CN113358792B - HPLC content analysis method of medicinal substance - Google Patents

Abstract

The invention relates to an HPLC content analysis method of a medical substance with pyridine substituent, which takes a silane bonding silica gel reverse phase chromatographic column with a long alkyl chain embedded with a polar amide group as a stationary phase; preparing a mobile phase from at least a mobile phase A and a mobile phase B, wherein the mobile phase A is selected from an aqueous solution containing a first modifying agent, and the mobile phase B is selected from an acetonitrile solution containing a second modifying agent; taking mobile phase B as a first diluent for diluting the medicinal substance with the pyridine substituent; the first modifier is greater than or equal to the mass volume percentage concentration (g/100 ml) of the second modifier in at least one period of time during which the mobile phase elutes the stationary phase adsorbed with the pharmaceutical substance.

Description

HPLC content analysis method of medicinal substance

Technical Field

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a HPLC content analysis method for a pharmaceutical substance.

Background

There are many kinds of medical substances having pyridine substituent groups, and it has been reported to establish a content analysis method of pyridine active substances based on HPLC technique, but the conventional methods do not consider the common nature of medical substances having pyridine substituent groups, and these established methods have at least the technical problem of poor durability. Although the prior art reports some methods for constructing chromatographic conditions for acidic and basic substances, no method for analyzing the HPLC content of a pharmaceutical substance having a substituent for selectively constructing pyridine has been reported.

Disclosure of Invention

One aspect of the present invention relates to a method for HPLC content analysis of a pharmaceutical substance having a pyridine substituent. In some embodiments, the method comprises at least the steps of:

taking a silane bonded silica gel reverse phase chromatographic column with a long alkyl chain embedded with a polar amide group as a stationary phase;

preparing a mobile phase from at least a mobile phase A and a mobile phase B, wherein the mobile phase A is selected from an aqueous solution containing a first modifying agent, and the mobile phase B is selected from an acetonitrile solution containing a second modifying agent;

taking mobile phase B as a first diluent for diluting the medicinal substance with the pyridine substituent;

the first modifier is greater than or equal to the mass volume percentage concentration (g/100 ml) of the second modifier in at least one period of time during which the mobile phase elutes the stationary phase adsorbed with the pharmaceutical substance.

The term "HPLC" includes High Performance Liquid Chromatography (High Performance Liquid Chromatography) and devices or methods that use Liquid as a mobile phase, pump a mobile phase such as a single solvent with different polarities or a mixed solvent and a buffer solution with different proportions into a chromatographic column containing a stationary phase using a High pressure infusion system, separate components in the column, and then detect the components in a detector, thereby analyzing a sample.

The term "content analysis" includes a quantitative method in which a medical substance having a pyridine substituent is used as an active ingredient, a qualitative method in which a medical substance having a pyridine substituent is used as a related substance, and other quantitative and qualitative methods equivalent to any of the foregoing methods.

The term "pharmaceutical substance with a pyridine substituent" is selected from but not limited to, nicotemam, isoniazid, isoniazone, propylthioisoniazid, 1,4-dihydro-2,6-dimethyl-4- (2-nitrophenyl) -3,5-dipicolinate dimethyl ester (nifedipine), (±) - (E) -2-Cyclohexyl-1- (6-methanesulfonyl-pyridin-3-yl) -cyclopropanecarboxylic acid thiazol-2-ylamide (±) - (E) -2-cyclohexenyl-1- (6-methanesulfonyl-pyridin-3-yl) -cyclopropenylcarboxylic acid thiazol-2-ylamide), (±) - (E) -2-cyclopentyl-1- [4- (pyridin-3-ylmethylsulfonyl) -phenyl ] -cyclopropanecarboxylic acid thiazol-2-ylamide, (±) - (E) -3- [ 2-Cyclohexyl-1- (thiazol-2-ylcarbamoyl) -cyclopropyl ] -N-pyridinecarboxamide, (±) - (E) -3- [ 2-Cyclohexyl-1- (pyridin-3-ylmethylsulfonyl) -phenyl ] -cyclopropanecarboxylic acid thiazol-2-ylamide, (±) - (E) -3- [ 2-Cyclohexyl-1- (thiazol-2-ylcarbamoyl) -cyclopropyl ] -N-3-methyl-3-pyridinecarboxamide, (±) - (E-cyclopentyl-2-ylcarboxamide Pyridin-2-yl-ethylsulfamoyl) -phenyl ] -cyclopropanecarboxylic acid thiazol-2-ylamide.

