CN104865323B - The assay method of hydrogen sulfide in a kind of blood and urine - Google Patents

Abstract

The invention discloses a kind of method that dispersed solid phase micro-extraction combines the hydrogen sulfide in high-performance liquid chromatogram determination blood and urine, utilize free H in serum and urine₂S、HS^‑、S^2‑With P-aminodimethylaniline hydrochlorate and ferric chloride derivative formation methylene blue in acid condition, by adding modified magnetic nano material, methylene blue is adsorbed, under the effect of externally-applied magnetic field, magnetic material is separated with matrix, remove the interference that in blood, urine, macromolecular complex confrontation measures.With a small amount of eluant, magnetic material being carried out eluting, eluent high performance liquid chromatography quantitative determines, and detection limit is up to 0.005 μ g/mL；Method has accuracy, the feature such as highly sensitive, simple and quick.

Description

A kind of determination method of hydrogen sulfide in blood and urine

technical field

The invention belongs to the technical field of drug detection in vivo, and in particular relates to a method for detecting hydrogen sulfide in blood and urine, in particular to a method for detecting hydrogen sulfide in blood and urine combined with dispersed solid-phase microextraction and high-performance liquid chromatography.

Background technique

In the cardiovascular system, hydrogen sulfide (H ₂ S) can open ATP-sensitive potassium ion channels, relax vascular smooth muscle, and lower blood pressure; the reduction of H ₂ S production in the body is closely related to the formation of spontaneous hypertension; exogenous administration H ₂ S can effectively protect myocardial ischemic injury. Since it can be produced in vivo and has various physiological functions, H ₂ S becomes the third gas signal molecule after NO and CO. More and more evidence shows that endogenous H ₂ S is closely related to various diseases. In many clinical diseases, the production and metabolism of endogenous H ₂ S are changed, and further affect the development of the disease The treatment with H ₂ S as the target has potential clinical application prospects; a reliable and accurate method for the determination of endogenous H ₂ S in blood and urine is useful for monitoring H ₂ S content in the body and predicting related diseases and diagnosis are important.

H ₂ S detection methods mainly include methylene blue spectrophotometry, sulfide ion selective electrode method, fluorescence derivatization method, gas chromatography, fluorescent probe and immunoassay. At present, the existing H ₂ S detection technology mainly focuses on environmental samples such as air and water, and its detection sensitivity is 1 mg/kg, while the content of endogenous hydrogen sulfide (H ₂ S) in the body is at the μmol level. The research team has previously used dispersion liquid-liquid microextraction combined with photometry, co-precipitation combined with high performance liquid chromatography to study the detection of H ₂ S in blood and urine, and achieved good results, and applied for invention patents (201410382081) and ( 201410062197). Further research found that due to the complex matrix of blood and urine, there are effects of color, protein, and fat, and there is a problem of incomplete matrix separation in coprecipitation and liquid-liquid microextraction.

Contents of the invention

The object of the present invention is to aim at the deficiencies of the prior art, to provide a method with good stability, little interference, accuracy, sensitivity and rapid determination of hydrogen sulfide content in blood and urine.

The invention utilizes sodium silicate-modified magnetic nanometer material (Fe ₃ O ₄ @SiO ₂ ) to adsorb methylene blue, a hydrogen sulfide derivative, to rapidly enrich the target substance, and can rapidly separate the matrix from the magnetic material under the action of a magnetic field. Due to the specificity of the magnetic adsorption between the magnetic adsorbent and the target, the influence of the suspended particles in the solution on the entire adsorption process is avoided, the matrix interference is reduced and the detection sensitivity is improved at the same time. The present invention utilizes magnetic material in conjunction with high-performance liquid chromatography (HPLC) method, the one, utilize magnetic material to efficiently separate the interfering substance in blood, urine; The amount of detaching agent is small, the elution is fast, and the detection limit can be 0.005 μg/mL; Fourth, the nano-magnetic material can be reused more than 3 times.

Unless otherwise stated, the percentages used in the present invention are all percentages by weight.

