CN110308218B - Detection method and application of hemoglobin adduct for evaluating in-vivo exposure of glycidol and ester thereof - Google Patents

Abstract

The invention discloses a method for detecting a hemoglobin adduct for evaluating in-vivo exposure of glycidol and esters thereof, which comprises the steps of preparing hemoglobin dry powder, and deriving and purifying the hemoglobin powder; setting chromatographic conditions for isocratic elution; setting mass spectrum conditions for mass spectrum quantitative detection; and (3) carrying out quantitative analysis on the sample by using the liquid phase-mass spectrum combined method and a standard curve method. The method adopts isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify the hemoglobin adduct of the glycidol, greatly shortens the analysis time, simultaneously leads Gly-Val-PTH spatial isomer to be better separated, realizes synchronous detection, and can more comprehensively evaluate the internal exposure of the glycidol.

Description

Detection method and application of hemoglobin adduct for evaluating in-vivo exposure of glycidol and ester thereof

Technical Field

The invention relates to a synchronous detection method for evaluating hemoglobin adducts of dietary glycidol and esters thereof exposed in vivo for a long time and application thereof, belonging to the field of food safety.

Background

The german risk assessment institute evaluated the contamination of glycidyl esters in infant milk powder and edible oil in 2009 (assuming 100% conversion of glycidyl esters to glycidyl in vivo), based on BMDL10 for the genotoxicity and carcinogenicity of glycidyl esters, the Exposure Margin (MOE) ratio of glycidyl esters in infant ingested milk powder was 670, and the health risk was extremely high. Attention is paid to contamination and exposure of 3-chloro-1, 2-propanediol (3-monochloropropane-1, 2-diol; 3-MCPD) or chloropropanol esters alone to underestimate the health risk of such compounds, and thus contamination of various types of chloropropanol esters and glycidyl esters in food products is also of concern. Research on rats by Appel and the like shows that glycidol palmitate is rapidly hydrolyzed into glycidol in the body of rats, mainly in the gastrointestinal tract, and is distributed in various tissues; the rate of glycidol hydrolysis from the glycidol ester binds to hemoglobin is only slightly slower compared to the control glycidol group; in both groups, the amount of glycidol bound to hemoglobin and the amount of mercaptouric acid excreted from the urine were comparable (Apel K E, Abraham K, Bergerris E, et al, relative biological availability of glycerol from glycerol fatty acid esters in rates [ J ]. Archives of geneticals,

2013,87(9):1649-1659.). This study shows that glycidyl esters are rapidly converted to glycidol in animals to exert toxic effects. Glycidol is classified as a class 2A carcinogen (highly carcinogenic to humans) by the international agency for research on cancer (IARC).

The german grease association (DGF)2011 issued a method for simultaneously measuring 3-MCPD esters and glycidyl esters in grease, which is currently widely accepted. Garbarllo-Rubio et al established a synchronous detection method for detecting 3-MCPD esters and glycidyl esters in fish oil by GC-MS-MS method (Garbarllo-Rubio A, Soto-Chinchilla J, Moreno A, et al. A novel method for the determination of carbohydrates and 3-monochloropropanodio esters in fish oil by gas chromatography method [ J ]. Talanta,2017,165: 267-273.). The method comprises the steps of analyzing substances to be detected in a sample by a liquid chromatography-mass spectrometry (such as LC-MS, LC-MS/MS, LC-TOF/MS and the like), directly determining glycidyl ester without a derivatization method, not hydrolyzing the sample, simply processing an oil sample, such as homogenizing, dissolving and diluting with a solvent and the like, and then directly injecting a sample. Blumhorst et al directly determined 7 glycidylesters in Vegetable Oils by LC-MS method (Blumhorst M R, Venkitasuramanian P, Collison M W.Direct Determination of Glycidyl Esters of Fatty Acids in Fatty Acids by LC-MS [ J ]. Journal of the American Oil Chemists Society,2011,88(9): 1275-1283.).

Domestic scholars also develop various methods for measuring glycidol and esters thereof, and a method for quantifying the glycidol ester in the oil and fat by adopting an epoxy compound to perform a specific reaction with sodium N, N-diethyldithiocarbamate, enabling a reaction product to have a maximum absorption wavelength at 278nm and analyzing the absorbance of the maximum absorption wavelength (a detection method of the glycidol ester in the edible oil and fat, CN102937580A, 2013); alcoholysis of 3-chloropropanediol fatty acid ester and epoxy glycerin fatty acid ester in edible oil by strong base alcoholysis method to convert into 3-alkoxy propylene glycol, and measuring 3-alkoxy propylene glycol content by gas chromatography or gas chromatography-mass spectrometry (a method for detecting 3-chloropropanediol fatty acid ester and epoxy glycerin fatty acid ester in edible oil, CN 1046988113, 2015); after ultrasonic extraction, transesterification, degreasing and derivatization, GC-MS analysis (a method for simultaneously detecting 3-chloropropanol ester and glycidyl ester in food, CN106501390A, 2017) is carried out. At the same time, researchers have studied reducing the production of glycidol and its esters during processing (a method of reducing the content of glycidol esters in edible oils with adsorbents, CN105053269A, 2015; reducing the content of glycidol esters in oils, CN102711496A, 2012).

