Method for detecting and analyzing pyridine compounds in 2-pyridone and application

Abstract

The invention discloses a method for detecting and analyzing pyridine compounds in 2-pyridone and application, and belongs to the technical field of medicine analysis. According to the detection and analysis method, reversed-phase high-performance liquid chromatography is adopted for detection, and chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, a buffer salt solution is used as a mobile phase A, and an organic solvent is used as a mobile phase B for gradient elution. The method disclosed by the invention can be used for detecting the content of the 5-chloro-2, 4-dihydroxypyridine or / and controlling the quality of the 5-chloro-2, 4-dihydroxypyridine. The method is scientific in design, ingenious in conception, simple and convenient in detection condition, high in applicability and short in analysis period; and the medicine quality and the medication safety are effectively ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a detection and analysis method for pyridine compounds in 2-pyridones and an application thereof. Background Art

[0002] The compound preparation Tegafur contains tegafur, gimeracil, and oteracil potassium, and is used to treat gastric cancer, colorectal cancer, and head and neck tumors. The parent nuclei of tegafur, gimeracil, and oteracil potassium all contain 2-pyridone compounds, which have the following balance:

[0003]

[0004] Therefore, monitoring and controlling pyridine compounds in products is the key to ensuring drug quality.

[0005] The prior art discloses a method for detecting impurities in the compound preparation of S-170, such as CN102368063A discloses a method for detecting related substances in a pharmaceutical composition containing tegafur, gimeracil (5-chloro-4-hydroxy-2(1H)-pyridone) and oteracil potassium, using HPLC for detection, gradient elution, a chromatographic column with octyl or octadecylsilane bonded silica as filler, mobile phase A is a salt solution containing an ion pair reagent or a mixed solution thereof with either or both of methanol and acetonitrile, and mobile phase B is a mixed solution of either or both of acetonitrile and methanol. CN 102253152 A discloses a method for determining S-170 capsules by high-performance liquid chromatography, using a chromatographic column with octadecylsilane bonded silica as filler, and using methanol as a mixed solvent of an organic phase and an aqueous phase as a mobile phase, wherein the aqueous phase contains a cation pair reagent. In the methods for detecting compound preparations containing gimeracil in the prior art, ion pair reagents are used in the mobile phase, which causes great damage to the chromatogram and requires a long equilibrium time. In addition, the prior art methods are all aimed at the compound preparations of Tegafur and related substances, and the intermediates and by-products of gimeracil (5-chloro-4-hydroxy-2(1H)-pyridone) are not controlled. Therefore, the methods cannot be directly used for the quality control of the raw material of gimeracil (5-chloro-4-hydroxy-2(1H)-pyridone).

[0006] Therefore, it is an urgent problem to be solved by those skilled in the art to provide a method for quickly and effectively detecting gimeracil (5-chloro-4-hydroxy-2(1H)-pyridone) and its analogs, which is simple and convenient to operate and has accurate results. Summary of the invention

[0007] One of the purposes of the present invention is to provide a detection and analysis method for pyridine compounds in 2-pyridones, which can achieve good separation of multiple pyridine compounds in 2-pyridones without using ion pair reagents, thereby improving the service life of the chromatographic column and shortening the analysis and detection time.

[0008] The second object of the present invention is to provide an application of the detection and analysis method in the detection of 5-chloro-4-hydroxy-2(1H)-pyridone content and / or the quality control of 5-chloro-4-hydroxy-2(1H)-pyridone.

[0009] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0010] The invention discloses a detection and analysis method for pyridine compounds in 2-pyridone, comprising the steps of selecting chromatographic conditions, preparing test sample and reference sample solutions, and injecting and detecting the chromatographic conditions, wherein the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, a buffered salt solution is used as a mobile phase A, and an organic solvent is used as a mobile phase B for gradient elution, wherein the pH value of the mobile phase A is 2.5-3.5.

[0011] The organic solvent in some embodiments of the present invention is selected from one or more of acetonitrile, methanol, ethanol and isopropanol, and the buffered saline solution is a phosphate buffered saline solution, preferably the phosphate buffered saline solution is one or more of potassium dihydrogen phosphate solution, sodium dihydrogen phosphate solution, sodium hydrogen phosphate solution, and potassium hydrogen phosphate solution.

[0012] The gradient elution program in some embodiments of the present invention is as follows: 0-5 min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-20 min, the volume of mobile phase A is from 92-97% to 5-25%, and the volume of mobile phase B is from 3-8% to 75-95%.

