CN105181828A - Method for measuring folic acid in human serum - Google Patents

Abstract

The invention provides a method for measuring the folic acid in human serum, and belongs to the field of medical detection. According to the method, human serum is separated by high performance liquid chromatography (HPLC) and then detected by a chemical luminous detection device. The method comprises steps of standard substance preparation, sample disposal, chromatography condition setting, preparation of reagents in a chemical luminous system, and detection. The HPLC with an efficient separating characteristic and a chemical luminous method having the advantages of high sensitivity and wide linear range are used together; and thus folic acid in human serum can be high-sensitively, high-accurately, and high-stably detected. A Fe<2+>-H2O2-luminol-NaOH chemical luminous system is established to rapidly and precisely measure folic acid in human serum.

Description

A kind of assay method of human serum folic acid

Technical field

The invention belongs to medical science, relate to a kind of assay method of human serum folic acid.

Background technology

Folic acid is a kind of water soluble vitamin, and it is not present in occurring in nature yet inactive, but for having the precursor of bioactive folate (folate).Folate extensively exists at nature, has in animals and plants, and liver, kidney, green vegetable, potato, wheat bran equal size enrich.Folic acid can transmit a carbon-based group (methyl or formyl) to deoxyuridylic acid, makes it to become deoxythymidylic acid, and then synthetic DNA.

Serum folic acid measures and is mainly used in the etiological diagnosis with cell anemia.The main manifestations of folic acid deficiency is anaemia, and other are as smooth in glossitis, glossalgia, atrophy of tongue papillae, lingual surface, angular stomatitis and anorexia.In the gestation previous moon and the pregnant junior three month, oral folic acid can prevent Fetal neurotubules malformation.Having of the method for laboratory diagnosis folic acid is multiple, mainly contains microbial method, chemiluminescence assay, high performance liquid chromatography, fluorescence method, radioimmunology etc.Light patent of invention CN100543462C discloses a kind of method for measuring human body red blood cell acidum folicum content, by chemiluminescence determination, patent of invention CN104807946A discloses that a kind of what measure health food Folic Acid is chromatographic process, measured by high efficiency liquid phase chromatographic analysis method, said determination method all adopts the obvious single analytical approach of limitation, separately all there is different defects, limit it and further develop and apply.

In available data, the folic acid method reported, " Fenton reagent oxidation-fluorometry measures the content of fruit tree Folic Acid " of delivering as Su Wenbin adopts Fe ²⁺-H ₂o ₂luminescence reagent system of determination folic acid; " content of high effective liquid chromatography for measuring Couteat of Folic Acid and related substance " that Wu Hongfu etc. deliver adopts mobile phase to be the liquid chromatography for measuring folate content of phosphate buffer, said method has carried out optimization in various degree respectively to chemoluminescence method and high effective liquid chromatography for measuring folate content, but does not occur the situation of conbined usage.Two kinds and two or more detection techniques being combined is the inexorable trend that modern technologies develop, paper that such as Wei Yue etc. deliver " high performance liquid chromatography and the application of chemiluminescence coupling technology in Pharmaceutical Analysis; just have and relate to the technology that two kinds of method conbined usage measure methylthiouracil (MTU) in human serum and propylthiouracil (PTU); the method that this application high performance liquid chromatography technology of combining with chemiluminescence measures human serum folate content is significant, has Study and appliance value.

Summary of the invention

The object of this invention is to provide a kind of assay method of highly sensitive, accuracy good, stability is strong human serum folic acid.

To achieve these goals, the present invention is achieved by the following technical solutions:

An assay method for human serum folic acid, is detected with chemiluminescence detector after high performance liquid chromatography is separated by human serum, specifically comprises the steps:

(1) standard items preparation: precision takes in the brown volumetric flask of 20mg folic acid reference substance to 100ml, be diluted to scale with 0.5% ammonia solvent, as Standard Reserving Solution, in accurate absorption 1ml Standard Reserving Solution to 10ml volumetric flask, scale is diluted to, as standard working solution with 0.5% ammonia solvent;

(2) sample disposal: gather fasting blood 4ml with clean vacuum test tube, in 30min, the centrifugal 10min separation of serum of 3000r/min, for subsequent use;

(3) chromatographic condition setting:

1. chromatographic column: adopt 5 μm of C18 chromatographic columns (250 × 4.6mm);

2. mobile phase: potassium dihydrogen phosphate 6.8g and 0.1molL ^-1potassium hydroxide solution 70mL, is diluted with water to 800mL, regulates pH to 6.3, adds methyl alcohol 80mL, be diluted with water to 1000mL, crosses the film of 0.45um, ultrasonic 20min, for subsequent use;

3. flow velocity: 1mL/min;

4. sample size: 20 μ L;

5. column temperature: normal temperature

(4) chemical luminous system reagent preparation

1. luminol (luminol) storing solution of 0.01mol/L: take 1.772g luminol, dissolve with the sodium hydroxide solution of 0.1mol/L, be settled to 1L, obtain luminol storing solution, keep in Dark Place;

2. the H of 0.05mol/L ₂o ₂solution: with massfraction 30%H ₂o ₂formulated with water;

3. the l ferrous ammonium sulfate solution of 0.01mol/L: take 4g iron ammonium sulfate, is dissolved in 50ml20% sulfuric acid, adds 950ml water and shakes up;

(5) detect: H ₂o ₂solution and l ferrous ammonium sulfate solution are respectively again to mix with the luminol solution of flow velocity 2.0ml/min after the mixing of the flow rate of 2.0ml/min, the mixed liquor obtained mixes with the mobile phase flowing through chromatographic column, pass through chemiluminescent vessel, the measuring instrument record luminous intensity of illuminator is as baseline, after baseline stability, by high performance liquid chromatography sampling valve sample introduction, after wash-out is separated, enter chemiluminescent vessel, produce chromatographic peak, record luminous signal.

