Determination of the content of active ingredients in traditional Chinese medicine (2)

3.

(1) Reaction principle: Fluorescent substance molecules have two characteristic spectra, fluorescence excitation spectrum and fluorescence emission spectrum

(2) Features: high sensitivity, generally 2 to 3 times greater than spectrophotometry; compared with spectrophotometry, fluorescence spectrophotometry has stronger selectivity in the determination, such as the absorption of tyrosine and tryptophan The peaks are similar, but under ultraviolet light, the emission peak of tyrosine is at 305nm , while the emission peak of tryptophan is at 348nm; the amount of sample is small, and some only need 10ug of sample in the micro cell; can provide More physical parameters and other characteristics

(3) Matters needing attention

1) Solvents and glassware used should be highly pure, and attention should be paid to prevent fluorescent pollution

2) The quartz sample cell used on the fluorescence instrument should be of good quality and should not contain fluorescent substances.

3) The fluorescent instrument opens the light gate during the measurement, and the light path should be closed immediately after reading to prevent the sample solution from reducing the fluorescence efficiency and fatigue and aging of the photoelectric cell of the photodetector due to the long-time illumination

4) Due to the differences in the sensitivity of different brands and models of fluorescent instruments, the concentration specified in the standard may not be completely appropriate.

5) If the excitation and emission wavelengths of the test solution of the new drug are unknown, a certain excitation wavelength (such as 250nm) can be preset according to the method specified by the instrument, the emission spectrum can be scanned and the maximum wavelength is found, and then the excitation spectrum can be scanned and recorded.

6) Measure the fluorescence intensity with several different concentrations of the test solution from dilute to concentrated, and find out its linear concentration range

7) Attention should be paid to the influence of temperature, pH and reagent purity on fluorescence intensity during measurement

(4) Calculation formula:

In the formula, Cx is the concentration of the test solution; Cr is the concentration of the reference solution; Rx is the fluorescence intensity of the test solution; Rxb is the fluorescence intensity of the test solution reagent blank; Rr is the fluorescence intensity of the reference solution ; Rrb is the fluorescence intensity of the reference substance solution reagent blank:

Due to the narrow linear range between the concentration of fluorescence analysis and the fluorescence intensity, the ratio (Rx-Rxb)/(Rr-Rrb) should be within 0.

(5) Determination of results: Fluorescence analysis is generally used for the determination of sample content, dissolution, content uniformity, etc.

The measurement results of 2 test samples should be within ±1.

(2) Ultraviolet-visible spectrophotometry (UV)

Commonly used content determination methods are: reference substance comparison method, computational spectrophotometry, absorption coefficient method, and colorimetric method

(3) High performance liquid chromatography

High-performance liquid chromatography is a chromatographic method that uses a high-pressure infusion pump to pump the specified mobile phase into a chromatographic column equipped with fillers to separate and determine the test product

1.
Analysis principle High performance liquid chromatography is a process of continuous multiple exchanges of solutes between the stationary phase and the mobile phase under high pressure conditions.
It uses the solute partition coefficient, affinity, adsorption capacity or molecular size between the two phases.
The difference in the exclusion effect caused by different causes the different solutes to be separated
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2.
Calculation formula

In the formula, A is the peak area value of the test solution; A is the peak area value of the reference solution; C is the concentration of the test solution; C is the concentration of the reference solution; M is the test The injection volume of the product solution (ul); M pair is the injection volume of the reference solution (pl)
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(4) Thin-layer scanning method

It means to irradiate the thin-layer plate with a certain wavelength of light to scan the spots in the thin-layer chromatogram that can absorb ultraviolet or visible light, or the spots that can emit fluorescence after excitation, and use the scanned spectrum and integral data For identification, inspection and content determination
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The measurement can be done according to the structural characteristics of different thin-layer scanners, and the measurement can be performed in a prescribed manner.
Generally, the reflection method is selected, and the absorption method or the fluorescence method is adopted
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Unless otherwise specified, commercially available thin-layer boards should be used for content determination
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1.
When TLC scanning is used for content determination, the linear regression two-point method is usually used for calculation.
If the linear range is very narrow, the multi-point method can be used to calibrate the polynomial regression calculation
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The test solution and the reference solution should be crossed on the same thin-layer plate.
The test solution should not be less than 2 points, and each concentration of the reference substance should not be less than 2
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When scanning, scan in the unfolding direction, not horizontally
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2.
Calculation formula TLC quantitative analysis can use external standard method and internal standard method, and external standard method is more commonly used
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When the working curve passes through the origin of the coordinate, the external standard one-point method can be used for quantification
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That is, with a reference substance of one concentration, point 3 to 4 test substances and 3 to 4 reference substances on the thin-layer plate at the same time, measure the respective peak areas, find the average value, and calculate it
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m _sample /m _standard =A _sample /A _standard .
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(Equation 14-13)

In the formula, m supply and m indicate the amount of test substance and reference substance; A sample and A indicate the peak area of ​​the test substance and reference substance
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When the working curve does not pass the origin, use the external standard two-point method to quantify
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That is, use a high and low concentration reference solution or a concentration of two sample amounts to compare and quantify the test solution
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The amount of components in the test product is:

m _supply =a+bA _supply .
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(Equation 14-14)

In the formula, are the two spot amounts (or concentrations) of the reference substance ; A ₁ and A ₂ are the peak areas of the reference substance
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In the formula, A is the peak area value of the test solution; A is the peak area value of the reference solution; C is the concentration of the test solution; C is the concentration of the reference solution; M is the test The spot amount of the sample solution (ul); M pair is the spot amount of the reference solution (ul)
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