CN1908005B - A compound protein precipitant - Google Patents

Abstract

A compound protein precipitating agent relates to a protein precipitating agent. It solves the problem of the low protein precipitation rate and poor extraction and separation effect of the existing single-component protein precipitant, the excessive acidity of 10% trichloroacetic acid and 6% perchloric acid is too strong to damage the chromatographic column, and the liquid-liquid extraction method is used for extraction. Drugs in biological samples have the defects of long time-consuming, cumbersome operation, and large sample loss. The composite protein precipitant is prepared by parts by weight from 1-1.5 parts of 5-sulfosalicylic acid with analytical purity, 75-85 parts of methanol with analytical purity and 15-25 parts of distilled water. The protein precipitation rate of the invented composite protein precipitation agent reaches over 99%, and the pH is weakly acidic, causing little damage to the chromatographic column. Using the composite protein precipitation agent of the present invention, protein precipitation and drug extraction and separation in biological samples are completed in one step, the operation is simple, the time is less, the loss of drug components in the sample is less, and the recovery rate of drug extraction is more than 70%, which greatly improves drug analysis in vivo The efficiency and accuracy can meet the needs of drug research.

Description

A compound protein precipitant

technical field

The invention relates to a protein precipitation agent.

Background technique

Most of the existing organic solvent protein precipitants, acidic or alkaline protein precipitants and heavy metal protein precipitants are single components, the protein precipitation rate is low, and the extraction and separation effect is poor. Although 10% trichloroacetic acid and 6% perchloric acid have a high protein precipitation rate, and the protein precipitation rate reaches 98.5% to 99.5% in the case of the same volume as the sample, but the acidity is too strong, which will damage the chromatographic column. However, the use of liquid-liquid extraction to extract drugs from biological samples is time-consuming, cumbersome, and the loss of drug components in the sample is large, which greatly affects the efficiency and accuracy of drug analysis in vivo. Meet the needs of providing experimental data quickly and accurately.

Contents of the invention

The purpose of the present invention is to solve the low protein precipitation rate of the existing single component protein precipitant, the poor extraction and separation effect, the excessive acidity of 10% trichloroacetic acid and 6% perchloric acid are too strong to the chromatographic column, the cost is high and the The liquid-liquid extraction method is used to extract the defects of long time-consuming, cumbersome operation and large sample loss of drugs in biological samples, thereby providing a composite protein precipitation agent. The composite protein precipitant is prepared by parts by weight from 1-1.5 parts of 5-sulfosalicylic acid with analytical purity, 75-85 parts of methanol with analytical purity and 15-25 parts of distilled water. The composite protein precipitation agent of the invention is simple to prepare, has few components, and has a high protein precipitation rate of over 99%. The pH of the composite protein precipitant of the present invention is weakly acidic, has little damage to the chromatographic column (sample pH<2.5 has great damage to the chromatographic column), and has low cost. Using the composite protein precipitation agent of the present invention, protein precipitation and drug extraction and separation in biological samples are completed in one step, the operation is simple, the time is less, the loss of drug components in the sample is less, and the recovery rate of drug extraction is more than 70%, which greatly improves drug analysis in vivo The efficiency and accuracy can meet the needs of drug research.

Description of drawings

Fig. 1 is the chromatogram of blank plasma treated with 6% perchloric acid, and Fig. 2 is the chromatogram of blank plasma treated with complex protein precipitant.

Detailed ways

Specific embodiment one: this embodiment is that the composite protein precipitating agent is by weight parts by 1～1.5 parts purity is analytical pure 5-sulfosalicylic acid, 75～85 parts purity is analytical pure methanol and 15～25 parts Made with distilled water.

In this embodiment, analytically pure 5-sulfosalicylic acid, analytically pure methanol and distilled water are mixed in parts by weight and dissolved before use.

In this embodiment, the composite protein precipitant is applied to the pretreatment of biological samples of cardiovascular drugs such as celiprolol hydrochloride, so as to conduct reversed-phase high-performance liquid chromatography analysis. Processing steps of biological samples (plasma):

(1) Mixing and shaking the biological sample and the composite protein precipitation agent of this embodiment at a volume ratio of 1:1;

(2) Centrifuging, protein precipitation, taking the supernatant and carrying out liquid chromatography analysis to analyze the drug components in the biological sample.

The precision of high-performance liquid chromatographic analysis of biological samples after precipitation by the composite protein precipitant of this embodiment is higher than that of other existing protein precipitants after liquid-liquid extraction. Several protein precipitants were carried out For comparison, the results are shown in Table 1.

Table 1

Precipitate the blank plasma biological sample with perchloric acid with a concentration of 6% and the composite protein precipitant of this embodiment as the protein precipitant respectively, and then perform liquid chromatography analysis, detection wavelength: 254nm, mobile phase: methanol: H ₂ O(65:35, chromatographic column: C ₁₈ column, flow rate: 1mL/min, running time: 10min, injection volume: 20 μ L. The chromatographic analysis results are shown in Figure 1 and Figure 2, and the peaks of plasma impurities are all at 4.5 It appeared within minutes, and there was no interference from any impurity peaks thereafter, indicating that the application of the composite protein precipitant is beneficial to the separation and determination of drug samples and impurities.

Specific embodiment two: the difference between this embodiment and specific embodiment one is: the composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1.1～1.4 parts by weight, 76～84 parts by weight Made of analytically pure methanol and 16 to 24 parts of distilled water. Other steps are the same as those in Embodiment 1.

Specific embodiment three: the difference between this embodiment and specific embodiment one is: the composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1.2～1.3 parts by weight, 78～82 parts by weight Made from analytically pure methanol and 18 to 22 parts of distilled water. Other steps are the same as those in Embodiment 1.

Specific embodiment four: the difference between this embodiment and specific embodiment one is: the composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1.2 parts by weight, and 80 parts by weight are analytically pure methyl alcohol and 20 parts distilled water. Other steps are the same as those in Embodiment 1.

Claims (4)

1. A composite protein precipitant is characterized in that the composite protein precipitant is analytically pure 5-sulfosalicylic acid, 75 to 85 parts of pure methanol and 15 parts by weight of the composite protein precipitant in parts by weight. ~25 parts distilled water. 2. composite protein precipitation agent according to claim 1, it is characterized in that composite protein precipitation agent is analytically pure 5-sulfosalicylic acid by 1.1～1.4 parts of purity, 76～84 parts of purity are analysis by parts by weight. Made from pure methanol and 16 to 24 parts of distilled water. 3. composite protein precipitant according to claim 1 is characterized in that composite protein precipitant is analytically pure 5-sulfosalicylic acid by 1.2～1.3 parts of purity, 78～82 parts of purity by weight. Made from pure methanol and 18-22 parts of distilled water. 4. composite protein precipitating agent according to claim 1, it is characterized in that composite protein precipitating agent is analytically pure 5-sulfosalicylic acid, 80 parts of purity are analytically pure methyl alcohol and Made with 20 parts distilled water.