CN111057067A - Multichannel mass spectrum derivative reagent for detecting sphingosine hexatriglycoside and preparation method and application thereof - Google Patents
Multichannel mass spectrum derivative reagent for detecting sphingosine hexatriglycoside and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of analytical chemistry, and particularly relates to a multichannel mass spectrum derivative reagent for detecting sphingosine hexaglycoside, and a preparation method and application thereof. The structural formula of the derivatization reagent is：
(ii) a The levofloxacin-based multichannel mass spectrometry derivation reagent is designed and synthesized for the first time, the derivation reagent has unique intramolecular permanent positive charges and isotope mass difference groups of 9 channels, and the method has the advantages of high analysis flux, good specificity, high accuracy and high sensitivity. The analysis method provided by the invention has good applicability and is of great significance for monitoring drug treatment.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a multichannel mass spectrometry derivation reagent for detecting sphingosine hexatriglycoside, a preparation method and application thereof, in particular to a high-throughput analysis method for performing labeling derivation by using the multichannel mass spectrometry derivation reagent based on levofloxacin, and combining virtual magnetic surface molecular imprinting extraction with ultra-high performance liquid chromatography triple quadrupole mass spectrometry.
Background
Fabry disease is an X-linked lysosomal storage disorder caused by α -galactosidase A deficiency, which leads to the accumulation of glycosphingolipids, mainly ceramide-hexosyl glycoside (Gb 3) and sphingosine-hexosyl glycoside (lyso-Gb3, CAS No.:126550-86-5), which are stored in human blood vessels, nerves, kidneys, heart, etc., causing structural and functional disorders of the respective tissue organs.
However, in actual detection, serious matrix interference exists in the sample, and the plasma lyso-Gb3 concentration of healthy subjects is extremely low, so that a detection analysis method with high sensitivity and high accuracy is necessary to be established. Breemen et al (Biochimica et Biophysica Acta,2011,1812:70-76) disclose a method for analyzing and detecting lyso-Gb3 in plasma by using o-phthalaldehyde as a derivatization reagent and using a high performance liquid chromatography-fluorescence method, but the method has a complex plasma sample processing process, directly performs chromatography-fluorescence detection after derivatization, and the plasma sample may contain other o-phthalaldehyde reactivations which interfere with the detection of lyso-Gb3The method has high detection limit and low sensitivity; gold et al (Clinical Chemistry,2013,59:547-13C5lysoGb3 as internal standard, an analytical method for detecting lyso-Gb3 in plasma combined with ultra performance liquid chromatography tandem mass spectrometry, but stable isotope internal standards are not all commercialized and expensive to purchase, harsh from synthesis conditions, and the purity of the synthesis determines the accuracy of the quantitative results. Therefore, it is very important to continuously develop a high-sensitivity and high-accuracy analysis method.
Disclosure of Invention
The invention aims to solve the defects of the prior detection technology, and designs and synthesizes a multichannel mass spectrum derivative reagent based on Levofloxacin (LFC) for the first time.
The invention also provides a preparation method of the multichannel mass spectrum derivation reagent based on Levofloxacin (LFC).