The term "silane-bonded silica reverse phase chromatography column with a polar amide group embedded in the long alkyl chain" refers to at least a chromatography column with a packing prepared by the following method, such as: silica particles are used as a support, alkyl chains with longer length (such as octadecyl chain, tetradecyl chain and octaalkyl chain) are used as a silane coupling agent, and amide groups are embedded in the silane coupling agent; for another example: on the basis of the long alkyl chain embedding the polar amide group of the previous method, the method also has diisopropyl, diisobutyl or isopropyl side groups; or may be selected from commercially available chromatographic columns: agilent InfinityLab Poroshell120, agilent Pursuit, XRs and XRs Ul tra, agilent Polaris HPLC columns, agilent HC-C18 (2) and TC-C18 (2) columns, ZORBAX Eclipse Plus C18, ZORBAX StableBond columns, agilent Zorbax Box RP, waters SunAir C18 reverse phase columns, CORTECS columns, XBri columns, watts PAH columns, μ Porasil/Porasil columns, DBS HYPERSIL C columns, symmetry C18 columns, ODStisil 5um columns.

The term "mobile phase is prepared from at least mobile phase a and mobile phase B", it is an object to facilitate direct use of a 2-channel, 4-channel liquid chromatograph, and although it is within the scope of some embodiments to mix mobile phase a and mobile phase B outside the liquid chromatograph to form the mobile phase, in other embodiments the mobile phase prepared by 2-channel, 4-channel liquid chromatograph exhibits unique characteristics, such as obtaining a more stable baseline, and a smaller tailing factor.

The term "the mobile phase a is selected from an aqueous solution containing a first modifier and the mobile phase B is selected from an acetonitrile solution containing a second modifier" means that the first modifier and the second modifier separately disposed in the mobile phase a and the mobile phase B have unique characteristics such as durability to maintain pH, durability to wavelength, and durability to column temperature, alone or in combination, in some embodiments, when the mobile phase is prepared.

The term "first modifier" means a substance having at least one physical or chemical property capable of changing a mobile phase or a stationary phase or a pharmaceutical substance having a pyridine substituent, such as a pH adjuster, an ion pair agent, and the like, selected from at least one or more of phosphates, alkylsulfonates, perfluoro-substituted linear organic acids, diethylamine, triethylamine, methylenediphosphonic acid (medronic acid);

the term "second modifier" means, for example, a substance having at least one physical or chemical property capable of changing a mobile phase or a stationary phase or a pharmaceutical substance having a pyridine substituent, such as a pH adjuster, an ion pair reagent, and the like, selected from at least one or more of alkyl sulfonate, perfluoro-substituted linear organic acid, diethylamine, triethylamine, methylene diphosphonic acid (meidronic acid);

the term "alkylsulfonate" is selected from at least one or more of sodium pentane sulfonate, sodium hexane sulfonate, sodium heptane sulfonate, sodium octane sulfonate, sodium decane sulfonate, sodium octane sulfonate;

the term "perfluoro substituted linear organic acid" is selected from at least one or more of trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid.

The term "diluent" refers to, for example, a solution containing a sample or a control for measurement, although some embodiments are within the scope of the present invention with mobile phase B as the diluent, however, the dilution with the first diluent also includes a mixture of mobile phase a and mobile phase B as the second diluent in a volume ratio of 40-60.

The term "at least one time period" refers to, for example, the entire retention time of the chromatogram, the time from the start of sample injection to the completion of sample elution, the time from baseline equilibration per needle to the end of sample analysis, in order to ensure that the pharmaceutical substance having a pyridine substituent in some embodiments is eluted under preset mobile and stationary phase conditions.

