and the starting materials used in the synthesis, it can be known that enantiomers exist in the synthetic route of this product.

[0005] Since enantiomers are difficult to separate on ordinary high performance liquid chromatography, a method for detecting the enantiomers of tedizolid phosphate is developed to detect and separate the enantiomers of tedizolid phosphate, thereby ensuring the effectiveness and safety of tedizolid phosphate products.

[0006] Isomers produced during the preparation of tedizolid phosphate need to be strictly controlled, whether in raw materials or preparations. At present, the quality standard of tedizolid phosphate is not included in the Chinese Pharmacopoeia, and there is a method for detecting enantiomers. It is reported in the literature that the chromatographic column used is an acidic glycoprotein column. Since the protein in the chromatographic column is easily denatured and inactivated, there are certain restrictions on the type and concentration of the organic phase when using it, and the maintenance of the chromatographic column is more troublesome when using it.

[0007] Therefore, it is necessary to develop a simple, feasible, sensitive and specific analysis and detection method for tedizolid phosphate and its enantiomers for the quality control of tedizolid phosphate, which is of great significance for achieving safety assurance of tedizolid phosphate. Summary of the invention

[0008] The present invention provides a high performance liquid chromatography separation and detection method for tedizolid phosphate and its enantiomers. The method has the advantages of simplicity, high efficiency and high accuracy, and can accurately reflect the content of isomers in the tedizolid phosphate raw material, thereby being able to better control and master product quality and improve the safety of clinical medication.

[0009] In view of the deficiencies in the background technology, the present invention provides a method for detecting the enantiomers of tedizolid phosphate, which has the advantages of simplicity, high efficiency and high accuracy, and can accurately reflect the content of the enantiomers in the tedizolid phosphate raw material, thereby being able to better control and master the product quality and improve the safety of clinical drug use.

[0010] The present invention adopts the following technical solutions:

[0011] The invention provides a high performance liquid chromatography for effectively separating tedizolid phosphate and its enantiomers.

[0012] Analysis method. The method comprises: determining according to high performance liquid chromatography, using a phenyl-hexyl bonded silica gel chromatography column, using phosphate buffer (take 5.6g of anhydrous disodium hydrogen phosphate, 5.7g of β-cyclodextrin, add 1000ml of water to dissolve by ultrasound, add 3ml of triethylamine, and adjust the pH value to 8.0 with phosphoric acid)-acetonitrile as the mobile phase, and performing isocratic elution under certain conditions. It comprises the following steps:

[0013] Detection on high performance liquid chromatography

[0014] Chromatographic column filler: phenyl-hexyl bonded silica gel

[0015] Column temperature: 15℃~20℃

[0016] Flow rate: 0.8mL / min-1.2mL / min

[0017] Detection wavelength: 300nm

[0018] Mobile phase: phosphate buffer-acetonitrile

[0019] Diluent: Mobile phase

[0020] Preparation of system suitability solution: Accurately weigh the tedizolid phosphate reference substance and the enantiomer reference substance, and dilute them with diluent to make each ml contain about 1 mg of tedizolid phosphate and 1 μg of enantiomer, as the system suitability solution;

[0021] Sample solution preparation: Take the tedizolid phosphate sample to be tested and use the diluent to prepare a solution with a concentration of 0.8mg-1.2mg / ml as the test solution;

[0022] The chromatographic column uses phenyl-hexyl bonded silica gel as a filler, preferably a GL.Inertsustain Phenylhexy (4.6 mm×250 mm, 5 μm) column.

[0023] The volume ratio of the mobile phase phosphate buffer (5.6 g of anhydrous disodium hydrogen phosphate and 5.7 g of β-cyclodextrin are added to 1000 ml of water and dissolved by ultrasound, 3 ml of triethylamine is added, and the pH value is adjusted to 8.0 with phosphoric acid) to acetonitrile is 960-1040:100-150.

[0024] The column temperature is 15-20°C, preferably 17°C.

[0025] The flow rate is 0.8 mL / min-1.2 mL / min, preferably 1.0 mL / min.

[0026] The wavelength is 300 nm.

[0027] The peaks in the system suitability solution can achieve baseline separation, with a separation degree of ≥1.0.

[0028] By adopting the detection method provided by the present invention, tedizolid phosphate and its enantiomers can be effectively separated, the effectiveness of the detection can be ensured, and the quality of tedizolid phosphate can be accurately and strictly controlled. The method has strong specificity, high sensitivity, good accuracy and durability. DETAILED DESCRIPTION

[0029] To explain the present invention in detail, the following is further described in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and do not limit the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The HPLC chromatogram of the solvent in Example 1 is

[0031] Figure 2 4 is the HPLC chromatogram of the system suitability solution in Example 1.

[0032] Figure 3 It is the high performance liquid chromatogram of the tedizolid phosphate test solution in Example 1.