The invention also provides an application of the multichannel mass spectrometry derivation reagent in detecting the ceramide hexatriglycoside, and an analysis method combining the virtual magnetic surface molecular imprinting extraction and the ultra-high performance liquid chromatography triple quadrupole mass spectrometry detection with high flux, high sensitivity, high accuracy and high specificity is established through internal standard method quantification. Accurate quantitative analysis of 8 actual samples can be simultaneously realized by one-time liquid chromatography-mass spectrometry sample introduction, and the analysis time is greatly shortened while the accuracy is improved.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a multichannel mass spectrum derivatization reagent based on levofloxacin, which has a structural formula as follows:
r is CH3、CH2D、CHD2、CD3、13CD3、C2H5、C2H3D2、C2H2D3Or C2D5。
The invention also provides a preparation method of the multichannel mass spectrometry derivation reagent, which comprises the following steps:
a. dissolving 1.5g levofloxacin in 100mL chromatographic acetonitrile, performing ultrasonic treatment for 2 minutes, adding 10.038g ammonium bicarbonate and 48.6mmol CH3I、CH2DI、CHD2I、CD3I、13CD3I、C2H5Br、C2H3D2I、C2H2D3I or C2D5And I, putting in magnetons, sealing, and stirring at room temperature for 150 hours. Drying the solid obtained by suction filtration in a vacuum oven at 50 ℃ for 7-8h to obtain a white solid;
b. dissolving 0.5g white solid in 5mL freshly distilled thionyl chloride, magnetically stirring, heating to 80 ℃, refluxing for 4h, distilling under reduced pressure to remove thionyl chloride, and cooling to room temperature to obtain a red solid which is CH3-LFC-Cl、CH2D-LFC-Cl、CHD2- LFC-Cl、CD3-LFC-Cl、13CD3-LFC-Cl、C2H5-LFC-Cl、C2H3D2-LFC-Cl、C2H2D3-LFC-Cl or C2D5-LFC-Cl。
C3H7The synthesis of-LFC-Cl was similar to that of the 9 mass spectrum derivation reagents described above, except that C was used in step a3H7Br。
The invention also provides a method for detecting sphingosine hexatriglycoside by using the multichannel mass spectrum derivation reagent based on levofloxacin, which comprises the following steps: carrying out derivatization reaction on a target by 9 levofloxacin-based multichannel mass spectrum derivatization reagents: wherein CH3-LFC-Cl and lyso-Gb3 derivative as internal standard substance for mass spectrum quantification, and the remaining 8 derivatization reagents CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5LFC-Cl labels 8 actual samples, respectively; c3H7And (3) taking a synthetic product of the-LFC-Cl and the lyso-Gb3 as a virtual template for synthesis of the magnetic surface molecularly imprinted material, extracting the 9 derivatives by using the virtual magnetic surface molecularly imprinted material, filtering eluent by using a filter membrane, and then analyzing and detecting by combining an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system.
The detection method provided by the invention specifically comprises the following steps:
a. and (3) a labeling and derivatization process: adding 50 μ L of standard solution or sample to be tested into centrifuge tube containing 200 μ L of sodium borate buffer solution with pH of 8.5-10.5, respectively injecting 200 μ L of CH with pH of 55-70 μ M3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Label derivatization with-LFC-Cl, wherein CH3taking-LFC-Cl and a derivative of lyso-Gb3 as internal standard substances, shaking up, sealing, and carrying out ultrasonic oscillation reaction for 1-4 minutes in a water bath at the temperature of 37-40 ℃;
b. the extraction process comprises the following steps: respectively sucking 100 mu L of solution from the 9 samples, uniformly mixing the solution in a centrifuge tube, adding 200 mu L of sodium borate buffer solution with pH7.5-9 and 8-15mg of virtual magnetic surface molecular imprinting material, shaking uniformly, sealing, violently shaking for 10-20 minutes at room temperature, finally realizing separation by using an external magnet, and adding 200 mu L of desorption solution for elution for 2-4 minutes;
c. and c, filtering the eluent in the step b by using a filter membrane, fixing the volume to 200 mu L, and carrying out quantitative analysis and detection by using an ultra-high performance liquid chromatography triple quadrupole mass spectrometry system.