The invention has the beneficial effect of providing an HPLC content analysis method for various medical substances with selectively constructed pyridine substituent groups.

Drawings

FIG. 1. A schematic representation of the general case where more of the first modifier is used than the second modifier;

FIG. 2. A schematic representation of a general case where the first modifier is used in an amount less than the second modifier;

FIG. 3 elution gradient screening liquid chromatogram

Wherein the ratio of A to B of the chromatogram is 70:30, 67:33;

FIG. 4 analysis method content determination liquid chromatogram

Wherein, sample diluent 1: the first step of acetonitrile, the second step of acetonitrile;

FIG. 5 elution gradient and sample size adjustment liquid chromatogram；

Wherein, the chromatogram is as follows from bottom to top in sequence: a, B =65, and 10 μ L of sample is injected; b =67:33, injecting 20 mu L of sample;

FIG. 6 is a liquid chromatogram of the nifedipine sample of example 6.

Detailed Description

The invention will now be illustrated with reference to specific examples, which should not be construed as limiting the invention.

In some embodiments, a silane-bonded silica gel reverse phase chromatography column with diisopropyl side groups with a polar amide group embedded in the long alkyl chain, the first modifier is present in a greater mass volume percent concentration (g/100 ml) than the second modifier, which is surprising. Specifically, the aqueous phase (mobile phase a) having a higher mass volume percentage and the corresponding acetonitrile phase (mobile phase B) having a lower mass volume percentage are mixed in a ratio (e.g., ± 5%) and subjected to gradient elution or single isocratic elution, and the mixture exhibits at least two or more levels of durability, such as durability against pH, durability against wavelength, and durability against column temperature, alone or in combination. Surprisingly, referring to fig. 1, it is shown that, in a range Z of a durability condition (± 5%) corresponding to a ratio of mobile phases (a represents mobile phase a, B represents mobile phase B), not only equilibrium of absorbance of the mobile phases (a square column corresponding to Z has a small change in the X-axis direction), but also at least one of a durability condition (± 0.2) of pH value, durability (± 5 ℃) of column temperature, durability (± 20%) of flow rate, and durability (± 5 nm) of wavelength (a square column corresponding to Z has a small change in the Y-axis direction, where a boundary line between a square column shadow and a blank may represent pH value and a position of the entire square column may represent column temperature) is ensured, and it is required to explain that fig. 1 reveals that a general case where a first modifier is greater than a mass volume percentage concentration (g/100 ml) of a second modifier, when the kinds of the first modifier and the second modifier are different, the difference in dosage is adjusted accordingly. Some specific examples are that the first modifier and the second modifier are both perfluoro substituted linear organic acid, and the two are used in different amounts to provide at least better pH durability, such as that mobile phase a is selected from an aqueous solution containing 0.06% perfluoro substituted linear organic acid, and mobile phase B is selected from a 0.04% acetonitrile solution of perfluoro substituted linear organic acid. In other specific embodiments, the mobile phase a is selected from a solution containing 0.06% methylene diphosphonic acid (mepronic acid) in water and the mobile phase B is selected from a solution containing 0.04% perfluoro substituted linear organic acid in acetonitrile, which has better pH durability and column temperature durability. In other specific embodiments, the mobile phase A is selected from a 0.06% alkyl sulfonate containing aqueous solution, and the mobile phase B is selected from a 0.04% perfluoro substituted linear organic acid acetonitrile solution, and also has better pH durability and column temperature durability. In other specific embodiments, the mobile phase A is selected from a solution containing 0.06% phosphate in water, and the mobile phase B is selected from a solution containing 0.04% perfluoro substituted linear organic acid in acetonitrile, and has better pH durability and column temperature durability. In other specific embodiments, the mobile phase a is selected from a solution containing 0.06% triethylamine in water, and the mobile phase B is selected from a solution containing 0.04% triethylamine in acetonitrile, and the solution has better pH durability and column temperature durability. Referring to fig. 2, the general case where the mass volume percentage concentration (g/100 ml) of the first modifier is smaller than that of the second modifier is disclosed, so that in a durability condition (± 5%) range Z corresponding to the ratio of mobile phases (a represents mobile phase a and B represents mobile phase B), but in the X-axis and Y-axis directions, only a part of the region where durability is significant is in the other sections, or the number of durability ranges is small, for example, the durability at column temperature in the Y-axis direction is good, but it is easy to find a region where the pH durability is stable. However, fig. 1 and 2 are schematic diagrams showing statistics of HPLC measurement of a pharmaceutical substance based on a pyridine substituent, and are not specific chromatographic conditions for a particular substance. It should be noted that, in the establishment of the liquid chromatography conditions, the conditions may interact with each other, and the adjustment of the durability of one index is usually regular, but if the durability of all indexes is adjusted at the same time, the adjustment of the durability of several indexes at the same time is irregular, so that it is meaningful to establish the liquid chromatography conditions.