[0033] Example 1

[0034] Specificity test

[0035] Adopt the method in the present invention.

[0036] Chromatographic column: GL.Inertsustain Phenylhexy (4.6mm×250mm, 5μm)

[0037] Mobile phase: phosphate buffer (take 5.6g of anhydrous disodium hydrogen phosphate, 5.7g of β-cyclodextrin, add 1000ml of water and ultrasonically dissolve, add 3ml of triethylamine, and adjust the pH value to 8.0 with phosphoric acid)-acetonitrile volume ratio is 1000:130

[0038] Flow rate: 1.0ml / min

[0039] Column temperature: 17°C

[0040] Detection wavelength: 300nm

[0041] Injection volume: 5 μl

[0042] Experimental steps:

[0043] Blank solvent: mobile phase

[0044] Preparation of system suitability solution: Accurately weigh the tedizolid phosphate reference substance and the enantiomer reference substance, and dilute them with diluent to make each ml contain about 1 mg of tedizolid phosphate and 1 μg of enantiomer, as the system suitability solution;

[0045] Preparation of sample solution: Take an appropriate amount of this product, weigh it accurately, place it in a volumetric flask, dissolve it with mobile phase and dilute it to make a 1 mg / ml solution.

[0046] Preparation of control solution: Take an appropriate amount of sample solution and dilute it with mobile phase to make a solution containing about 1 μg / ml.

[0047] Take each of the above solutions, inject them into a high performance liquid chromatograph respectively, and record the chromatogram.

[0048] The results of blank solvent are shown in the attached Figure 1 .

[0049] The results of the system suitability solution are attached. Figure 2 .

[0050] The results of the sample solution are shown in the attached Figure 3 .

[0051] Results: The blank solvent did not interfere with the detection of tedizolid phosphate and its enantiomers, and the method had good specificity. Figure 2 The peaks from left to right are enantiomers and tedizolid phosphate, with a resolution of 1.3, which is greater than 1.0 and can achieve effective separation.

[0052] Example 2

[0053] Limit of detection and limit of quantitation tests.

[0054] The chromatographic method in Example 1 was used.

[0055] Experimental steps:

[0056] Blank solvent: mobile phase

[0057] Detection limit and quantification limit solution: Take appropriate amount of tedizolid phosphate reference substance and enantiomer reference substance, add mobile phase to prepare a series of concentration solutions, and measure according to the law. The quantification limit and detection limit of the substance are determined by the signal-to-noise ratio. The results are shown in the table below:

[0058] Limit of Quantitation Results

[0059]

[0060] Detection limit results

[0061] name Detection limit concentration (μg / ml) Equivalent to the percentage of test sample concentration (%) limit(%) S / N Enantiomers 0.0715 0.0071 0.10 6.3 Tedizolid phosphate 0.0821 0.0082 / 8.5

[0062] Example 3

[0063] Linear Relationship and Range

[0064] The chromatographic method in Example 1 was used.

[0065] Experimental steps:

[0066] Blank solvent: mobile phase

[0067] Linear solution: Take appropriate amount of tedizolid phosphate reference substance and enantiomer reference substance, add anhydrous ethanol to prepare solutions of a series of concentrations, and measure according to the method.

[0068] Results: The linear equation of the enantiomers of tedizolid phosphate was y=0.2806x+0.0152, r=0.9998 (n=6) in the concentration range of 0.2483-2.9790 μg / ml; the linear equation of tedizolid phosphate was y=0.3211x+0.0139, r=0.9988 (n=6) in the concentration range of 0.2740-3.2880 μg / ml; the linear results were good.

[0069] Example 4

[0070] Recovery test

[0071] The chromatographic method in Example 1 was used.

[0072] Experimental steps:

[0073] Accurately weigh an appropriate amount of tedizolid phosphate, place it in a volumetric flask, accurately add an appropriate amount of tedizolid phosphate enantiomer stock solution, dissolve it with anhydrous ethanol and dilute it to the scale, prepare solutions with relative concentrations of 50%, 100%, and 150%, shake well, and use them as test solutions. Prepare 3 copies of each concentration in parallel. Accurately weigh an appropriate amount of tedizolid phosphate, dissolve it with ethanol and dilute it to the scale, shake well, and use it as a blank sample solution. Accurately measure an appropriate amount of tedizolid phosphate enantiomer stock solution, quantitatively dilute it with the mobile phase to prepare a solution of appropriate concentration as the reference solution. Accurately measure 5μl of each of the above solutions, inject them into the liquid chromatograph respectively, and record the chromatogram and peak area. Calculate the recovery rate.

[0074] Results: The recovery rates of tedizolid phosphate enantiomers at each concentration were in line with the requirements of the pharmacopoeia. The accuracy of this method was good. The specific data are shown in the table below:

[0075] Tedizolid phosphate enantiomer recovery results

[0076]

[0077] The recoveries of the sample at the limit values ​​of 50%, 100% and 150% were 93.7% to 95.2% for the enantiomers of tedizolid phosphate, respectively. The results showed that the method had good accuracy.