Glycidol and esters thereof are used as hazards in food, and the evaluation of in vivo risks of the glycidol and esters thereof is mainly carried out by adopting an evaluation mode of meal external exposure, namely, the meal level evaluation is carried out by investigating the content of the glycidol and the esters thereof in various foods. The strategy has strong operability and is widely applied to public health systems in China. However, the evaluation method has certain limitations, and cannot visually reflect the metabolic mode and exposure level of glycidol and esters thereof after entering the human body. Therefore, by adopting the evaluation mode of in vivo metabolism and exposure, attention needs to be paid, and the in vivo research method of the glycidol and the ester thereof is necessary to research, namely, the risk evaluation of the in vivo metabolism biomarker of the glycidol and the ester thereof is carried out through detection. In vivo strategies allow a more accurate assessment of the daily uptake of glycidol and its esters than in vitro. At present, international research and application of glycidyl ester and its ester in vivo hemoglobin adduct are mainly focused on europe and japan, and development bottlenecks for its detection method mainly exist in blood pretreatment and instrument analysis. The blood pretreatment includes two aspects, namely hemoglobin preparation on one hand and derivative purification on the other hand. Wherein, various reported derivatization modes are similar, and different derivatization agents are selected, such as pentafluorophenyl thioisocyanate (PFPITC), Phenyl Isothiocyanate (PITC) or Fluorescein Isothiocyanate (FITC) and the like. Honda et al, by GC-MS-MS, determine the hemoglobin adduct of glycidol in human blood (Honda H, Onishi M, Fujii K, et al.Measurement of glycerol hemoglobin adducts in humans of the same inorganic sources of glycerol fatty acids esters, 2011,49(10): 2536-; aasa et al, derivatized with an adduct of FITC and hemoglobin, measured for hemoglobin content by LC-MS-MS (Aasa J, Abramsson-Zettererg L, Carlsson H, et al, the generic toxicity of hemoglobin esterified from microorganisms and hemoglobin additives in mice [ J ]. Food & Chemical society, 2016,100.).

The medium-and long-term exposure of dietary glycidol and its esters in vivo is mainly based on N- (2, 3-dihydroxypropyl) -valine (Gly-Val) as the main biomarker. Because of its specificity of binding to the valine of erythrocyte hemoglobin in blood, it is common to use derivatization methods to bind and remove hemoglobin, and to indirectly detect the derivatization by quantitative analysis methods to achieve detection and analysis of hemoglobin adducts. The method has the disadvantages of long pretreatment operation, low derivatization efficiency and purification efficiency, and very limited detection sensitivity by adopting a gas chromatography method, and is not beneficial to detection of large-batch biological samples and detection of samples with low exposure content at a dietary level.

Disclosure of Invention

The invention aims to provide a detection method and application of hemoglobin adduct for evaluating long-term in vivo exposure in dietary glycidol and ester thereof.

In order to solve the technical problems, the invention provides a method for detecting a hemoglobin adduct for evaluating in-vivo exposure of glycidol and esters thereof, which comprises the step 1) of preparing dry hemoglobin powder, and is characterized by further comprising the following steps:

2) derivatization and purification:

adding 0.8-3 mL of formamide and 20-100 mu L of 1mol/L NaOH solution into 20-100 mg of hemoglobin powder, adding 5-30 mu L of a derivative, namely pentafluorophenyl thioisocyanate (pentafluorophenyl isothiocyanate), and uniformly mixing in a vortex manner; oscillating for 12-24 h at room temperature for derivatization, and then continuing derivatization in a water bath at 30-60 ℃ for 0.5-6 h;

after water bath derivatization is finished, adding 0.2-2 mL of 20% (mass%) NaCl solution (for precipitating hemoglobin) and 20 muL of isotope internal standard-d 5-Gly-Val-PTH solution (the solvent is methanol), vortex mixing uniformly, centrifuging at the rotating speed of 8000-12000 rpm for 2-15 min, and taking supernatant;

activating and balancing a solid phase extraction column (activating by 3mL of methanol in advance, balancing by 3mL of pure water), taking a supernatant fluid, leaching by using 2-3 mL of leacheate (for removing impurities), then eluting by using 2-3 mL of methanol, and collecting an eluent; drying the eluent at 40 deg.C with nitrogen, diluting to 1mL with mobile phase, dissolving for 1min by vortex, filtering with 0.22 μm microporous membrane, and analyzing by sample injection;

the leacheate is a methanol water solution with the volume concentration of 5-10%;

the mobile phase is mobile phase A: mobile phase B: the volume ratio of 40: 60-60: 40;

3) and chromatographic conditions:

the instrument comprises the following steps: ultra High Performance Liquid Chromatography (UHPLC); flow rate: 0.2 mL/min; column temperature: 40 ℃; mobile phase A: 0-1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile; isocratic elution ratio: a and B are 40: 60-60: 40; sample introduction volume: 5-10 mu L; sample introduction time: 10-15 min.