[0013] In some embodiments of the present invention, the gradient elution program is: 0-5 min, mobile phase A volume is 95%, mobile phase B volume is 5%, 5-20 min, mobile phase A volume from 95% to 10%, mobile phase B volume from 5% to 90%.

[0014] In some embodiments of the present invention, the gradient elution program is: 0-5 min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-15 min, the volume of mobile phase A is from 92-97% to 65-75%, the volume of mobile phase B is from 3-8% to 25-35%, 15-20 min, the volume of mobile phase A is from 65-75% to 5-25%, and the volume of mobile phase B is from 25-35% to 75-95%.

[0015] In some embodiments of the present invention, the gradient elution program is: 0-5min, the volume of mobile phase A is 95%, the volume of mobile phase B is 5%, 5-15min, the volume of mobile phase A is from 95% to 70%, the volume of mobile phase B is from 5% to 30%, 15-20min, the volume of mobile phase A is from 70% to 20%, and the volume of mobile phase B is from 30% to 80%.

[0016] In some embodiments of the present invention, the gradient elution program is: 0-5 min, the volume of mobile phase A is 95%, the volume of mobile phase B is 5%, 5-15 min, the volume of mobile phase A is from 95% to 70%, the volume of mobile phase B is from 5% to 30%, 15-20 min, the volume of mobile phase A is from 70% to 20%, and the volume of mobile phase B is from 30% to 90%.

[0017] In some embodiments of the present invention, the gradient elution program is: 0-5min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-10min, the volume of mobile phase A is from 92-97% to 80-90%, the volume of mobile phase B is from 3-8% to 10-15%, 10-15min, the volume of mobile phase A is from 80-90% to 65-75%, the volume of mobile phase B is from 10-15% to 25-35%, 15-20min, the volume of mobile phase A is from 65-75% to 5-25%, and the volume of mobile phase B is from 25-35% to 75-95%.

[0018] In some embodiments of the present invention, the gradient elution program is: 0-5min, the volume of mobile phase A is 95%, the volume of mobile phase B is 5%, 5-10min, the volume of mobile phase A is from 95% to 88%, the volume of mobile phase B is from 5% to 12%, 10-15min, the volume of mobile phase A is from 88% to 70%, the volume of mobile phase B is from 12% to 30%, 15-20min, the volume of mobile phase A is from 70% to 20%, and the volume of mobile phase B is from 30% to 80%.

[0019] Some embodiments of the present invention further include a chromatographic column equilibration program after 20 minutes, wherein the chromatographic column equilibration program starts at 20 minutes, the volume of mobile phase A decreases from 92-97% to 5-25%, and the volume of mobile phase B decreases from 3-8% to 75-95% to equilibrate the chromatographic column.

[0020] In some embodiments of the present invention, the gradient volume changes in the gradient elution program at a uniform or non-uniform rate.

[0021] In some embodiments of the present invention, the buffered saline solution is a phosphate buffered saline solution with a concentration of 0.01 to 0.05 mol / L, and preferably the concentration of the potassium dihydrogen phosphate solution or sodium dihydrogen phosphate solution buffered saline solution is 0.01 to 0.02 mol / L.

[0022] In some embodiments of the present invention, an ultraviolet detector is used, preferably, the detection wavelength is 220 nm.

[0023] In some embodiments of the present invention, the column temperature of the chromatographic column is 30-40° C., and the flow rate of the mobile phase is 0.8-1.2 ml / min; preferably, the flow rate of the mobile phase is 1.0 ml / min.

[0024] The present invention discloses an application of pyridine compounds in the above-mentioned 2-pyridones, and the method is used for detecting the content of 5-chloro-4-hydroxy-2(1H)-pyridone and / or controlling the quality of 5-chloro-4-hydroxy-2(1H)-pyridone.

[0025] In some embodiments of the present invention, a method for detecting and analyzing pyridine compounds in 2-pyridones is provided, wherein the pyridine compounds in the 2-pyridones include at least one of the following compounds:

[0026] 5-Chloro-4-hydroxy-2(1H)-pyridinone

[0027] 5-Chloro-2-hydroxy-3-cyano-4-methoxypyridine,

[0028] 5-Chloro-2,4-dihydroxy-3-cyanopyridine,

[0029] 2,4-Dihydroxypyridine,

[0030] 5-Chloro-2,4-dihydroxypyridine-3-carboxamide,

[0031] 5-Chloro-2,4-dihydroxypyridine-3-carboxylic acid,

[0032] 5-Chloro-4-methoxy-2-hydroxypyridine,

[0033] 5-Chloro-4-hydroxy-1-methyl-2(1H)pyridinone.

