CN100422735C - A kind of method of high performance liquid chromatography analysis phenylpropanol raw material and preparation thereof - Google Patents

Abstract

The present invention discloses a method for analyzing phenyl propanol raw materials and preparations thereof by a high performance liquid chromatogram (HPLC). The method can well separate substances, such as phenyl propanol, benzyl alcohol, acetyl benzene, etc., control the quality of the phenyl propanol, accurately measure the content of phenyl propanol raw materials and preparations thereof and indicate the stability of the phenyl propanol raw materials and the preparations thereof. The adopted chromatographic columns comprises a reversed-phase column, a CN base column and an NH2 base column; the temperature of the columns is from a room temperature to 50 DEG C; a moving phase is formed by methanol-water, ethanol-water, acetonitrile-water or the ternary or quaternary mixture of methanol, ethanol, acetonitrile and water; the flow speed is 1 ml/min; the elution mode can be constant eltion or gradient elution; the detection wavelength is the maximum absorption wavelength, which is 215(+/-)2 nm or 258(+/-)2 nm, of the phenyl propanol, THe HPLC method established by the present invention has the characteristics of simple operation, high precision, strong specificity, etc.

Description

A kind of method of high performance liquid chromatography analysis phenylpropanol raw material and preparation thereof

Technical field:

The invention relates to the technical field of medicines, in particular to a method for analyzing phenylpropanol raw materials and preparations thereof by high performance liquid chromatography.

Background technique:

Phenylpropanol, also known as: cholesterol, is recorded in the Chinese Pharmacopoeia 2000 edition, and is a basic drug in my country, and its capsules are OTC varieties. Phenylpropanol was first synthesized by Warrb and Cortese in 1927, and was first listed in Germany. my country started production in 1977. This product is an oily liquid, pungent and sweet in taste, easily soluble in methanol, ethanol and chloroform, slightly soluble in water. After oral administration, this product is mainly distributed in the intestine, liver, gallbladder, kidney and other organs, metabolized in the liver, and excreted in bile and urine in the form of metabolites and part of the original drug. This product is a bile secretion accelerator. Bile secretion begins 10 minutes after taking the medicine, reaches the peak within 1-2 hours, and disappears after 3-5 hours, and bile secretion increases by 2 times on average. It can relieve abdominal distension, abdominal pain, nausea, oily and other symptoms, increase appetite and promote digestion. It can relax the sphincter of Oddis and have the effect of removing stones, but it cannot dissolve stones. It can accelerate the process of converting cholesterol into bile acid and lower cholesterol. It is clinically used in the adjuvant treatment of cholecystitis, biliary tract infection, cholelithiasis, postoperative biliary tract syndrome and chronic hepatitis.

The main raw material for preparing phenylpropanol is propiophenone, which often contains propiophenone. Since the boiling points of phenylpropanol and propiophenone are close, the former is 219°C, and the latter is 218°C. It is difficult to use simple distillation techniques. Separate the two. The method for checking propiophenone contained in the Chinese Pharmacopoeia 2000 edition is ultraviolet spectrophotometry, because both phenylpropanol and propiophenone have ultraviolet absorption at the specified wavelength, the method is nonspecific, and there are also phenylethyl alcohol and phenylacetone in the raw materials. Acetophenone and other related substances cannot be distinguished by ultraviolet spectrophotometry. In addition, the method for determining the content of phenylpropanol contained in the 2000 edition of the Chinese Pharmacopoeia is a volumetric analysis method, which uses an acid anhydride to react with hydroxyl groups in phenylpropanol to calculate the content of phenylpropanol. This method is cumbersome to operate and has poor specificity. Although there are literatures that adopt gas chromatography to measure phenylpropanol content in phenylpropanol capsules (gas chromatography such as Shi Dajun measures phenylpropanol content "Journal of Pharmaceutical Analysis" 2000, 20 (6): 428～429; Li Ruiping Gas Chromatography- Determination of phenylpropanol content in phenylpropanol capsules by mass spectrometry "(Journal of Pharmaceutical Analysis" 2003, 23 (3): 170～172), but so far, there is no use of high performance liquid chromatography (HPLC) to measure phenylpropanol at home and abroad. The content of alcohol raw material and preparation thereof.In addition, adopted acetophenone as internal standard in the gas chromatography of bibliographical information, because containing acetophenone impurity in phenylpropanol, so adopting acetophenone as internal standard is not suitable, At the same time, there is no mention in the literature of the separation of related substances benzyl alcohol, acetophenone, and phenylethanol by the established method. The solvent carbon tetrachloride used is extremely toxic to the operator and is not suitable for routine analysis. Technology. The HPLC we have established can not only determine the content of phenylpropanol raw materials and its preparations, but also can simultaneously separate phenylpropanol, phenylacetone, benzyl alcohol, acetophenone, phenylethyl alcohol and other components, so that phenylpropanol The determination of the content of raw materials and preparations thereof is uninterrupted, and the solvent used is a conventional solvent.Methodological research results show that the diluent vegetable oil of blank excipients and soft capsules has no interference, and the precision, recovery, and stability of the established HPLC method and other indicators meet the analysis requirements.