[0078] Example 4 Durability test of the method of the present invention

[0079] The tolerance of the test method was investigated by changing parameters such as flow rate, column temperature, and mobile phase ratio. Only one parameter was changed in each test, and the results were compared with the results when the parameters were not changed. The separation of enantiomers and tedizolid phosphate was not less than 1.0, which met the requirements. The specific results are shown in the table below:

[0080]

[0081] Example 5 Sample detection test

[0082] 1. Instruments and conditions:

[0083] High performance liquid chromatograph: Thermofisher Dixonex U3000, UV detector

[0084] Chromatographic column: Phenyl-hexyl bonded silica gel GL.Inertsustain Phenylhexy (4.6mm×250mm, 5μm)

[0085] Mobile phase: phosphate buffer [5.7 g of β-cyclodextrin and 5.6 g of anhydrous disodium hydrogen phosphate were dissolved in water by ultrasonication and diluted to 1000 ml, then 3 ml of triethylamine was added and the pH value was adjusted to 8.0 with phosphoric acid], the volume ratio was 1000:130

[0086] Flow rate: 1.0ml / min

[0087] Column temperature: 17°C

[0088] Injection volume: 5 μl

[0089] Detection wavelength: 300nm

[0090] 2. Experimental methods and results

[0091] Diluent: Mobile phase

[0092] Preparation of system suitability solution: Accurately weigh the tedizolid phosphate reference substance and the enantiomer reference substance, and dilute them with the mobile phase to make each ml contain about 1 mg of tedizolid phosphate and 1 μg of enantiomer, as the system suitability solution;

[0093] Sample solution preparation: Take the tedizolid phosphate sample to be tested and use the diluent to prepare a solution with a concentration of 1.0 mg / ml as the test solution; take 5 μl of each of the above solutions, inject them into the liquid chromatograph in sequence, and record the chromatogram.

[0094] Table 4 Tedizolid phosphate isomer detection experimental results

[0095] batch number Enantiomers TD-20200701 Not detected TD-20200702 Not detected TD-20200703 Not detected

Claims

1. A detection and analysis method for tedizolid phosphate and its enantiomers, characterized in that: phenyl-hexyl bonded silica gel is used as filler, the mobile phase is a normal phase mixed solvent of phosphate buffer-acetonitrile, and the volume ratio of [5.7 g of β-cyclodextrin and 5.6 g of anhydrous disodium hydrogen phosphate is added to water and ultrasonically dissolved and diluted to 1000 ml, then 3 ml of triethylamine is added, and the pH value is adjusted to 8.0 with phosphoric acid]-acetonitrile is 960-1040:100-150.

2. The method for detecting the enantiomers of tedizolid phosphate as claimed in claim 1, characterized in that: The volume ratio of [5.7 g of β-cyclodextrin and 5.6 g of anhydrous disodium hydrogen phosphate are added to water and ultrasonically dissolved and diluted to 1000 ml, then 3 ml of triethylamine is added and the pH value is adjusted to 8.0 with phosphoric acid] is 1000:

130.

3. The method for detecting tedizolid phosphate and its enantiomers as claimed in claim 2, characterized in that: The high performance liquid chromatography detection adopts the following optional chromatographic conditions: the mobile phase flow rate is 0.8-1.2 ml / min; the chromatographic column temperature is 15-20° C.; the detector adopts an ultraviolet detector, and the detection wavelength is 300 nm.

4. It is characterized by: The flow rate of the mobile phase is 1.0 ml / min; the temperature of the chromatographic column is 17°C.

5. The method for detecting the enantiomers of tedizolid phosphate according to any one of claims 1 to 4, characterized in that: The detection method includes three steps: preparation of the test solution, preparation of the system suitability solution and determination: The preparation of the test solution comprises: taking a sample of tedizolid phosphate to be tested, and using a diluent to prepare a solution with a concentration of 0.8 mg to 1.2 mg / ml as the test solution; The preparation of the system suitability solution comprises: accurately weighing the tedizolid phosphate reference substance, the diastereomer and the enantiomer The reference substance was diluted with diluent to prepare a solution containing about 1 mg of tedizolid phosphate and 1 μg of the enantiomer per 1 ml as the system suitability solution; The determination steps include: taking 5 μl of the system suitability solution and injecting it into a high performance liquid chromatograph, and the separation degree of the tedizolid phosphate peak and the enantiomer peak must not be less than 1.5; then taking 10 μl of the test solution and injecting it into the liquid chromatograph, and recording the chromatogram.

6. The separation and detection method according to any one of claims 1 to 5, characterized in that In the method, the separation degree between the peaks of tedizolid phosphate and the enantiomeric derivatives is not less than 1.0, and the repeatability, accuracy and sensitivity of the method can meet the detection requirements.

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