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A method for detecting related substances in tauroursodeoxycholic acid

A technology of tauroursodeoxycholic acid and related substances, which is applied in the field of high-performance liquid chromatography-evaporative light scattering detection method, can solve the problems of low sensitivity, low accuracy, and interference of components to be measured, and achieve a reduction in chromatographic The effect of peak broadening behavior, good specificity and accuracy, and improved quality controllability

Active Publication Date: 2020-06-16
YAOPHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The ultraviolet absorption of bile acids is weak, or only at the end. When using ultraviolet detectors for detection, low wavelengths should be used for detection, but many organic solvents have absorption in the wavelength range of 190nm~220nm (the cut-off wavelength of common organic solvents: Methanol 205nm, acetonitrile 190nm, isopropanol 205nm), there is interference in the components to be measured, there are problems such as baseline fluctuations, large noise, low sensitivity, poor linearity, and low accuracy
Moreover, the existing literature reports only detect 4 kinds of impurities at most, which cannot control the quality of tauroursodeoxycholic acid well.
Among the 8 known impurities of tauroursodeoxycholic acid, there are components with large difference in polarity and components with close polarity. Multiple chromatographic systems are usually used for detection, especially taurine- 7-ketocholic acid and taurochenodeoxycholic acid, cholic acid and ursodeoxycholic acid, 7-ketocholic acid and chenodeoxycholic acid are close in polarity, it is difficult to effectively separate; tauroursodeoxycholic acid Cholic acid has a large polarity difference with cholic acid and gallstone acid. Under the same chromatographic conditions, the retention time of the components differs greatly. The peak shape of the strong retention component will broaden with the extension of the retention time, which reduces the detection sensitivity. Even some components cannot be eluted, and it is difficult to quickly detect 8 known impurities and other unknown impurities of tauroursodeoxycholic acid at the same time using one chromatographic condition

Method used

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  • A method for detecting related substances in tauroursodeoxycholic acid
  • A method for detecting related substances in tauroursodeoxycholic acid
  • A method for detecting related substances in tauroursodeoxycholic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1 Determination of chromatographic column

[0047] Preliminary mobile phase: mobile phase A-mobile phase B;

[0048] Mobile phase A is 50mmol / L ammonia water (adjust the pH to 3.5 with formic acid), and mobile phase B is acetonitrile;

[0049] The column temperature is 40°C;

[0050] The flow rate is 1.0ml / min;

[0051] Evaporative light scattering detector, drift tube temperature is 115°C, carrier gas flow rate is 2.5L / min;

[0052] The test solution: take an appropriate amount of tauroursodeoxycholic acid raw material, and prepare a solution of about 10 mg / ml to obtain the product;

[0053]System Suitability Solution: Take Tauroursodeoxycholic Acid, Tauro-7-ketocholic Acid, Taurochenodeoxycholic Acid, Taurocholic Acid, Cholic Acid, Ursodeoxycholic Acid, 7-Ketocholic Acid Appropriate amounts of cholelithic acid, chenodeoxycholic acid, and cholelithic acid reference substances were prepared into a mixed solution containing about 250 μg / ml of each componen...

Embodiment 2

[0058] The selection of embodiment 2 mobile phase

[0059] Accurately measure tauroursodeoxycholic acid reference solution 1 (2.5 μg / ml) or tauroursodeoxycholic acid reference solution 2 (5.0 μg / ml) and system suitability solution respectively, under different mobile phase conditions Next, inject into the high performance liquid chromatograph and record the chromatogram. Investigate the effects of different types and concentrations of buffer salts and different pH regulators on component separation (the number of system suitability solution peaks) and detection sensitivity. The results are shown in Table 2:

[0060] Table 2 Buffer salt selection results

[0061]

[0062] Column: The chromatographic column number is the same as in Table 1.

[0063] Above-mentioned result shows: adopt 2.5mmol / L~50mmol / L ammonium salt buffer solution, adjust pH value to in the scope of 2.5~3.5 with formic acid, acetic acid or trifluoroacetic acid, can obtain higher sensitivity, can guarantee...

Embodiment 3

[0077] Example 3 Determination of Evaporative Light Scattering Detector Conditions

[0078] Chromatographic conditions:

[0079] Chromatographic column: with the chromatographic column 3 in embodiment 1;

[0080] Mobile phase: with buffer salt 6 in embodiment 2;

[0081] Gradient elution conditions:

[0082]

[0083] Flow rate: 1.0ml / min;

[0084] The injection volume is 10 μl;

[0085] Investigate the influence of evaporative light scattering detector conditions, drift tube temperature in the range of 70-115 °C, and carrier gas flow rate in the range of 2.5-4.0 L / min, take 10 μg / ml tauroursodeoxycholic acid reference solution 10 μl respectively Inject into a liquid chromatograph, record the chromatogram, and investigate the sensitivity of tauroursodeoxycholic acid response under different conditions. Other chromatographic conditions are as above, and the results are shown in Table 7.

[0086] Table 7 Effect of evaporative light scattering detector conditions on sensit...

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Abstract

The invention relates to a method for detecting related substance in tauroursodeoxycholic acid, and especially relates to a method using a HPLC-ELSD technology to simultaneously detect eight related substances in tauroursodeoxycholic acid. According to the method, an octyl bonded silica gel chromatographic column and an evaporative light-scattering detector are adopted; and a mobile phase (A) and a mobile phase (B) are mixed to carry out isocratic elution or gradient elution, wherein the mobile phase (A) is an ammonium salt buffer solution with a concentration of 2.5 to 50 mmol / L, and the mobile phase (B) is methanol or acetonitrile. The provided method has the advantages of simpleness, rapidness, accuracy, reliability, and good resolution of chromatographic peaks, and is capable of simultaneously detecting eight related substances in tauroursodeoxycholic acid quantitatively or qualitatively.

Description

technical field [0001] The invention relates to a high-performance liquid chromatography-evaporative light scattering detection method (HPLC-ELSD) for detecting related substances in tauroursodeoxycholic acid, belonging to the technical field of chemical drug analysis. Background technique [0002] The chemical name of tauroursodeoxycholic acid (TUDCA) is 2-[[(3α,5β,7β)-3,7-dihydroxy-24-oxocholan-24-yl]amino]ethyl Alkanesulfonic acid is a combined bile acid formed by condensation between the carboxyl group of ursodeoxycholic acid and the amino group of taurine. Compared with ursodeoxycholic acid, there is one more taurine group, and the polarity is greatly increased. The ability to dissolve cholesterol is also greatly improved. Tauroursodeoxycholic acid can increase the concentration of bile acids in bile, inhibit the formation of cholesterol crystals, and promote the dissolution of cholesterol gallstones, which is of great significance in reducing the recurrence rate of ga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/74
Inventor 李睿钟家庆曾加智周莎
Owner YAOPHARMA CO LTD
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