[0034] The chemical formula of the above compounds and the corresponding numbers in the present invention are shown in the following table:

[0035] Table 1

[0036]

[0037] The impurities described in the present invention are all prior art.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The invention is scientifically designed and ingeniously conceived. The detection conditions of the method are simple and convenient, the applicability is strong, and the analysis cycle is short. The method can not only be used for detecting the content of 5-chloro-4-hydroxy-2(1H)-pyridone, but also can realize the control of 7 known compounds in 5-chloro-4-hydroxy-2(1H)-pyridone, thereby effectively ensuring the quality of the medicine and the safety of the medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Attached Figure 1 It is the mixed chromatogram of Example 1;

[0041] Attached Figure 2 It is the mixed solution chromatogram of Example 2;

[0042] Attached Figure 3 It is the mixed solution chromatogram of Example 3;

[0043] Attached Figure 4 It is the mixed solution chromatogram of Example 4;

[0044] Attached Figure 5 It is the mixed solution chromatogram of Comparative Example 1;

[0045] Attached Figure 6 It is the mixed solution chromatogram of Comparative Example 2;

[0046] Attached Figure 7 It is the mixed solution chromatogram of Comparative Example 3;

[0047] Attached Figure 8 It is the mixed solution chromatogram of Comparative Example 4;

[0048] Attached Fig. 9 It is the mixed solution chromatogram of Comparative Example 4;

[0049] Attached Fig.10 It is the chromatogram of the mixed solution of Comparative Example 5. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0051] Example 1

[0052] This embodiment discloses a method for measuring M-1, (M-1)-Z02, Z01, Z02, Z03, Z04, and Z05 in 5-chloro-4-hydroxy-2(1H)-pyridone by high performance liquid chromatography. The detection method is as follows:

[0053] Solution preparation

[0054] Single-label positioning solution: Take 5-chloro-4-hydroxy-2(1H)-pyridone pyridine reference substance and use acetonitrile-water (10:90) as solvent to prepare 5-chloro-4-hydroxy-2(1H)-pyridone positioning solution with a concentration of 10 μg / ml;

[0055] The positioning solutions of M-1, (M-1)-Z02, Z01, Z02, Z03, Z04 and Z05 were prepared in the same way.

[0056] Mixed solution: Take M-1 reference substance, (M-1)-Z02 reference substance, Z01 reference substance, Z02 reference substance, Z03 reference substance, Z04 reference substance, and Z05 reference substance respectively, use acetonitrile-water (10:90) as solvent, and prepare a mixed solution containing 0.5 mg / ml of 5-chloro-4-hydroxy-2(1H)-pyridone and 10 μg / ml of the remaining substances.

[0057] Chromatographic conditions: C18 column, 4.6×150 mm, 3.5 μm, 0.02 mol / L KH2PO4 buffer solution (pH adjusted to 2.5 with phosphoric acid) as mobile phase A, acetonitrile as mobile phase B for gradient elution, flow rate of 1.0 ml / min; column temperature: 35°C; detection wavelength: 220 nm, gradient elution program is as shown in Table 2:

[0058] Table 2

[0059] Time / min 0 5 15 20 24 24.1 30 Mobile phase A / % 95 95 70 20 20 95 95 Mobile phase B / % 5 5 30 80 80 5 5

[0060] Take 20 μl of each single-label positioning solution and mixed solution, inject them into the liquid chromatograph, measure them according to the chromatographic conditions, and record the chromatogram. The results are shown in the attached figure. Figure 1 shown.

[0061] The experimental results show that the separation between 5-chloro-2-hydroxy-3-cyano-4-methoxypyridine and 7 analogues is good, baseline separation is achieved, and the peak symmetry is good.

[0062] Example 2

[0063] Compared with Example 1, the gradient elution procedure of this embodiment is different, and the other conditions are the same. The gradient elution procedure in this embodiment is shown in Table 3 below:

[0064] Table 3

[0065] Time / min 0 5 10 15 20 24 24.1 30 Mobile phase A / % 95 95 88 70 20 20 95 95 Mobile phase B / % 5 5 12 30 80 80 5 5

[0066] The results are attached Figure 2 As shown in the figure, the separation between gimeracil and several other compounds is good, baseline separation is achieved, and the peaks are symmetrical.