The kind and specification of the chromatographic column are ACQUTITY

HSST3 (2.1X 150mm i.d.,1.8 μm) or ACQUTITY

BEH C18(2.1×150mm i.d.,1.7μm)；

4) And mass spectrum conditions:

the instrument comprises the following steps: a triple quadrupole tandem mass spectrometer; the mass spectrum quantification method comprises the following steps: multiple Reaction Monitoring (MRM); an ion source: electrospray (ESI) negative ion scanning mode; temperature of sheath gas: 375 ℃; the flow rate of the sheath gas is 8L/min; nozzle voltage: 500V; atomizer pressure: 45 psi; capillary voltage: 2000-3000V; temperature of the drying gas: 300 ℃; flow rate of the dryer: 5L/min; carrier gas: nitrogen gas; collision gas: nitrogen gas; the total ion flow and the graph of each ion channel are shown in FIG. 2;

TABLE 1 Mass Spectrometry parameters of Gly-Val-PTH and its isotopes

Note:^ain order to quantify the ion channels,^bis a qualitative ion channel;

5) and the result is as follows:

and (3) carrying out quantitative analysis on the sample by using the liquid phase-mass spectrum combined method and a standard curve method.

As an improvement of the inventive method for the detection of hemoglobin adducts evaluating the in vivo exposure of glycidol and its esters: the isotope internal standard in the step 2),

when the sample is a human blood sample, the concentrations of isotope internal standard d5-Gly-Val-PTH are all 1 mug/mL;

when murine blood samples were used, the concentration of the isotopic internal standard d5-Gly-Val-PTH was 10. mu.g/mL.

The invention comprises a pretreatment method of blood (rat blood and human blood), a chromatographic condition of a detection method and a mass spectrum condition. Wherein the pretreatment comprises preparation, derivation and column purification of hemoglobin powder. The preparation of the hemoglobin powder relates to the dosage and dilution times of red blood cells, quick freezing time, thawing water bath temperature, centrifugal speed and time and the dosage of an organic solvent; the derivatization conditions relate to the dosage of the hemoglobin powder, the added solvent and volume, the derivatization reaction time and temperature; the column-passing purification relates to a precipitated protein solvent, the type and specification of a solid-phase extraction column and the volume of column-passing leacheate; the detected chromatographic conditions relate to the type and specification of a chromatographic column, the type and proportion of a mobile phase, the sample injection volume and a gradient elution program; the detected mass spectral conditions relate to capillary voltage, cone-hole voltage and collision voltage.

The volume of the unfrozen red blood cells is 0.5-1 mL;

the dilution multiple of the red blood cells is 2-10 times;

the quick-freezing time at-80 ℃ is 0.5-3 h;

the temperature of the unfreezing water bath is 20-55 ℃;

the volume of the acidified isopropanol (50mmol/L hydrochloric acid) is 15-30 mL;

the preparation method of the acidified isopropanol solution (50mmol/L hydrochloric acid) comprises the following steps: adding concentrated hydrochloric acid with the molar concentration of 12mol/L into the isopropanol solvent of 0.83mL to 200mL, and uniformly stirring;

the centrifugal rotating speed is 3500-6000 rpm;

the centrifugation time is 2-15 min;

the time for standing and precipitating the protein at 4 ℃ is 0.5-4 h;

the volume of the ethyl acetate is 10-30 mL;

the volume of the n-hexane is 10-30 mL;

the dosage of the hemoglobin powder is 20-100 mg;

the volume of the formamide is 0.8-3 mL;

the volume of the NaOH solution (1mol/L) is 20-100 mu L;

the volume of the pentafluorophenyl thioisocyanate (PFPITC) is 5-30 mu L;

the derivatization reaction time is 12-24 h;

the temperature of the derivatization water bath is 30-60 ℃;

the reaction time of the derivative water bath is 0.5-6 h;

the volume of the 20% NaCl solution is 0.2-2 mL;

the type and specification of the solid phase extraction column are Supelclean^TMLC-18 cartridge (3cc,500 mg; Supelco, Bellefonte, USA), oasISR HLB cartridge (3cc,60mg, Waters, Milford, MA) and Extrelut^TM NT cartridge(3cc,1g；Merck,Darmstadt,Germany)；

The leacheate is a 5-10% methanol aqueous solution (v/v);

the volume of the leacheate is 2-3 mL;

the kind and specification of the chromatographic column are ACQUTITY

HSST3 (2.1X 150mm i.d.,1.8 μm) and ACQUTITY

BEH C18(2.1×150mm i.d.,1.7μm)；

The mobile phase is 0-0.5% formic acid aqueous solution (v/v) and acetonitrile, and the ratio of the mobile phase to the acetonitrile is 40: 60-60: 40;

the sample injection volume is 5-10 mu L;

the capillary voltage under the mass spectrum condition is 2000-3000V;

the cone voltage and the collision voltage are shown in table 1.