Beneficial effect of the present invention: by having the high efficiency liquid phase chromatographic analysis method of efficient stalling characteristic and the chemoluminescence method coupling highly sensitive, the range of linearity is wide, achieve the mensuration of human serum folic acid high sensitivity, high accuracy, stiff stability; Set up Fe ²⁺-H ₂o ₂-luminol-NaOH chemical luminous system is used for measuring human serum folic acid quickly and accurately.

Accompanying drawing explanation

Fig. 1 is human serum folic acid method flow schematic diagram of the present invention.

Embodiment

In conjunction with the embodiments and accompanying drawing illustrate and further the present invention described in detail, but not to be construed as limiting the invention.

Embodiment

As shown in Figure 1, a kind of assay method of human serum folic acid, comprises the steps:

(1) standard items preparation: precision takes in the brown volumetric flask of 20mg folic acid reference substance to 100ml, be diluted to scale with 0.5% ammonia solvent, as Standard Reserving Solution, in accurate absorption 1ml Standard Reserving Solution to 10ml volumetric flask, scale is diluted to, as standard working solution with 0.5% ammonia solvent;

(2) sample disposal: gather fasting blood 4ml with clean vacuum test tube, in 30min, the centrifugal 10min separation of serum of 3000r/min, for subsequent use;

(3) chromatographic condition setting:

1. chromatographic column: adopt 5 μm of C18 chromatographic columns (250 × 4.6mm);

2. mobile phase: potassium dihydrogen phosphate 6.8g and 0.1molL ^-1potassium hydroxide solution 70mL, is diluted with water to 800mL, regulates pH to 6.3, adds methyl alcohol 80mL, be diluted with water to 1000mL, crosses the film of 0.45um, ultrasonic 20min, for subsequent use;

3. flow velocity: 1mL/min;

4. sample size: 20 μ L;

5. column temperature: normal temperature

(4) chemical luminous system

1. luminol (luminol) storing solution of 0.01mol/L: take 1.772g luminol, dissolve with the sodium hydroxide solution of 0.1mol/L, be settled to 1L, obtain luminol storing solution, keep in Dark Place;

2. the H of 0.05mol/L ₂o ₂solution: with massfraction 30%H ₂o ₂formulated with water;

3. the l ferrous ammonium sulfate solution of 0.01mol/L: take 4g iron ammonium sulfate, is dissolved in 50ml20% sulfuric acid, adds 950ml water and shakes up;

(5) detect: H ₂o ₂solution and l ferrous ammonium sulfate solution are respectively again to mix with the luminol solution of flow velocity 2.0ml/min after the mixing of the flow rate of 2.0ml/min, the mixed liquor obtained mixes with the mobile phase flowing through chromatographic column, pass through chemiluminescent vessel, the measuring instrument record luminous intensity of illuminator is as baseline, after baseline stability, by high performance liquid chromatography sampling valve sample introduction, after wash-out is separated, enter chemiluminescent vessel, produce chromatographic peak, record luminous signal.

This method technical parameter:

Detectability 0.4ng/ml;

Average recovery rate 98.75%;

40 samples divide four groups to carry out replicate determination, and in group, relative standard deviation is less than 3%, and between group, relative standard deviation is less than 5%.

Claims (1)

1. an assay method for human serum folic acid, is detected with chemiluminescence detector after high performance liquid chromatography is separated by human serum, it is characterized in that: comprise the steps:

(1) standard items preparation: precision takes in the brown volumetric flask of 20mg folic acid reference substance to 100ml, be diluted to scale with 0.5% ammonia solvent, as Standard Reserving Solution, in accurate absorption 1ml Standard Reserving Solution to 10ml volumetric flask, scale is diluted to, as standard working solution with 0.5% ammonia solvent;

(2) sample disposal: gather fasting blood 4ml with clean vacuum test tube, in 30min, the centrifugal 10min separation of serum of 3000r/min, for subsequent use;

(3) chromatographic condition setting:

1. chromatographic column: adopt 5 μm of C18 chromatographic columns (250 × 4.6mm);

2. mobile phase: potassium dihydrogen phosphate 6.8g and 0.1molL ^-1potassium hydroxide solution 70mL, is diluted with water to 800mL, regulates pH to 6.3, adds methyl alcohol 80mL, be diluted with water to 1000mL, crosses the film of 0.45um, ultrasonic 20min, for subsequent use;

3. flow velocity: 1mL/min;

4. sample size: 20 μ L;

5. column temperature: normal temperature

(4) chemical luminous system reagent preparation

1. luminol (luminol) storing solution of 0.01mol/L: take 1.772g luminol, dissolve with the sodium hydroxide solution of 0.1mol/L, be settled to 1L, obtain luminol storing solution, keep in Dark Place;

2. the H of 0.05mol/L ₂o ₂solution: with massfraction 30%H ₂o ₂formulated with water;

3. the l ferrous ammonium sulfate solution of 0.01mol/L: take 4g iron ammonium sulfate, is dissolved in 50ml20% sulfuric acid, adds 950ml water and shakes up;

(5) detect: H ₂o ₂solution and l ferrous ammonium sulfate solution are respectively again to mix with the luminol solution of flow velocity 2.0ml/min after the mixing of the flow rate of 2.0ml/min, the mixed liquor obtained mixes with the mobile phase flowing through chromatographic column, pass through chemiluminescent vessel, the measuring instrument record luminous intensity of illuminator is as baseline, after baseline stability, by high performance liquid chromatography sampling valve sample introduction, after wash-out is separated, enter chemiluminescent vessel, produce chromatographic peak, record luminous signal.