[0067] Example 3

[0068] Compared with Example 1, this example has different mobile phase pH value and gradient elution program, and the other conditions are the same. The mobile phase in this example: phase A: 10mmol / L potassium dihydrogen phosphate (pH3.0), phase B: acetonitrile, and the gradient elution program is shown in Table 4 below:

[0069] Table 4

[0070] Time / min 0 5 20 20.5 25 Phase B / % 5 5 90 5 5 Phase A / % 95 95 10 95 95

[0071] The results are attached Figure 3 As shown, the components can be effectively separated.

[0072] Example 4

[0073] Compared with Example 1, the mobile phase and gradient elution procedure of this comparative example are different, and the other conditions are the same. The mobile phase in this comparative example: phase A: 10mmol / L potassium dihydrogen phosphate (pH2.5), phase B: methanol, and the gradient elution procedure is shown in Table 5 below:

[0074] Table 5

[0075] Time / min 0 5 15 20 20.1 25 Phase B / % 5 5 30 90 5 5 Phase A / % 95 95 70 10 95 95

[0076] The results are attached Figure 4 As shown, the components can be effectively separated.

[0077] Comparative Example 1

[0078] Compared with Example 1, the mobile phase pH value and gradient elution program of this comparative example are different, and the other conditions are the same. The mobile phase in this comparative example: phase A: 10mmol / L potassium dihydrogen phosphate (pH6.5), phase B: acetonitrile, and the gradient elution program is shown in Table 6:

[0079] Table 6

[0080] Time / min 0 5 20 25 25.5 30 Phase B / % 5 5 90 90 5 5 Phase A / % 95 95 10 10 95 95

[0081] Results attached Figure 5 As shown, the detection separation requirements cannot be met.

[0082] Comparative Example 2

[0083] Compared with Example 1, the mobile phase and gradient elution procedure of this comparative example are different, and the other conditions are the same. The mobile phase A in this comparative example is: 10mmol / L potassium dihydrogen phosphate (pH3.0), phase B is: methanol, and the gradient elution procedure is shown in Table 7 below:

[0084] Table 7

[0085] Time / min 0 5 20 20.5 25 Phase B / % 5 5 90 5 5 Phase A / % 95 95 10 95 95

[0086] The results are attached Figure 6 As shown, Z02 and Z03 are not separated.

[0087] Comparative Example 3

[0088] Compared with Example 1, the mobile phase and gradient elution procedure of this comparative example are different, and the other conditions are the same. The mobile phase in this comparative example: phase A: 10mmol / L potassium dihydrogen phosphate (pH3.5), phase B: methanol, and the gradient elution procedure is shown in Table 8 below:

[0089] Table 8

[0090] Time / min 0 5 15 20 20.1 25 Phase B / % 5 5 30 90 5 5 Phase A / % 95 95 70 10 95 95

[0091] The results are attached Figure 7 As shown, Z01 and (M-1)-Z02 were not separated, and the peak shape of Z04 deteriorated.

[0092] Comparative Example 4

[0093] Compared with Example 1, this comparative example is the oxidative damage sample detection of Example 1. Specifically, about 12.5 mg of gimeracil is taken, placed in a 25 ml volumetric flask, 1 ml of 0.3% hydrogen peroxide is added, dissolved with a solvent and diluted to the scale, placed at room temperature, filtered, and the filtrate is taken and measured under the conditions of Example 1. Impurities cannot be separated from the main component baseline, as shown in FIG. Figure 8 , the separation effect was good when the conditions of Example 2 were used. Fig. 9 .

[0094] Comparative Example 5

[0095] Compared with Example 1, the column temperature was changed to 30°C and 40°C respectively, and the other conditions were not changed. Fig.10 ,The results showed that changing the column temperature did not affect the detection and separation of the above compounds.

[0096] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for detecting and analyzing pyridine compounds in 2-pyridones, comprising selecting chromatographic conditions, preparing test and reference solutions, and injecting and detecting the chromatographic conditions, wherein the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler, a buffered salt solution as a mobile phase A, an organic solvent as a mobile phase B, and eluting with a gradient program, wherein the pH value of the mobile phase A by volume is 2.5 to 3.

5.

2. The method for detecting and analyzing pyridine compounds in 2-pyridones according to claim 1, characterized in that: The organic solvent is selected from one or more of acetonitrile, methanol, ethanol and isopropanol, and the buffered saline solution is a phosphate buffered saline solution. Preferably, the phosphate buffered saline solution is one or more of potassium dihydrogen phosphate solution, sodium dihydrogen phosphate solution, sodium hydrogen phosphate solution and potassium hydrogen phosphate solution.