Invention content:

The object of the invention is to provide a kind of method of high performance liquid chromatography analysis phenylpropanol raw material and preparation thereof. The chromatographic columns used include: reversed-phase column or CN-based column or NH2-based column; column temperature: room temperature to 50°C; mobile phase: methanol-water, acetonitrile-water, or a ternary mixture of methanol, acetonitrile and water; The flow rate is 1ml/min (can be appropriately adjusted according to the specific situation); the elution method can be isocratic; the detection wavelength is the maximum absorption wavelength of phenylpropanol 215±2nm, 258±2nm, or other reasonable wavelengths. The reversed-phase column is C ₁₈ or C ₈ . Said mobile phase on the C ₁₈ and C ₈ columns: the ratio of methanol-water is 35:65-60:40, and the ratio of acetonitrile-water is 30:70-60:40. The mobile phase on the CN base column: the ratio of methanol-water is 15:85, the ratio of acetonitrile-water is 5:95, and the ratio of methanol-acetonitrile-water is 5:5:90; the mobile phase on the NH2 base column : The ratio of methanol-water is 2:98, the ratio of acetonitrile-water is 5:95, and the ratio of methanol:acetonitrile:water is 2:3:95. Said ternary or quaternary mixed mobile phase is: methanol-acetonitrile-water; ethanol-acetonitrile-water; methanol-ethanol-water; methanol-ethanol-acetonitrile-water, etc.

The advantages of the present invention are: the method can well separate substances such as phenylpropanol, propiophenone, benzyl alcohol, phenylethyl alcohol, acetophenone, control the quality of phenylpropanol, and accurately measure the content of phenylpropanol raw materials and preparations thereof , indicating the impurity and stability of phenylpropanol raw material and its preparation. The HPLC method established by the invention has the characteristics of simple operation, high accuracy, strong specificity and the like.

Description of drawings:

Fig. 1 is the ultraviolet scanning figure that extracts phenylpropanol peak with diode array detector (DAD).

Fig. 2 is the impurity inspection chromatogram when the phenylpropanol concentration in the phenylpropanol raw material is 5mg/ml.

Fig. 3 is after phenylpropanol is made clathrate, impurity inspection chromatogram when phenylpropanol concentration is 5mg/ml.

Figure 4 is a chromatographic overlay of possible impurities in the phenylpropanol bulk drug, namely benzyl alcohol, phenylethyl alcohol, acetophenone, and propiophenone under the same chromatographic conditions.

Fig. 5 is the chromatogram of phenylpropanol crude drug after illumination 5 days under the 4500LX illumination environment.

Fig. 6 is the chromatogram of the clathrate of phenylpropanol after being illuminated for 5 days under the environment of 4500LX illumination.

Detailed ways:

The determination of embodiment 1 detection wavelength

Take 20ul of 0.5mg/ml phenylpropanol solution prepared with 95% ethanol and inject it into the chromatograph, use methanol-water (60:40) as the mobile phase, and carry out on the DIKMA Diamonsil ODS (200×4.6mm, 5μm) column Separation, extract the ultraviolet scanning figure of phenylpropanol peak with diode array detector (DAD), see accompanying drawing 1. It can be seen from Figure 1 that the maximum absorption wavelength of phenylpropanol is 215±2nm and 258±2nm, and there are also absorption peaks at 252±2nm and 264±2nm. The detection wavelength can be selected according to the specific situation. In principle, 215±2nm or 258±2nm is used as the detection wavelength.