The scheme of the invention specifically comprises the following steps: a synchronous detection method for evaluating hemoglobin adducts exposed in vivo for a long time in dietary glycidol and esters thereof comprises the following steps:

preparation of hemoglobin dry powder

Centrifuging the whole blood sample in an anticoagulation tube at 3500rpm for 5min, removing supernatant (blood plasma) and middle layer (white membrane), adding PBS solution or normal saline into the erythrocyte part at the lower layer, blowing with a dropper, sucking out the supernatant, centrifuging at 3000rpm for 5min, and repeatedly washing for 3 times to obtain erythrocytes.

Taking a certain amount of red blood cells (0.5-1 mL), transferring the red blood cells into a centrifuge tube, adding pure water to dilute the red blood cells by a certain multiple (2-10 times), performing vortex shaking for 10min, and quickly freezing for a period of time (0.5-3 h) in a refrigerator at the temperature of-80 ℃. And then, thawing in a water bath at a certain temperature (20-55 ℃) to obtain the hemolytic liquid. Adding a certain amount of acidified isopropanol solution (15-30 mL) into the hemolytic liquid, and centrifuging for a certain time (2-15 min) at a certain rotating speed (3500-6000 rpm). And transferring the dark red supernatant into another centrifuge tube, adding a certain amount of ethyl acetate (10-30 mL), carrying out vortex oscillation for 10min, placing the centrifuge tube in a refrigerator at 4 ℃ for a period of time (0.5-4 h) to precipitate the protein, then centrifuging the centrifuge tube at a certain rotating speed (3500-6000 rpm) for a period of time (2-15 min), and discarding the supernatant. And adding a certain amount of ethyl acetate (10-30 mL) into the precipitate, repeatedly washing the precipitate for 2 times according to the steps, adding a certain amount of n-hexane (10-30 mL) into the precipitate, washing the precipitate once, standing the precipitate overnight in a ventilated kitchen, drying the precipitate, grinding the dried precipitate into powder to obtain the hemoglobin powder, and storing the hemoglobin powder at the temperature of-20 ℃.

(II) derivatization and purification

Taking a certain amount of hemoglobin powder (20-100 mg), adding a certain amount of formamide (0.8-3 mL) and a certain amount of 1mol/L NaOH solution (20-100 mu L), adding a certain amount of derivative pentafluorophenyl thioisocyanate (5-30 mu L), and uniformly mixing by vortex. And (3) oscillating for a certain time (12-24 h) at room temperature for derivatization, and then continuing to derivatize for a certain time (0.5-6 h) in a water bath (30-60 ℃) at a certain temperature. After water bath derivatization, a certain amount of 20% NaCl solution (0.2-2 mL) is added into the solution to precipitate hemoglobin. For human blood samples, 20. mu.L of an isotope internal standard (d5-Gly-Val-PTH, concentrations 1. mu.g/mL) was added; and adding 20 mu L of isotope internal standard (d5-Gly-Val-PTH with the concentration of 10 mu g/mL) into a rat blood sample, uniformly mixing by vortex, centrifuging for a certain time (2-15 min) at the rotation speed of 10000rpm, and taking the supernatant for later use.

Activating the solid-phase extraction column with 3mL of methanol in advance, balancing with 3mL of pure water, after the solution is drained, loading the supernatant to the solid-phase extraction column, draining, leaching with a certain volume (2-3 mL) of leacheate (5% -10% methanol aqueous solution), removing impurities and discarding the leacheate. And finally, eluting with a certain volume (2-3 mL) of methanol, and collecting the eluent. Drying the eluent at 40 ℃ with nitrogen, diluting to 1mL with initial mobile phase solution, dissolving for 1min by vortex, filtering with 0.22 μm microporous membrane, and analyzing by sample injection.

Remarks explanation:

1. the processing method is suitable for sample preparation of rat blood and human blood;

2. the type and specification of the solid phase extraction column are Supelclean^TM LC-18 cartridge(3cc,500mg；Supelco,Bellefonte,USA)、

HLB cards (3cc,60mg, Waters, Milford, MA) and Extrelut^TM NT cartridge(3cc,1g；Merck,Darmstadt,Germany)；

3. The rat blood sample and the human blood sample have different dosages of the added internal standard due to different contents of the glycidol hemoglobin adduct;

4. as for the preparation of Gly-Val-PTH and d5-Gly-Val-PTH, the following publicly published documents can be referred to: carlsson H, Von Stedingk H, Nilsson U, et al LC-MS/MS Screening Stratagene for Unknown additives to N-Terminal Valine in Hemoglobin Applied to Smokers and Nonsmokers [ J ]. Chemical Research in morphology 2014,27(12): 2062. 2070.