The purpose of the present invention is achieved through the following technical solutions:

Using the prepared sodium silicate-modified magnetic nanomaterial (Fe ₃ O ₄ @SiO ₂ ) as the solid-phase extraction agent, add 0.2% p-aminodimethyl Add 100-200 μL of aniline hydrochloride solution, then add 50-100 μL of 20% ferric chloride solution by mass percentage, mix well, and let stand for 5-10 minutes; add 5mol/L NaOH solution to adjust the pH of the sample to neutral, 3000r/ Min centrifuge for 2-5min, absorb the supernatant; add 1-3mg solid phase extractant to the supernatant, vortex for 1-2min to disperse the solid-phase extractant evenly in the water, then let it stand for 5-10min, then use an external magnetic field Collect the solid phase extraction agent with the target substance, remove the supernatant, elute the solid phase extraction agent with the eluent, collect the eluent, and use the set high performance liquid chromatography conditions for content determination.

The preparation of the sodium silicate modified magnetic nanomaterial (Fe ₃ O ₄ @SiO ₂ ) includes the following steps:

①Preparation of Fe ₃ O ₄ magnetic nanomaterials: Dissolve the ferric salt solution and the ferrous salt solution in 5 to 10 times the In deionized water with the volume of valent iron salt and divalent iron salt solution, under the protection of nitrogen, add ammonia water dropwise to make the pH of the solution system 9-12, heat in a water bath at 60-80°C, stirring speed 400-800rpm, and stir at a constant temperature After reacting for 1-3 hours, wash with deionized water until neutral, and vacuum-dry at 40-80°C for 6-12 hours to prepare ferroferric oxide magnetic nanomaterials;

②Preparation of Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials: Disperse the Fe ₃ O ₄ magnetic nanomaterials prepared in step ① in deionized water at the ratio of 1.0 g of Fe ₃ O ₄ magnetic nanomaterials to 100 mL of deionized water, And under the protection of nitrogen, drop 1.0 mol/L sodium silicate 10 mL into the Fe ₃ O ₄ nano-dispersion to make the pH of the system 6-7. After stirring and reacting for 2-4 hours, wash with deionized water for 4 times, and vacuum-dry at 40-80° C. for 6-12 hours to prepare Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials.

The ferric salt is FeCl ₃ .7H ₂ O or Fe ₂ (SO ₄ ) ₃ .9H ₂ O, and the ferric salt is FeCl ₂ .4H ₂ O or FeSO ₄ .7H ₂ O.

The treated blood refers to taking 5 mL of fresh blood, centrifuging at 2000 r/min for 10 min, transferring the serum to a centrifuge tube and distilling it to 5 mL with distilled water.

The 0.2% p-aminodimethylaniline hydrochloride solution is prepared by dissolving 0.1 g of p-aminodimethylaniline hydrochloride in 50 mL of 3mol/L hydrochloric acid.

The ferric chloride solution is prepared by dissolving 10g of ferric chloride in 50mL of distilled water.

One of the eluents methanol, acetonitrile and acetone is used in an amount of 300-600 μL.

The chromatographic conditions for the HPLC determination are as follows: the stationary phase is an analytical chromatographic column of C18, the eluent is methanol:water (volume ratio 50:50), and the elution method is isocratic elution; the flow rate is 1.0mL /min; the injection volume was 10 μL, the column temperature was 25 °C; the detection wavelength of the ultraviolet-visible light diode array detector was 665 nm.

Compared with the prior art, the present invention has the following significant advantages:

1. S ^2- is derivatized with p-aminodimethylaniline hydrochloride and ferric chloride under acidic conditions to form methylene blue, and the methylene blue is adsorbed by adding modified magnetic nanomaterials, and the magnetic material and Matrix separation, using a small amount of eluent to elute the magnetic material; not only the S ^2- derivatives are separated from the complex matrix, but also the sulfur ions are concentrated and enriched, and the detection limit can reach 0.005μg/mL;

2. The dispersive solid-phase microextraction process has the characteristics of simplicity, speed, good stability and easy operation. The flow chart is shown in Figure 1. The detection of hydrogen sulfide in the body has the status of large matrix interference and low content, providing fast, simple and intuitive qualitative and quantitative determination.