The present invention is explained below in more specific examples, which are to be construed as illustrating the full HPLC chromatogram in the drawings, wherein the undeveloped views include other chromatograms that meet the specifications but belong to the repeat test, non-meeting chromatograms, and non-meeting views including various HPLC chromatograms having non-meeting tailing factors, non-meeting retention time, non-meeting resolution, inaccurate quantification (peak area fluctuation), and the like. It should be noted that each chromatographic condition of the standard is listed in the general examples, and the durability condition for the durability test is shown in parentheses. Finally, the specifications of the main drug and the types of the auxiliary materials per minimum dose given in the test sample, but these examples are not to be considered as limiting the specific preparation process and the types of the auxiliary materials, and are all within the selection range of the invention based on other auxiliary materials and the known capsule process. For example, the capsule is nifedipine capsule or (±) - (E) -2-cyclohexyl-1- (6-methanesulfonyl-pyridin-3-yl) -cyclopropanecarboxylic acid thiazol-2-ylamide or nifedipine capsule, and the capsule comprises the following auxiliary materials in percentage by weight: 0.5 to 5.0 percent of cross-linked carboxymethyl cellulose, 20 to 40 percent of microcrystalline cellulose 102, 20 to 40 percent of lactose and 0.5 to 2.0 percent of sodium stearyl fumarate. Moreover, the dosage adjustment of the auxiliary materials is within the protection scope of the invention.

Example 1

Content determination of (+/-) - (E) -2-cyclohexyl-1- (6-methylsulfonyl-pyridin-3-yl) -cyclopropanecarboxylic acid thiazole-2-ylamide capsule (compound I capsule (containing main ingredient 80mg, sodium stearyl fumarate 5mg, and cross-linked carboxymethylcellulose, microcrystalline cellulose 102, and lactose) for short

Chromatographic conditions are as follows:

a chromatographic column: agilent Zorbax Box RP (4.6 mm. Times.150mm, 3.5 μm) (durability test columns: agilent InfinityLab Poroshell120, waters SunAire C18 reverse phase column, ZORBAX Eclipse Plus C18, ZORBAX stableBox column)

A mobile phase A:0.05% aqueous trifluoroacetic acid; and (3) mobile phase B:0.05% trifluoroacetic acid acetonitrile solution

Column temperature: 40 deg.C (+ -5 deg.C)

Flow rate: 1.5mL/min (+ -20%)

Wavelength: 242nm (± 5 nm)

Sample introduction amount: 20 μ L

Diluent agent: comparison products: 50% acetonitrile solution; sample preparation: see the preparation process of the test solution.

Gradient:

T(min)	A％	B％
				0	95	5
50	35	65
			60	5	95
65	5	95
			65.1	95	5
74	95	5

sample preparation: the 5-pellet contents were placed in a 250mL brown vial, each capsule shell was washed with 25mL acetonitrile, the washes were incorporated into the vial, diluted to 4/5 volume with acetonitrile, sonicated for 10min, and shaken intermittently. Standing at room temperature for 30min, diluting with acetonitrile to scale, and shaking. Centrifuge at 8000 rpm for at least 5 minutes. The supernatant was precisely measured in a 10mL to 50mL brown volumetric flask, diluted to the mark with acetonitrile-water (30).