In the detection process, the used virtual magnetic surface molecularly imprinted material is prepared by the following method:
1g of ferric chloride hexahydrate, 2g of anhydrous sodium acetate and 6.5g of 1, 6-hexanediamine were dispersed in 30mL of ethylene glycol in this order with vigorous stirring, and stirring was continued at room temperature for 30 minutes. The solution was transferred to a polytetrafluoroethylene autoclave and reacted at 200 ℃ for 24 hours. Washing the obtained product with water and ethanol for three times respectively, and vacuum drying at 60 ℃ for 24 hours to obtain a black solid Fe3O4@NH2；
b.100mg Fe3O4@NH2200mg of 4-formylphenylboronic acid and 250mg of sodium cyanoborohydride are dispersed in 25mL of anhydrous methanol, ultrasonic treatment is carried out for 20 minutes, reflux is carried out for 24 hours at 65 ℃, water and methanol are respectively washed for three times, vacuum drying is carried out for 24 hours at 60 ℃, and the obtained black solid is Fe3O4@NH2@FPBA；
c.0.4mg lyso-Gb3, 10mL dichloromethane and 20. mu.L pyridine were mixed well in an ice bath, 0.3587mg C3H7-LFC-Cl is dissolved in 10mL dichloromethane, poured into a constant pressure funnel, slowly added dropwise to the reaction flask, after 2 hours the ice bath is removed, stirring is continued for 2 hours at room temperature, 5mL sodium carbonate buffer pH 9.5 is added for reaction for 0.5 hours, filtered to obtain the solid product: c3H7-LFC-Cl-lyso-Gb3；
d.0.6mg C3H7-LFC-Cl-lyso-Gb3、50mg Fe3O4@NH2@ FPBA was dissolved in 20mL of 20mM phosphate buffer pH 8. Stirring at 25 deg.C for 1 hr, rinsing twice with phosphate buffer, washing twice with water and ethanol, and vacuum drying at 60 deg.C for 24 hr. Dissolving the product with 10mL of acetonitrile as a solvent, adding 0.1422mg of acrylamide, carrying out ultrasonic treatment for 15min, and carrying out dark treatment for 12 h. 1.9822mg of ethylene glycol dimethacrylate and 0.1mg of azobisisobutyronitrile are uniformly dispersed in the prepolymerization solution, ultrasonic treatment is carried out for 15min, 70mL of acetonitrile is added, nitrogen purification is carried out, reaction is carried out for 8h at 60 ℃ under mechanical stirring, reaction is carried out for 2h at 75-80 ℃, separation is carried out under the action of an external magnet, water and ethanol are respectively washed for three times, vacuum drying is carried out for 24h at 60 ℃, soxhlet extraction is carried out for 24h at 80 ℃, except for a template, water and ethanol are respectively washed for three times, vacuum drying is carried out for 24h at 60 ℃, and the obtained solid powder is a virtual magnetic surface molecularly imprinted.
Further, the desorption solution is methanol/H of acetonitrile, methanol, ethanol, acetone and 0.01-0.20% formic acid2O (v/v,1:1) solution. Optimally, the desorption solution is 0.1 percent formic acid in methanol/H2O (v/v,1:1) solution.
The ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system used in the invention uses an Agilent SB C18 chromatographic column for chromatographic separation: 2.1mm × 50mm, 1.8 μm, a sample injection volume of 2 μ L, a column temperature of 30 deg.C, and a gradient elution method.
The gradient elution method described above, the time was 2.0min, the flow rate was 0.2mL/min, the mobile phase A was a 5% acetonitrile aqueous solution containing 0.1% formic acid, the mobile phase B was acetonitrile containing 0.1% formic acid, the mobile phase composition at 0min was 40% A +60% B, 15% A +85% B at 0.5min, 8% A +92% B at 1.5min, 2% A +98% B at 1.6min, 0% A +100% B at 2.0 min; each fraction in the mobile phase is a volume fraction.
The conditions of the mass spectrum when the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system used in the invention is used for analysis and detection are as follows: the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the air pressure of the sprayer is 40psi, the temperature of the sheath gas is 280 ℃, the flow rate is 11L/min, and the voltage of the capillary tube is 3.5 kV.