Example 2 Retention behavior of phenylpropanol in columns of different reversed-phase filler brands.

With methanol-water (60:40) as the mobile phase, the flow rate is 1.0ml min-1, the injection volume is 20 μl, and the chromatogram is recorded at 257nm to investigate the effect of phenylpropanol on different reversed-phase _C18 packed columns. Chromatographic behavior, see Table 1:

Table 1. Retention behavior of phenylpropanol on different ODS columns

As can be seen from Table 1, all existing _C18 brands can be used for the determination of phenylpropanol. Diamonsil ODS was selected as the chromatographic column, and the column temperature was investigated at 20, 30, 40, and 50°C. The results showed that both phenylpropanol and propiophenone could be separated. The retention time of the main peak shortened with the increase of column temperature, and the resolution was qualified. . In practical applications, a suitable column temperature may be in the range of 20-50°C or lower or higher than this range.

The retention behavior of embodiment 3 phenylpropanol in C _column

The chromatogram was recorded at 215nm when the injection volume was 20μl with methanol-water (55:45) as the mobile phase, the flow rate was 1.0ml·min ^-1 , and the results are shown in Table 2.

Table 2. Retention behavior of phenylpropanol in different C8 columns

Example 4 investigates the parameters of phenylpropanol in the chromatographic column CenturySIL C ₈ BDS column and in different mobile phases, see Table 3.

Table 3: Retention behavior of phenylpropanol in different mobile phases

Example 5 In the CN-based column and _NH2- based column, the separation of the mobile phase sample phenylpropanol and impurity propiophenone peaks with different ratios of "acetonitrile-water", "methanol-water" or "methanol-acetonitrile-water" Effect. The results are shown in Table 4.

Table 4: Retention behavior of phenylpropanol and propiophenone

Example 6 investigates the separation effect of the mobile phase sample phenylpropanol and impurity propiophenone peaks in a DIKMA Diamonsil C ₁₈ column with different acetonitrile-water ratios. The results are shown in Table 5.

Table 5: Retention behavior of phenylpropanol in various ratios of acetonitrile-water

Example 7 investigates the separation effect of the mobile phase sample phenylpropanol and the impurity propiophenone peaks of different acetonitrile-methanol-water ratios in the DIKMA Diamonsil C ₁₈ column. The results are shown in Table 6.

Table 6: Retention behavior of phenylpropanol in various ratios of acetonitrile-methanol-water

Example 8 investigates the separation effect of the mobile phase sample phenylpropanol and impurity propiophenone peaks in a DIKMA Diamonsil C ₁₈ column with different methanol-water ratios. The results are shown in Table 7.

Table 7: Retention behavior of phenylpropanol in various ratios of methanol-water

Example 9 further used methanol-water (55:45) as the mobile phase to investigate the separation of phenylpropanol and various impurities in the Diamonsil C18 column. The results are shown in Table 8 and the accompanying drawings.

Table 8: Retention Behavior of Phenylpropanol and Related Compounds

Example 10 Methodological investigation of the determination of phenylpropanol content The standard curve uses ethanol as a solvent, accurately prepares a phenylpropanol reference substance stock solution with a concentration of 10 mg×mL-1, and stores it in a refrigerator at 4°C. Accurately draw 0.2, 0.4, 0.6, 0.8, 1.0mL of the phenylpropanol reference substance stock solution respectively, put them in a 10mL measuring bottle, make the volume up to the mark with the mobile phase, shake well, and obtain the concentration of 200, 400, 600, 800, 1000μg For a series of solutions of mL ^-1 , inject 20 μL respectively, take the concentration (C) as the abscissa, and the peak area (A) as the ordinate, and the regression equation is: C=31.38A(10-4)+0.3678, r=0.9997 , with a linear range of 200-1000 μg·mL ^-1 .