The experimental results are as follows: the durability of the Agilent Zorbax Box RP based column temperature, the durability of flow rate, the durability of wavelength and the durability of pH value are all good, but the detection time of the analysis method is long, and the durability based on other chromatographic columns is poor.

Example 2

Content determination of Compound I Capsule (containing Main drug 80 mg)

Mobile phase A:0.06% aqueous trifluoroacetic acid; and (3) mobile phase B:0.04% trifluoroacetic acid in acetonitrile, which differs from example 1 and comprises the testing of each test once under two gradients (isocratic elution): gradient 1: a: B =70 (pH = 2.8); gradient 2: a: B =67 (pH = 3.0);

the experimental results are as follows: the column temperature based on Agilent Zorbax Box RP and Agilent InfinityLab Poroshell120 has better durability, flow rate durability, wavelength durability and pH value durability. However, referring to FIG. 3 (FIGS. 3-5 in the drawings of the specification are all based on the Agilent Zorbax Box RP), the gradient 1: peak shape difference. Gradient 2: the peak shape is good, the separation degree of impurities and a main peak is high, the peak-off time is about 7min, but the capsule content value measured by an analysis method is low, and the durability based on other chromatographic columns is poor.

Example 3

In contrast to example 2 (gradient 2), 4 tests were performed with the following four sample diluents.

Sample diluent 1: first acetonitrile, second acetonitrile: water =30:50.

sample diluent 2: first step acetonitrile: water =1:1, second acetonitrile: water =1:1.

sample diluent 3: first step acetonitrile, second step 0.05% tfa- (acetonitrile: water = 30) solution.

Sample diluent 4: a first step of 0.05% TFA-acetonitrile, a second step of 0.05% TFA- (acetonitrile: water = 30) solution.

The experimental results are as follows: the column temperature based on Agilent Zorbax Box RP and Agilent InfinityLab Poroshell120 has better durability, flow rate durability, wavelength durability and pH value durability. The effect of diluent extraction efficiency was examined (results are shown in table 1). Diluent 1: peak shape difference (see figure 4 for spectrum); diluent 2: the content value is low (the result is shown in table 2); diluent 3: the content value is low (the result is shown in table 3); diluent 4: compliance with the acceptance criteria (results are shown in table 4) is based on the poor durability of other columns.

Table 1 sample diluent 1 (sample diluent: first step acetonitrile, second step acetonitrile: water =30

TABLE 2 sample Diluent 2 (sample Diluent: first acetonitrile: water =1:1, second acetonitrile: water = 1:1)

Table 3 sample diluent 3 (sample diluent: first step acetonitrile, second step 0.05% trifluoroacetic acid (acetonitrile: water = 30))

Table 4 sample diluent 4 (sample diluent: first step 0.05% trifluoroacetic acid acetonitrile, second step 0.05% trifluoroacetic acid (acetonitrile: water =30 = 50))

Example 4

In contrast to example 3 (sample diluent 4), 4 tests were performed using four extraction times as follows:

extraction time 1: sonicate for 5min, shake for 20min.

Extraction time 2: sonicate for 10min, shake for 10min.

Extraction time 3: sonicate for 10min, shake for 20min.

Extraction time 4: and (5) performing ultrasonic treatment for 20min.

The experimental results are as follows: the column temperature based on Agilent Zorbax Box RP and Agilent InfinityLab Poroshell120 has better durability, flow rate durability, wavelength durability and pH value durability. The extraction time is prolonged from the original 10min to 20min, but the tailing factor is high, and the durability of other chromatographic columns is poor. See tables 5 and 6.

TABLE 5 extraction time screening

TABLE 6 extraction time screening

Example 5

In contrast to example 4 (extraction time 4), the following chromatographic conditions were varied:

elution gradient: a: B =65

Sample injection amount: 10 μ L

And (4) experimental conclusion: the column temperature based on Agilent Zorbax Box RP and Agilent InfinityLab Poroshell120 has better durability, flow rate durability, wavelength durability and pH value durability. The peak shape was better (see fig. 5, table 7). And (4) conclusion: the elution gradient was adjusted to a: B =65, and the sample size was adjusted to 10 μ L.