Description of drawings

Fig. 1 is the schematic diagram of dispersion solid-phase microextraction process;

Fig. 2 is a chromatogram comparing H ₂ S enrichment and non-enrichment by Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials in blood.

detailed description

The present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

Embodiment 1: Utilize this method to detect the content of hydrogen sulfide in human urine

1. Preparation of Fe ₃ O ₄ magnetic nanomaterials: 2mol/L FeCl ₃ 7H ₂ O 12mL and 1mol/L FeCl ₂ 4H ₂ O 12mL were mixed in 120mL deionized water, and under nitrogen protection, 50mL ammonia water was added dropwise to make The pH of the system is 9, heated in a water bath at 60°C, and the stirring speed is 400 rpm. After stirring and reacting at a constant temperature for 3 hours, it is washed with deionized water until neutral, and vacuum-dried at 70°C for 8 hours to obtain Fe ₃ O ₄ nanomaterials;

2. Preparation of Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials: Disperse 1.0 g of Fe ₃ O ₄ magnetic nanomaterials prepared in step 1 in 100 mL deionized water, and add 1.0 mol/L silicon Add 10 mL of Na2SO4 to the Fe ₃ O ₄ nano-dispersion liquid to make the pH of the system 6. After reacting for 2 hours with constant temperature stirring at 70°C and a stirring speed of 800 rpm, wash with deionized water 4 times, and vacuum-dry at 40°C for 12 hours to prepare Obtain Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials;

3. Urine detection: Take 5mL urine into a 10mL centrifuge tube, add 0.2% p-aminodimethylaniline hydrochloride solution (0.1g p-aminodimethylaniline hydrochloride dissolved in 50mL 3mol/L hydrochloric acid (obtained) 200 μL, then add 50 μL of 20% ferric chloride solution, mix well, and let it stand for 5 minutes; then add 5mol/L NaOH solution to adjust the pH of the sample to neutral, centrifuge at 3000r/min for 2min, absorb the supernatant; Add 2 mg of Fe ₃ O ₄ @SiO ₂ magnetic material to the solution, vortex and mix for 1 min, and let stand for 5 min; use a magnet close to the wall of the test tube to separate the magnetic material from the sample solution, and pour out the supernatant; For elution, the eluent is filtered with a 0.45 μm filter membrane; into high performance liquid phase, the chromatographic conditions for determination are: the stationary phase is an analytical chromatographic column of C18, and the eluent is a mixture of methanol and water with a volume ratio of 50:50 The elution method is isocratic elution; the flow rate is 1.0mL/min; the injection volume is 10μL, the column temperature is 25°C; the detection wavelength of the ultraviolet-visible light diode array detector is 665nm, and the content of hydrogen sulfide in urine is obtained 0.065μg/mL (see Figure 2).

Embodiment 2: Utilize this method to detect the content of hydrogen sulfide in human urine

1. Preparation of Fe ₃ O ₄ magnetic nanomaterials: Mix 2mol/L FeCl ₃ 7H ₂ O 12mL and 1mol/L FeSO ₄ 7H ₂ O 15mL in 150mL deionized water, and add 50mL ammonia water dropwise under nitrogen protection to make The pH of the system was 10, heated in a water bath at 70°C, and the stirring speed was 600 rpm. After stirring at a constant temperature for 1.5 hours, it was washed with deionized water until neutral, and vacuum-dried at 40°C for 12 hours to obtain Fe ₃ O ₄ nanomaterials. ;

2. Preparation of Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials: Disperse 1.0 g of Fe ₃ O ₄ magnetic nanomaterials prepared in step 1 in 100 mL deionized water, and add 1.0 mol/L silicon Add 10 mL of sodium bicarbonate into the Fe ₃ O ₄ nano-dispersion liquid to make the pH of the system 6.5. After stirring and reacting at a constant temperature for 2.5 hours at 75°C and a stirring speed of 600 rpm, wash with deionized water for 4 times, and vacuum-dry at 50°C for 10 hours. Prepare Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials;

3. Urine detection: Take 5mL urine into a 10mL centrifuge tube, add 0.2% p-aminodimethylaniline hydrochloride solution (0.1g p-aminodimethylaniline hydrochloride dissolved in 50mL 3mol/L hydrochloric acid (obtained) 150 μL, then add 80 μL of 20% ferric chloride aqueous solution by mass volume ratio, mix well, let stand for 7 minutes, add 5mol/L NaOH solution to adjust the pH of the sample to neutral, centrifuge at 3000r/min for 3min, and absorb the supernatant , add 1 mg of Fe ₃ O ₄ @SiO ₂ magnetic material to the supernatant, vortex and mix for 1.5 min, and let stand for 8 min; use a magnet to close the test tube wall, separate the magnetic material from the sample solution, and pour out the supernatant. The magnetic material was eluted with 500 μL of methanol, and the eluate was filtered with a 0.45 μm filter membrane, and then entered into high performance liquid phase for measurement at a wavelength of 665 nm (the conditions were the same as in Example 1), and the content of hydrogen sulfide in the urine was 0.035 μg /mL.