3. The method for detecting and analyzing pyridine compounds in 2-pyridones according to claim 2, characterized in that The gradient elution program is: 0-5min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-20min, the volume of mobile phase A is from 92-97% to 5-25%, and the volume of mobile phase B is from 3-8% to 75-95%. The preferred gradient elution is: 0-5min, the volume of mobile phase A is 95%, the volume of mobile phase B is 5%, 5-20min, the volume of mobile phase A is from 95% to 10%, and the volume of mobile phase B is from 5% to 90%.

4. The method for detecting and analyzing pyridine compounds in 2-pyridones according to claim 2, characterized in that The gradient elution program is as follows: 0-5min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-15min, the volume of mobile phase A is from 92-97% to 65-75%, the volume of mobile phase B is from 3-8% to 25-35%, 15-20min, the volume of mobile phase A is from 65-75% to 5-25%, and the volume of mobile phase B is from 25-35% to 75-95%; The preferred gradient elution program is: 0-5min, mobile phase A volume is 95%, mobile phase B volume is 5%, 5-15min, mobile phase A volume is from 95% to 70%, mobile phase B volume is from 5% to 30%, 15-20min, mobile phase A volume is from 70% to 20%, mobile phase B volume is from 30% to 80%; Or gradient elution program: 0-5min, mobile phase A volume is 95%, mobile phase B volume is 5%, 5-15min, mobile phase A volume from 95% to 70%, mobile phase B volume from 5% to 30%, 15-20min, mobile phase A volume from 70% to 20%, mobile phase B volume from 30% to 90%.

5. The method for detecting and analyzing pyridine compounds in 2-pyridones according to claim 2, characterized in that: The gradient elution program is as follows: 0-5min, the volume of mobile phase A is 92-97%, the volume of mobile phase B is 3-8%, 5-10min, the volume of mobile phase A is from 92-97% to 80-90%, the volume of mobile phase B is from 3-8% to 10-15%, 10-15min, the volume of mobile phase A is from 80-90% to 65-75%, the volume of mobile phase B is from 10-15% to 25-35%, 15-20min, the volume of mobile phase A is from 65-75% to 5-25%, and the volume of mobile phase B is from 3-8% to 10-15%. The volume of phase B is from 25-35% to 75-95%. The preferred gradient elution program is: 0-5min, the volume of mobile phase A is 95%, the volume of mobile phase B is 5%, 5-10min, the volume of mobile phase A is from 95% to 88%, the volume of mobile phase B is from 5% to 12%, 10-15min, the volume of mobile phase A is from 88% to 70%, the volume of mobile phase B is from 12% to 30%, 15-20min, the volume of mobile phase A is from 70% to 20%, and the volume of mobile phase B is from 30% to 80%.

6. The method for detecting and analyzing pyridine compounds in 2-pyridones according to any one of claims 1 to 5, characterized in that The buffered saline solution is a phosphate buffered saline solution with a concentration of 0.01 to 0.05 mol / L, and preferably the potassium dihydrogen phosphate solution or sodium dihydrogen phosphate solution buffered saline solution has a concentration of 0.01 to 0.02 mol / L.

7. A method for detecting and analyzing pyridine compounds in 2-pyridones according to any one of claims 1 to 6, characterized in that It further comprises a chromatographic column balancing procedure after 20 minutes, wherein the chromatographic column balancing procedure starts from 20 minutes, the volume of mobile phase A decreases from 92-97% to 5-25%, and the volume of mobile phase B decreases from 3-8% to 75-95% to balance the chromatographic column.

8. The method for detecting and analyzing pyridine compounds in 2-pyridones according to claims 1-7, characterized in that: The pyridine compound in the 2-pyridones includes at least one of the following compounds: 5-Chloro-4-hydroxy-2(1H)-pyridinone 5-Chloro-2-hydroxy-3-cyano-4-methoxypyridine, 5-Chloro-2,4-dihydroxy-3-cyanopyridine, 2,4-Dihydroxypyridine, 5-Chloro-2,4-dihydroxypyridine-3-carboxamide, 5-Chloro-2,4-dihydroxypyridine-3-carboxylic acid, 5-Chloro-4-methoxy-2-hydroxypyridine, 5-Chloro-4-hydroxy-1-methyl-2(1H)pyridinone.

9. The use of a method for detecting and analyzing pyridine compounds in 2-pyridones according to any one of claims 1 to 8, characterized in that: The method is used for 5-chloro-4-hydroxy-2(1H)-pyridone content detection and / or 5-chloro-4-hydroxy-2(1H)-pyridone quality control.

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