(III) chromatographic conditions:

the instrument comprises the following steps: ultra High Performance Liquid Chromatography (UHPLC); flow rate: 0.2 mL/min; column temperature: 40 ℃; mobile phase A: 0-1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile; isocratic elution ratio: a and B are 40: 60-60: 40; sample introduction volume: 5-10 mu L; sample introduction time: 10-15 min.

The kind and specification of the chromatographic column are ACQUTITY

HSST3 (2.1X 150mm i.d.,1.8 μm) or ACQUTITY

BEH C18(2.1×150mm i.d.,1.7μm)。

(IV) mass spectrometry conditions:

the instrument comprises the following steps: a triple quadrupole tandem mass spectrometer; the mass spectrum quantification method comprises the following steps: multiple Reaction Monitoring (MRM); an ion source: electrospray (ESI) negative ion scanning mode; temperature of sheath gas: 375 ℃; the flow rate of the sheath gas is 8L/min; nozzle voltage: 500V; atomizer pressure: 45 psi; capillary voltage: 2000-3000V; temperature of the drying gas: 300 ℃; flow rate of the dryer: 5L/min; carrier gas: nitrogen gas; collision gas: nitrogen gas. The total ion flow and the individual ion channels are shown in FIG. 2.

TABLE 1 Mass Spectrometry parameters of Gly-Val-PTH and its isotopes

Note:^ain order to quantify the ion channels,^bfor the qualitative ion channel

Note that when the method uses mass spectrometry, Gly-Val-PTH is the adduct to be analyzed, d5-Gly-Val-PTH is its isotopic internal standard (for internal standard quantitation), table 1 shows a total of 4 mass spectrometric detection channels, i.e. each compound has 2 channels, the first is a quantitative ion channel, and the second is a qualitative ion channel (to aid quantitation and confirmation). For example, for Gly-Val-PTH, 396.86 is the molecular ion peak of Gly-Val-PTH, and 189.96 and 376.86 are both characteristic fragment ion peaks of Gly-Val-PTH. 396.86>189.96 was selected as the quantitative ion channel, 396.86>376.86 was selected as the qualitative ion channel, and so on. And the two parameters of the cone hole voltage and the collision voltage are main parameters for determining the quantitative and qualitative ion response values of the mass spectrum.

The invention is mainly different from the prior art in that:

firstly, the method prepares the blood hemoglobin powder, selects a specific derivative agent to derive and combine a target object, adopts a pretreatment method such as an SPE column mode to purify a sample, and the like, so that a glycidyl hemoglobin adduct with very low content in rat blood and human blood is convenient to detect and can reach a lower detection limit;

secondly, the chiral structure of Gly-Val-PTH is successfully separated at the baseline level by optimizing the condition of the ultra-high performance liquid chromatography. Gly-Val-PTH has two configurations, levorotatory (L) and dextrorotatory (D), and the two stereoisomers are determined for the first time in the invention.

Thirdly, the invention adopts an isotope dilution method ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) to develop a method for detecting the glycidol hemoglobin. The chromatographic separation efficiency is improved, the analysis time is reduced, the sample treatment is simple, and higher sensitivity and lower quantitative limit can be reached.

The invention has the following technical advantages:

1. the invention develops a pretreatment step suitable for detecting the glycidol hemoglobin adduct in rat blood and human blood.

The invention optimizes the preparation process of the hemoglobin powder, enables the blood of rats and human bodies to release hemoglobin to the maximum extent, and obtains the hemoglobin dry powder with higher purity by cleaning with an organic solvent. The adopted derivatization agent combines the glycidyl adduct at the tail end of the peptide chain of the hemoglobin, and removes protein, thereby eliminating the interference of protein in a solution to be detected and reducing the matrix effect of mass spectrum detection. The SPE mode is adopted to purify the sample, so that on one hand, the interference of impurities in the sample is avoided, on the other hand, a solvent formamide (with a high boiling point and not beneficial to volume fixing after nitrogen blow drying) for dissolving hemoglobin is separated from a target object, the interference of the solvent is reduced, and the SPE mode has important significance for instrument detection;

2. the invention realizes the chromatographic separation of the glycidol hemoglobin adduct and simultaneously separates the chiral isomer of Gly-Val-PTH at the baseline level.

The method achieves better separation of the glycidol hemoglobin adduct by optimizing chromatographic conditions such as a chromatographic column, a flow line, elution conditions and the like. Where Gly-Val-PTH has a chiral structure, it is successfully separated under the chromatographic conditions of the present invention and will be quantified identically.

3. The chromatographic separation and the synchronous quantitative analysis of the glycidol hemoglobin adduct are realized. The method has important significance for comprehensively evaluating the level detection of each hemoglobin adduct of the dietary glycidol and the ester thereof after the in vivo middle-term exposure.