The minimum detection amount is to take an appropriate amount of phenylpropanol reference substance stock solution, dilute it with mobile phase to different concentrations, take 20 μL, and inject it into the chromatograph, so that the peak height is three times the baseline noise of the instrument, and the solution concentration is 3 μg mL ^-1 , the minimum detectable amount is 60ng. Quantitative limit Take an appropriate amount of phenylpropanol reference stock solution, dilute it to different concentrations with mobile phase, take 17 μL, and inject it into the chromatograph, so that the signal-to-noise ratio is 10:1. At this time, the solution concentration is 10 μg·mL ^-1 , and the quantitative limit is 170ng. Three concentrations of the above solutions (200, 600, and 1000 μgmL ^-1 (low, medium, and high) were used for the precision, and samples were injected at different times to calculate the intra-day precision and inter-day precision: the intra-day RSDs were 1.89%, 1.65%, and 1.64, respectively. %; daytime RSDs were 1.38%, 1.66% and 1.87%, respectively.

Determination of Phenylpropanol Content Recovery Rate Add phenylpropanol according to ±20% of the prescription ratio, dissolve in ethanol, dilute with mobile phase, shake well, and prepare sample solution. Inject 20 μL of samples respectively, measure the peak area, convert it into the actual measured concentration on behalf of the standard curve, and calculate the recovery rate. The result is that the recovery rate is 99% to 101%.

Example 11 Determination of phenylpropanol content Chromatographic conditions Chromatographic column: DIKMA Diamonsil C ₁₈ (4.6mm×200mm, 5 μm Dima Company); mobile phase: methanol-water (55:45); column temperature: room temperature; UV detection wavelength: 258nm. Injection volume: 20 μL, the number of theoretical plates is not less than 1000 based on the peak of phenylpropanol. Sample preparation: commercially available soft capsules, homemade phenylpropanol β-cyclodextrin inclusion compound, homemade phenylpropanol β-cyclodextrin hard capsules, prepared with ethanol to a solution of about 0.5mg/ml, external standard method Determination of the content of each sample, the results

Table 9: Retention Behavior of Phenylpropanol and Related Compounds

Example 12 Determination of the purity of the phenylpropanol raw material and its light stability The phenylpropanol raw material and the phenylpropanol β-cyclodextrin clathrate were placed in the environment of 4500LX illuminance together, and the light test was carried out for 5 days, and the test was carried out with "implementation Example 10 " chromatographic conditions carry out content determination and impurity inspection to the sample, the results show that the content of phenylpropanol raw material has a large decline, which is 94.6% of the 0 time, and the impurity propiophenone content increases, from 0.38% of the 0 time to 5.12%; while the content of phenylpropanol β-cyclodextrin inclusion compound remained basically unchanged, which was 99.7% of 0 time, and the content of impurity propiophenone did not increase, indicating that phenylpropanol was made into β-cyclodextrin inclusion compound After that, the stability of the drug is greatly improved. See attached drawing 6.

Claims (3)

1. the method for efficient liquid phase chromatographic analysis phenylpropanol raw material and preparation thereof, the chromatographic column of employing is: reversed-phase column or CN pilum or NH2 pilum is characterized in that: adopt column temperature: room temperature～50 ℃; Moving phase: the ternary mixed flow phase of methanol-water, acetonitrile-water or methyl alcohol-acetonitrile-water; Moving phase on the reversed-phase column: the ratio of methanol-water is that the ratio of 35: 65～60: 40 or acetonitrile-water is 30: 70～60: 40, the moving phase on the CN pilum: the ratio of methanol-water is that 15: 85, the ratio of acetonitrile-water are that the ratio of 5: 95 or methyl alcohol-acetonitrile-water is 5: 5: 90; Moving phase on the NH2 pilum: the ratio of methanol-water is that the ratio of 2: 98 or acetonitrile-water is 5: 95 or methyl alcohol: acetonitrile: the ratio of water is 2: 3: 95; Flow velocity 1ml/min; Type of elution is an isocratic elution; Detecting wavelength is the maximum absorption wavelength of phenylpropanol.

2. the method for a kind of efficient liquid phase chromatographic analysis phenylpropanol raw material according to claim 1 and preparation thereof is characterized in that: said reversed-phase column is C18 or C8 post.

3. the method for a kind of efficient liquid phase chromatographic analysis phenylpropanol raw material according to claim 1 and preparation thereof is characterized in that: said maximum absorption wavelength 215 ± 2nm or 258 ± 2nm.

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