The invention provides a detection and analysis method of lyso-Gb3 in a sample to be detected, which comprises the steps of firstly carrying out label derivatization on lyso-Gb3 based on a multichannel mass spectrum derivatization reagent of levofloxacin, then carrying out virtual magnetic surface molecular imprinting extraction, filtering an obtained derivative eluent by a filter membrane, and then carrying out analysis and detection by using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system, so that high-throughput analysis and detection of an actual sample can be realized.
The invention relates to 9 mass spectrum derivation reagents based on levofloxacin and analogues C thereof3H7The chemical structure and synthesis conditions of-LFC-Cl are shown below:
the invention designs and synthesizes a levofloxacin-based multichannel mass spectrum derivative reagent containing one intramolecular permanent positive charge for the first time: CH (CH)2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Derivatization of lyso-Gb3, CH from 8 actual samples with-LFC-Cl, respectively3-LFC-Cl and a derivative of lyso-Gb3 as internal standard. Lyso-Gb3 by derivatization of the label to give the corresponding derivative, C3H7The synthetic product of-LFC-Cl and lyso-Gb3 is used as a virtual template to synthesize the magnetic surface molecularly imprinted material, the virtual magnetic surface molecularly imprinted extraction and the internal standard method are combined to accurately quantify, a mass spectrum multi-reaction monitoring mode is connected in series, the daughter ions of the levofloxacin structure marked by mass difference groups are specifically generated, the mass spectrum detection sensitivity is obviously improved, and the analysis flux is greatly improved. The detection method has the remarkable advantages of high flux, high sensitivity, high accuracy and high specificity. With CH3The derivatization reaction process of LFC-Cl serving as a mass spectrum derivatization reagent is shown as follows:
the invention adopts a virtual magnetic surface molecular imprinting material to extract and enrich various derivatives C3H7the-LFC-Cl-lyso-Gb 3 is used as a virtual template to synthesize the molecularly imprinted material, so that the problem of inaccurate quantification caused by template molecule leakage is effectively solved. The material has the advantages that the magnetic surface molecularly imprinted material has large specific surface area and high mass transfer speed, and can complete extraction and enrichment with a small amount of adsorbent and short balance time; modified by boric acid groups and a molecular imprinting layer, the material synthesized by the method can realize specific selective extraction on derivatives of lyso-Gb 3; in addition, the material can be easily separated by only one external magnet, and has excellent reusability. The virtual magnetic surface molecular imprinting technology is a good pretreatment technology for enriching trace components, reducing matrix interference and improving method sensitivity, is combined with ultra-high performance liquid chromatography triple quadrupole mass spectrometry to simultaneously perform quantitative analysis on lyso-Gb3 in 8 actual samples, and has remarkable advantages.
The invention has the advantages and beneficial effects that:
1. the levofloxacin-based multichannel mass spectrometry derivative reagent is designed and synthesized for the first time, the derivative reagent has unique intramolecular permanent positive charges and an isotope mass difference group of 9 channels, and the chromatographic resolution, the detection sensitivity and the analysis flux of an analyte are obviously improved.
2. The pretreatment technology for the virtual magnetic surface molecular imprinting extraction provided by the invention specifically captures and releases the derivative of lyso-Gb3 through the dual combination of borate affinity and a molecular imprinting space matching cavity, and combines an ultra-high performance liquid chromatography triple quadrupole mass spectrometry detection means, so that the advantages of good specificity, high accuracy and high sensitivity are achieved.
3. The analysis method provided by the invention has good applicability and is of great significance for monitoring drug treatment.
Drawings
FIG. 1 is an overview of multichannel mass spectrometry derivatizing reagents and methods of making and using the same.
FIG. 2 is a diagram showing the separation by mass spectrometry of 9 lyso-Gb3 derivatives in example 1.
FIG. 3 shows CH in example 13Schematic representation of mass spectrometric cleavage mechanism of-LFC-Cl-lyso-Gb 3 derivatives.