4. The method is characterized in that a standard substance synthesized by a derivative is used for isotopic tracing UHPLC-MS-MS quantitative detection of a glycidol hemoglobin adduct for the first time, HSS T3 chromatographic columns are successfully utilized to realize baseline separation of Gly-Val-PTH isomer, and the method is suitable for blood determination of rats and humans.

The method adopts isotope dilution ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to quantify the hemoglobin adduct of the glycidol, greatly shortens the analysis time, simultaneously leads Gly-Val-PTH spatial isomer to be better separated, realizes synchronous detection, and can more comprehensively evaluate the internal exposure of the glycidol. The detection limit of the method can reach the level of 10pmol/g Hb, and the method has the advantages of high sensitivity and high detection sensitivity; the limit of detection quantitation of the prior art is greater than 10pmol/g Hb, and is about 15pmol/g Hb. In addition, by means of the ultra-high performance liquid chromatography, the column pressure borne by the ultra-high performance liquid chromatography column is many times higher than that borne by a common liquid phase column, so that the detection efficiency is greatly improved. The analysis time of the method only needs 10-15 minutes. Therefore, the method has the advantages of simplicity, detection accuracy and the like.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a total ion flowsheet and a sub-ion flowsheet of Gly-Val-PTH;

the chromatogram of 396.86>376.86 qualitative ion channel of Gly-Val-PTH is sequentially arranged from top to bottom; 396.86>189.96 chromatogram of Gly-Val-PTH quantifying ion channels; total ion flowsheet of Gly-Val-PTH.

FIG. 2 is a total ion flow graph of Gly-Val-PTH in murine blood;

FIG. 3 is a total ion flow graph of Gly-Val-PTH in human blood.

FIG. 4 is a graph comparing the separation performance of different columns.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1, a method for the simultaneous assay of hemoglobin adducts evaluating long-term in vivo exposure in dietary glycidol and its esters, the assay subject being rat blood, the following steps are performed in sequence:

1) preparing hemoglobin dry powder:

centrifuging 5ml of the whole blood sample in an anticoagulation tube at 3500rpm for 5min, removing supernatant (blood plasma) and middle layer (white membrane), adding the erythrocyte part left in the lower layer into PBS solution or 3ml of normal saline, sucking the supernatant after uniformly blowing with a dropper, centrifuging at 3000rpm for 5min, and repeatedly washing for 3 times to obtain 2ml of erythrocytes.

Taking 1mL of red blood cells, transferring the red blood cells into a centrifuge tube, adding 5mL of pure water to dilute the red blood cells by 6 times, vortexing and shaking for 10min, and quickly freezing the red blood cells in a refrigerator at the temperature of-80 ℃ for 2 h. Then, the resulting mixture was thawed in a water bath at 37 ℃ to obtain a hemolyzed solution. 20mL of acidified isopropanol solution was added to the hemolysis solution and centrifuged at 5000rpm for 10 min. Transferring the dark red supernatant to another centrifuge tube, adding 20mL ethyl acetate, vortexing and shaking for 10min, standing in a refrigerator at 4 ℃ for 2h to precipitate protein, centrifuging at 4500rpm for 10min, and discarding the supernatant. Adding 15mL of ethyl acetate into the precipitate, repeatedly washing the precipitate for 2 times according to the steps, adding 20mL of n-hexane for washing the precipitate once, standing the precipitate in a ventilated kitchen overnight, drying the precipitate, grinding the dried precipitate into powder to obtain 150-200 mg of hemoglobin powder, and storing the hemoglobin powder at-20 ℃.

2) Derivatization and purification:

50mg of hemoglobin powder is taken, 1.5mL of formamide and 40 mu L of 1mol/L NaOH solution are added, 10 mu L of derivatization agent pentafluorophenyl thioisocyanate is added, and vortex mixing is carried out. Derivatization was carried out at room temperature with shaking for 16h, and then continued for 2h in a water bath at 45 ℃. After the derivation in the water bath, 0.5mL of a 20% (mass%) NaCl solution was added to the solution to precipitate hemoglobin. Adding 20 μ L mixed isotope internal standard (d5-Gly-Val-PTH, concentration of 10 μ g/mL, solvent methanol), vortex mixing well, centrifuging at rotation speed of 10000rpm for 15min, and collecting supernatant for use.

Supelclean is selected as solid phase extraction column^TMLC-18 cartridge (3cc,500 mg; Supelco, Bellefonte, USA). Activating with 3mL of methanol in advance, balancing with 3mL of pure water, after the solution is drained, loading the supernatant to a solid phase extraction column, draining, leaching with 2mL of 10% methanol aqueous solution, removing impurities and discarding the leaching solution. Finally, the mixture is eluted by 2mL of methanol, and the eluent is collected. Drying the eluent at 40 deg.C with nitrogen, diluting to 1mL with mobile phase solution (as shown in step 3), dissolving for 1min by vortex, filtering with 0.22 μm microporous membrane, and analyzing by sample injection.