Embodiment 3: Using the present invention to detect the content of hydrogen sulfide in human blood

1. Preparation of Fe ₃ O ₄ magnetic nanomaterials: 2mol/L Fe ₂ (SO ₄ ) ₃ 9H ₂ O 10mL and 1mol/L FeSO ₄ 7H ₂ O 12mL were mixed in 150mL deionized water, under nitrogen protection , add 50mL of ammonia water dropwise to make the pH of the system 11, heat in a water bath at 80°C, stir at 500rpm, stir and react at a constant temperature for 2h, wash with deionized water until neutral, and dry in vacuum at 50°C for 10h to obtain trioxide ferromagnetic nanomaterials;

2. Preparation of Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials: Disperse 1.0 g of Fe ₃ O ₄ magnetic nanomaterials prepared in step 1 in 100 mL deionized water, and add 1.0 mol/L silicon Add 10 mL of sodium bicarbonate into the Fe ₃ O ₄ nano-dispersion liquid, make the pH of the system 7, stir at 85°C and 400 rpm at a constant temperature for 3 hours, wash with deionized water for 4 times, and dry in vacuum at 60°C for 8 hours to prepare Obtain Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials;

3. Blood test: take 5mL of fresh blood into a 10mL centrifuge tube, centrifuge at 2000r/min for 10min, transfer the serum to a new centrifuge tube, dilute to 5 mL with distilled water, add 0.2% p-aminodimethylaniline hydrochloride Solution (prepared by dissolving 0.1g p-aminodimethylaniline hydrochloride in 50mL 3mol/L hydrochloric acid) 100μL, then add 100μL of ferric chloride aqueous solution with a mass volume ratio concentration of 20%, mix well, let stand for 10min, add 5mol /L NaOH solution to adjust the pH of the sample to neutral, centrifuge at 3000r/min for 5min, absorb the supernatant, add 3mg of Fe ₃ O ₄ @SiO ₂ magnetic material to the supernatant, vortex and mix for 2min, let it stand for 10min, and use a magnet Close to the test tube wall, separate the magnetic material from the sample solution, pour out the supernatant, elute the magnetic material with 600 μL acetone, filter the eluate with a 0.45 μm filter membrane, enter the high performance liquid phase and measure it at a wavelength of 665nm ( The conditions are the same as in Example 1), and the content of hydrogen sulfide in the blood is 0.0082 μg/mL.

Example 4: Using the present invention to detect the content of hydrogen sulfide in human blood

1. Preparation of Fe ₃ O ₄ magnetic nanomaterials: Mix 2mol/L FeCl ₃ 7H ₂ O 10mL and 1mol/L FeSO ₄ 7H ₂ O 11mL in 160mL deionized water, and add 50mL ammonia water dropwise under nitrogen protection Make the pH of the system 12, heat in a water bath at 60°C, stir at 800 rpm, stir and react at a constant temperature for 1 hour, wash with deionized water until neutral, and dry in vacuum at 60°C for 8 hours to prepare ferroferric oxide magnetic nanomaterials ;

2. Preparation of Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials: Disperse 1.0 g of Fe ₃ O ₄ magnetic nanomaterials prepared in step 1 in 100 mL deionized water, and add 1.0 mol/L silicon Add 10mL of Na2SO4 to the nano-dispersion of Fe ₃ O ₄ to make the pH of the system 7. After reacting at 80°C with a stirring speed of 500rpm at a constant temperature for 3h, wash 4 times with deionized water and dry in vacuum at 80°C for 6h to obtain Fe ₃ O ₄ @SiO ₂ magnetic nanomaterials;