FIG. 4 is a graph showing the separation of lyso-Gb3 derivative from plasma of a simulated Fabry patient in example 3 by mass spectrometry.
Detailed Description
The present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The derivatization extract is filtered by a filter membrane of 0.22 mu m and then is analyzed and detected by an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system, and fig. 1 is an overview chart of a multichannel mass spectrometry derivatization reagent, and a preparation method and application thereof.
The synthesis method of the levofloxacin-based multichannel mass spectrum derivation reagent comprises the following steps: a. dissolving 1.5g of levofloxacin in 100mL of chromatographic acetonitrile, carrying out ultrasonic treatment for 2 minutes, adding 10.038g of ammonium bicarbonate and 48.6mmol of CH3And I, putting in magnetons, sealing, and stirring at room temperature for 150 hours. The solid obtained by suction filtration is dried for 7-8h in a vacuum oven at 50 ℃. The white solid obtained was: CH (CH)3-LFC。
CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5LFC synthesis method and CH3Synthesis of-LFC is similar, using CH2DI、CHD2I、CD3I、13CD3I、C2H5Br、C2H3D2I、C2H2D3I and C2D5I replaces CH3I; b. take 0.5g CH3dissolving-LFC in 5mL of freshly distilled thionyl chloride, magnetically stirring, heating to 80 ℃, refluxing for 4h, evaporating thionyl chloride under reduced pressure, cooling to room temperature to obtain a red solid, namely CH3-LFC-Cl。 CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Synthesis method of-LFC-Cl and CH3Synthesis of-LFC-Cl was similar using CH2D-LFC、CHD2-LFC、CD3-LFC、13CD3-LFC、C2H5-LFC、C2H3D2-LFC、 C2H2D3-LFC and C2D5-LFC instead of CH3-LFC。C3H7The synthesis of-LFC-Cl was similar to that of the 9 mass spectrum derivation reagents described above, except that C was used in step a3H7Br for CH3I, C synthesized in a is used in b step3H7-LFC instead of CH3-LFC。
In the application test performed in the embodiment of the present invention, the plasma used includes two types: defatted plasma and normal human plasma. The lyso-Gb3 to be detected is not found in the fat-free plasma, and the labeled sample is used for establishing an analysis method; normal plasma was obtained at the local hospital, spiked to simulate fabry disease patients, and the spiked concentrations were controlled to simulate different stages of the disease.
Example 1
Chromatographic separation and mass qualitative and quantitative analysis of 9 lyso-Gb3 derivatives:
adding defatted plasma to methanol-containing plasmaIn the heart tube, the volume ratio of the plasma sample to the methanol is 1:4, the insoluble protein is removed by centrifugation after vortexing for two minutes, and the supernatant is taken for standby. Lyso-Gb3 (purchased from Sigma) prepared from a 50% acetonitrile/water/0.1% formic acid solution to give a Lyso-Gb3 standard stock solution having a concentration of 2000nM, diluting the stock solution to give Lyso-Gb3 standard solutions having different concentrations of 0.3 to 1800nM, and adding 10. mu.L of the Lyso-Gb3 standard solutions having different concentrations to 8 parts of each 50. mu.L of the supernatant of the defatted plasma to obtain standard-added defatted plasma samples having different concentrations of 0.05 to 300 nM; CH (CH)3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5LFC-Cl was dissolved in acetonitrile to give 60. mu.M solution of derivatization reagent in acetonitrile. Taking 50 μ L of 5nM lyso-Gb3 standard solution and 8 parts of each 50 μ L of standard defatted plasma supernatant, adding into a centrifuge tube containing 200 μ L sodium borate buffer solution (pH 9.5), and respectively injecting 200 μ L of 9 derivatization reagents with 60 μ M molar concentration, wherein CH is CH3-LFC-Cl labeling of the derivative obtained from the lyso-Gb3 standard solution as an internal