The mobile phase solution was 0.1% aqueous formic acid: 0.1% formic acid acetonitrile 50: 50;

0.1% formic acid aqueous solution, which means that the volume concentration of formic acid in the mixed solution of formic acid and water is 0.1%;

0.1% formic acid acetonitrile means that the volume concentration of formic acid in the mixture of formic acid and acetonitrile is 0.1%.

3) And chromatographic conditions:

the instrument comprises the following steps: ultra High Performance Liquid Chromatography (UHPLC); a chromatographic column: ACQUTITY

HSS T3(2.1 × 150mm i.d.,1.8 μm); flow rate: 0.2 mL/min; column temperature: 40 ℃; mobile phase A: 0.1% aqueous formic acid; mobile phase B: 0.1% formic acid acetonitrile; isocratic elution ratio: a, B is 50: 50; sample introduction volume: 5 mu L of the solution; sample introduction time: and (4) 11 min.

4) And mass spectrum conditions:

the instrument comprises the following steps: a triple quadrupole tandem mass spectrometer; the mass spectrum quantification method comprises the following steps: multiple Reaction Monitoring (MRM); an ion source: electrospray (ESI) negative ion scanning mode; temperature of sheath gas: 375 ℃; the flow rate of the sheath gas is 8L/min; nozzle voltage: 500V; atomizer pressure: 45 psi; capillary voltage: 2500V; temperature of the drying gas: 300 ℃; flow rate of the dryer: 5L/min; carrier gas: nitrogen gas; collision gas: nitrogen gas. The total ion flow path is shown in figure 2.

The mass spectral parameters of the metabolites and their isotopes are shown in table 2.

TABLE 2 Mass Spectrometry parameters of Gly-Val-PTH and its isotopes

Note:^ain order to quantify the ion channels,^bfor the qualitative ion channel

5) As a result:

the samples were quantitatively analyzed by a standard curve method. A series of standard solutions containing 200ng of isotopic internal standard (d5-Gly-Val-PTH) at concentrations of 0.125, 0.625, 1.25, 2.5, 6.25 and 12.5nmol/g Hb were prepared for analysis. And taking the ratio of the concentration of the standard substance to the concentration of the isotope as a horizontal coordinate (X), and taking the ratio of the peak area of the concentration of the standard substance to the peak area of the internal standard substance of the isotope as a vertical coordinate (Y), so as to obtain a corresponding linear regression equation and obtain a detection result.

The linear equation is Y-1.09177X +0.00704912, r is 0.999, r 2 is 0.999.

The detected spectrum is shown in FIG. 2. In this example, blood from a toxicological test of Sprague Dawley rats was used as a sample, and the lowest limit of detection (LOD) was 1.82pmol/g Hb (Gly-Val-PTH); the lowest limit of quantitation (LOQ) was 5.46pmol/g Hb (Gly-Val-PTH).

The method achieves high precision. Blood samples of 5 SD rats (males) are selected for detection, and the content of the glycidyl hemoglobin adduct is 35.27-47.62 pmol/g Hb (Gly-Val-PTH). The method specifically comprises the following steps: 5 adult SD rats were fed with basal diet and 30 days later were sacrificed to obtain blood samples. The mouse blood was processed and analyzed by chromatography-mass spectrometry as described in example 1, and finally the ion channel spectra of the metabolite and its isotope were obtained, see fig. 2. And integrating the peak areas of the channels to obtain the corresponding peak areas of the compounds. The concentration corresponding to the peak area of the isotope is known, the concentration corresponding to the peak area of the target metabolite can be obtained by standard curve calibration calculation (the above linear regression equation), and the unit conversion is performed to obtain the result.

Verification experiment 1,

3 days 3 levels (three spiked doses high, medium and low) of murine blood 6 were validated by parallel methodology with the following specific results:

example 2, a method for the simultaneous assay of hemoglobin adducts of dietary glycidol and its esters for long-term in vivo exposure, the assay subject being human blood, sequentially performed the following steps:

1) preparing hemoglobin dry powder:

the method for collecting the blood of the human group in an anticoagulation tube and preparing the erythrocyte for pretreatment and preparing the hemoglobin dry powder is equivalent to the step 1) of the embodiment 1.

2) Derivatization and purification:

the concentration of d5-Gly-Val-PTH in 20. mu.L of internal mixed isotope was 1. mu.g/mL, which was identical to step 2) of example 1.

3) Chromatographic conditions

Equivalent to step 3 of example 1).

4) Conditions of Mass Spectrometry

Equivalent to step 4 of example 1).

5) Results

The samples were quantitatively analyzed by a standard curve method. A series of standard solutions containing 20ng of isotopic internal standard at concentrations of 12.5, 25, 62.5, 125 and 250pmol/g Hb were prepared for analysis. And taking the ratio of the concentration of the standard substance to the concentration of the isotope as a horizontal coordinate (X), and taking the ratio of the peak area of the concentration of the standard substance to the peak area of the internal standard substance of the isotope as a vertical coordinate (Y), so as to obtain a corresponding linear regression equation and obtain a detection result.