TABLE 7 elution gradient and sample size adjustment

Example 6

1,4-dihydro-2,6-dimethyl-4- (2-nitrophenyl) -3,5-dimethyl pyridinedicarboxylate (nifedipine, also known as compound II):

a chromatographic column: agilent Zorbax Box RP,4.6 mm. Times.150mm, 3.5 μm;

a detector: an ultraviolet detector;

wavelength: 237nm;

mobile phase: 0.06% aqueous trifluoroacetic acid-0.04% trifluoroacetic acid in acetonitrile (55;

flow rate: 1.0mL/min;

column temperature: 35 ℃;

sample introduction amount: 10 mu L of the solution;

diluent agent: comparison products: a water-acetonitrile solution containing 0.05% trifluoroacetic acid (1:1);

needle washing: a water-acetonitrile solution containing 0.05% trifluoroacetic acid (1:1); (ii) a

Target concentration: 0.16mg/mL;

operating time: 30min;

control solution: operation in dark place; precisely weighing a proper amount of nifedipine reference substance, adding a diluent solution, and ultrasonically preparing a solution containing 0.03mg of nifedipine in every 1mL to serve as a reference substance solution;

test solution: taking 20 tablets of the product, precisely weighing, grinding, precisely weighing a proper amount (about equal to 30mg of nifedipine) and placing the weighed amount into a mortar, adding 2ml of chloroform, grinding, quantitatively transferring into a 100ml measuring flask by absolute ethyl alcohol in batches, diluting to a scale by absolute ethyl alcohol, shaking up, filtering, discarding an initial filtrate, precisely weighing 5ml of a subsequent filtrate, placing the subsequent filtrate into a 50ml measuring flask, adding absolute ethyl alcohol to dilute to a scale, and shaking up.

As shown in FIG. 6, the liquid chromatogram of the sample is shown.

The above-mentioned embodiments are merely preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention made by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (1)

1. An HPLC content analysis method of (+/-) - (E) -2-cyclohexyl-1- (6-methanesulfonyl-pyridin-3-yl) -cyclopropanecarboxylic acid thiazol-2-ylamide, characterized in that the method comprises the following steps:

equipment: HPLC liquid chromatograph;

the chromatographic column is as follows: agilent Zorbax Box RP,4.6 mm. Times.150mm, 3.5 μm;

the mobile phase is as follows: the volume ratio is 65:35 of 0.06% trifluoroacetic acid in water and 0.04% trifluoroacetic acid in acetonitrile;

a detector: an ultraviolet detector;

wavelength: 285nm;

flow rate: 1.5mL/min;

column temperature: 40 ℃;

sample introduction amount: 10 mu L of the solution;

diluent (b): control diluent: the volume ratio is 1:1 water and acetonitrile solution containing 0.05% trifluoroacetic acid; sample diluent: preparing a test solution;

needle washing: the volume ratio is 1:1 water-acetonitrile solution containing 0.05% trifluoroacetic acid;

target concentration: 0.16mg/mL;

operating time: 15min;

control solution: precisely weighing a proper amount of a compound I capsule reference substance, adding a diluent solution, and performing ultrasonic treatment to prepare a solution containing about 0.16mg of the compound I capsule reference substance in lmL as a reference substance solution;

test solution: 80mg of standard test solution: taking 20 granules of the product, precisely weighing, calculating average loading, uniformly mixing the contents, precisely weighing an appropriate amount, placing the mixture into a 100mL volumetric flask, adding 0.05% trifluoroacetic acid acetonitrile solution to 4/5 of the volumetric flask, carrying out ultrasonic treatment for 20min, intermittently shaking, cooling to room temperature, diluting the 0.05% trifluoroacetic acid acetonitrile solution to a scale mark, shaking uniformly, centrifuging for 10min, taking 5mL of supernatant, placing the supernatant into a 25mL volumetric flask, and using the volumetric ratio of 30: diluting 50 acetonitrile containing 0.05% trifluoroacetic acid and water solution, placing scale marks, and shaking up to obtain the final product.

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