3. Blood test: take 5mL of fresh blood into a 10mL centrifuge tube, centrifuge at 2000r/min for 10min, transfer the serum to a new centrifuge tube, dilute to 5 mL with distilled water, add 0.2% p-aminodimethylaniline hydrochloride Solution (0.1g of p-aminodimethylaniline hydrochloride dissolved in 50mL 3mol/L hydrochloric acid) 100μL, then add 50μL of ferric chloride aqueous solution with a mass volume ratio concentration of 20%, mix well, let stand for 8min, add 5mol /L NaOH solution to adjust the pH of the sample to neutral, centrifuge at 3000r/min for 4min, absorb the supernatant, add 1mg of Fe ₃ O ₄ @SiO ₂ magnetic material to the supernatant, vortex and mix for 2 minutes, let stand for 8min, and use A magnet is attached to the wall of the test tube, the magnetic material is separated from the sample solution, and the supernatant is poured out. The magnetic material was eluted with 300 μL of acetonitrile, and the eluate was filtered with a 0.45 μm filter membrane. Enter high performance liquid phase and measure at a wavelength of 665nm (the conditions are the same as in Example 1), and the content of hydrogen sulfide in the blood is 0.049 μg/mL.

Claims (4)

1. the assay method of hydrogen sulfide in a blood and urine, it is characterised in that: receive with the magnetic that the sodium silicate of preparation is modified Rice material is solid extracting agent, adds 0.2% P-aminodimethylaniline hydrochlorate by every 5mL urine or the blood that processed Solution 100～200 μ L, adds the ferric chloride aqueous solutions 50～100 μ L of mass volume ratio concentration 20%, mix homogeneously, stands 5～10min；Being subsequently adding 5mol/L NaOH solution regulation pH the most neutral, 3000r/min is centrifuged 2～5min, Aspirate supernatant； Adding 1～3mg solid extracting agent in supernatant, vortex 1～2min makes solid extracting agent be dispersed in water, then stands After 5～10min, collect the solid extracting agent with object by external magnetic field, remove supernatant, use eluent Solid-Phase Extraction Agent, collects eluent, uses high performance liquid chromatograph to carry out assay；

The preparation of the magnetic Nano material that described sodium silicate is modified comprises the following steps:

①Fe₃O₄The preparation of magnetic Nano material: will according to the ratio that trivalent iron salt and divalent iron salt mol ratio are 2.0～1.6:1 Ferric salt solution and divalent iron salt solution are dissolved in the trivalent iron salt of 5～10 times and the deionized water of divalent iron salt liquor capacity In, under nitrogen protection, dropping ammonia makes solution system pH be 9～12, at 60～80 DEG C, mixing speed 400～800 rpm Under, heating in water bath, after constant temperature stirring reaction 1～3h, it is washed with deionized to neutrality, 40～80 DEG C of vacuum drying 6～12h, Prepare ferroferric oxide magnetic nano-material；

②Fe₃O₄@SiO₂The preparation of magnetic Nano material: by 1.0g Fe₃O₄Magnetic Nano material adds 100 mL deionized waters The Fe that 1. step is prepared by ratio₃O₄Magnetic Nano material disperses in deionized water, and under nitrogen protection, drips 1.0 Mol/L sodium silicate 10 mL to Fe₃O₄In nano dispersion fluid, the system pH of making is 6～7,70～85 DEG C, mixing speed 400～ Under 800rpm, after constant temperature stirring reaction 2～4h, it is washed with deionized 4 times, 40～80 DEG C of vacuum drying 6～12h, prepares Fe₃O₄@SiO₂Magnetic Nano material；

The chromatographic condition of described high-performance liquid chromatogram determination is: fixing the analysis chromatographic column mutually for C18, eluent is volume ratio The mixed liquor of the first alcohol and water of 50 50, type of elution is isocratic elution；Flow velocity is 1.0mL/min；Sample size is 10 μ L, column temperature 25℃；The detection wavelength of ultraviolet-visible photodiode array detector is 665nm；

Described eluant is the one in methanol, acetonitrile, acetone.

The assay method of hydrogen sulfide in blood the most according to claim 1 and urine, it is characterised in that: described ferric iron Salt is FeCl₃·6H₂O or Fe₂(SO₄)₃·9H₂O, divalent iron salt is FeCl₂·4H₂O or FeSO₄·7H₂O。

The assay method of hydrogen sulfide in blood the most according to claim 1 and urine, it is characterised in that: treated Blood refers to take 5mL fresh blood, and 2000r/min is centrifuged 10min, will be settled to 5 with distilled water in serum transfers to centrifuge tube ML prepares.

The assay method of hydrogen sulfide in blood the most according to claim 1 and urine, it is characterised in that: described 0.2% pair of ammonia Base dimethylaniline dihydrochloride solution be 0.1g P-aminodimethylaniline hydrochlorate be dissolved in 50mL 3mol/L hydrochloric acid prepare.