The linear equation Y is 1.03897X +0.140978, r has a value of 0.999, r ^ r²The value of (A) is 0.998.

The detected spectrum is shown in FIG. 3. In this example, blood from a diet population was used as a sample, and the lowest limit of detection (LOD) was 1.57pmol/g Hb (Gly-Val-PTH); the lowest limit of quantitation (LOQ) was 4.71pmol/g Hb (Gly-Val-PTH). The method achieves high precision. The content of the glycidol hemoglobin adduct is 5.76-8.06 pmol/g Hb (Gly-Val-PTH) detected in 5 male non-smoking people.

Verification experiment 2, 3-day 3-level (three high, medium and low labeled doses) 6 parallel methodological verification of human blood, and the specific results are as follows:

comparative experiment one, the "mobile phase B" in example 1 was changed from "0.1% formic acid acetonitrile" to "acetonitrile", and the rest was identical to example 1.

The results obtained are that the response of the mass spectrum is 5-10 times worse than in example 1, which finally affects the LOD and LOQ of the process, so example 1 is selected for the invention.

In comparative experiment two, the capillary voltage in example 1 was changed from "2500V" to "3500V", and the rest was the same as example 1.

The results obtained are that the response of the mass spectrum is 3-7 times worse than that of example 1, which finally affects the LOD and LOQ of the process, so example 1 is selected for the invention.

Comparing experiment three, adopting different chromatographic columns, and obtaining the result as shown in figure 4; the first is HSST3, the separation effect is best, and baseline separation is achieved; the second is a C18 column, which has a comparable separation effect, but does not achieve baseline separation. Several other columns are not effective in reproducing the present invention.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the invention should be considered as within the scope of the invention

Claims (2)

1. The detection method for evaluating the hemoglobin adduct exposed in vivo by glycidol and ester thereof comprises the step 1) of preparing the hemoglobin dry powder, and is characterized by further comprising the following steps:

2) derivatization and purification:

adding 0.8-3 mL of formamide and 20-100 mu L of 1mol/L NaOH solution into 20-100 mg of hemoglobin powder, adding 5-30 mu L of derivative, namely pentafluorophenyl thioisocyanate, and uniformly mixing by vortex; oscillating for 12-24 h at room temperature for derivatization, and then continuing derivatization in a water bath at 30-60 ℃ for 0.5-6 h;

after water bath derivatization is finished, adding 0.2-2 mL of 20% NaCl solution and 20 mu L of isotope internal standard-d 5-Gly-Val-PTH solution, mixing uniformly by vortex, centrifuging at the rotating speed of 8000-12000 rpm for 2-15 min, and taking supernatant;

activating and balancing the solid phase extraction column, taking a supernatant fluid to sample, leaching with 2-3 mL of leacheate, eluting with 2-3 mL of methanol, and collecting an eluent; drying the eluent at 40 deg.C with nitrogen, diluting to 1mL with mobile phase, dissolving for 1min by vortex, filtering with 0.22 μm microporous membrane, and analyzing by sample injection;

the leacheate is a methanol water solution with the volume concentration of 5-10%;

the mobile phase is mobile phase A: mobile phase B: the volume ratio of 40: 60-60: 40;

3) and chromatographic conditions:

the instrument comprises the following steps: ultra-high performance liquid chromatography; flow rate: 0.2 mL/min; column temperature: 40 ℃; mobile phase A: 0-1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile; isocratic elution ratio: a and B are 40: 60-60: 40; sample introduction volume: 5-10 mu L; sample introduction time: 10-15 min;

the chromatographic column is ACQUTITY

HSS T3；

4) And mass spectrum conditions:

the instrument comprises the following steps: a triple quadrupole tandem mass spectrometer; the mass spectrum quantification method comprises the following steps: monitoring the MRM by multiple reactions; an ion source: an electrospray ESI negative ion scanning mode; temperature of sheath gas: 375 ℃; the flow rate of the sheath gas is 8L/min; nozzle voltage: 500V; atomizer pressure: 45 psi; capillary voltage: 2500V; temperature of the drying gas: 300 ℃; flow rate of the dryer: 5L/min; carrier gas: nitrogen gas; collision gas: nitrogen gas;

TABLE 1 Mass Spectrometry parameters of Gly-Val-PTH and its isotopes

Note:^ain order to quantify the ion channels,^bis a qualitative ion channel;

5) and the result is as follows:

and (3) carrying out quantitative analysis on the sample by using the liquid phase-mass spectrum combined method and a standard curve method.

2. The method of claim 1, wherein the method comprises the steps of: the isotope internal standard in the step 2),

when the sample is a human blood sample, the concentrations of isotope internal standard d5-Gly-Val-PTH are all 1 mug/mL;

when murine blood samples were used, the concentration of the isotopic internal standard d5-Gly-Val-PTH was 10. mu.g/mL.

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