standard. Shaking up, sealing, and carrying out ultrasonic oscillation reaction for 1 minute in water bath at the temperature of 40 ℃; respectively sucking 100 μ L of the above 9 samples, mixing in a centrifuge tube, adding 200 μ L sodium borate buffer solution (pH 8.5) and 10mg virtual magnetic surface molecular imprinting material, shaking, sealing, shaking at room temperature for 10 min, separating with external magnet, adding 200 μ L0.1% formic acid methanol/H2O (v/v,1:1) solution (0.1% formic acid in methanol/H)2O (v/v,1:1) solution as desorption solution, and the desorption solution is replaced by methanol/H of acetonitrile, methanol, ethanol, acetone, 0.01% -0.20% formic acid2In O (v/v,1:1) solution, the analyte extraction efficiency was at 0.1% formic acid in methanol/H259.7-99.6% of O (v/v,1:1) solution as desorption solution) for 2 minutes; filtering the eluent with a filter membrane, metering to 200 mu L, and carrying out UHPLC-MS/MS (MRM) analysis and detection. The gradient elution method is characterized in that the time is 2.0min, the flow rate is 0.2mL/min, the mobile phase A is 5 percent acetonitrile aqueous solution containing 0.1 percent formic acid, and the mobile phase A is mobilePhase B is acetonitrile containing 0.1% formic acid, mobile phase composition of 40% A +60% B at 0min, 15% A +85% B at 0.5min, 8% A +92% B at 1.5min, 2% A +98% B at 1.6min, 0% A +100% B at 2.0 min; the conditions of mass spectrum are: the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the air pressure of the sprayer is 40psi, the temperature of the sheath gas is 280 ℃, the flow rate is 11L/min, and the voltage of the capillary tube is 3.5 kV.
Better resolution was obtained according to the gradient elution procedure described above, and FIG. 2 is a mass spectrometric separation profile of 9 lyso-Gb3 derivatives, with good resolution and relatively compact retention times among the 9 derivatives. Mass spectrometry experimental results show that 9 derivatives of lyso-Gb3, which all fragment at the amide bond and thus have a consistent mass fragmentation pattern, produce characteristic product ions of m/z359.16, 360.17, 361.18, 362.18, 363.19, 373.18, 375.19, 376.20, 378.21 for use as the quantifier ions in a multiple reaction monitoring mode. With CH3-LFC-Cl-lyso-Gb3 as an example, CH in FIG. 33The mass spectrum cleavage mechanism of-LFC-Cl-lyso-Gb 3 is shown in the specification, and the parent ion is [ M]+m/z 1143.60 was able to generate a quantifier ion of m/z 359.16. The parameters of the MRM mode were optimized, the retention time of 9 LFC-Cl-lyso-Gb3, MRM ion pair, optimized fragmentation voltage (V) and collision energy (eV), and the linear range, correlation coefficient, detection limit and quantitative limit of the analytical method are shown in Table 1.
TABLE 1
Example 2
The detection and analysis of lyso-Gb3 in normal human plasma comprises the following operation steps:
eight normal human plasma samples (male: 4, female: 4; different age stages) were collected from the local hospital, then added to a centrifuge tube containing methanol at a volume ratio of 1:4, vortexed for two minutes, centrifuged to remove insoluble proteins, and the supernatant was taken for use; the stock solution in example 1 was diluted with 50% acetonitrile/water/0.1% formic acid solution to give a solution of lyso-Gb3 standard at a concentration of 5 nM. Taking 50 mu L of molar concentration5nM lyso-Gb3 standard solution and 8 portions of 50. mu.L each of normal plasma supernatant were added to a centrifuge tube containing 200. mu.L of sodium borate buffer solution (pH 10.5), and 200. mu.L of CH with a molar concentration of 70. mu.M was injected3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Label derivatization with-LFC-Cl, wherein CH3-LFC-Cl labeling of the derivative obtained from the lyso-Gb3 standard solution as an internal standard. Shaking up, sealing, and carrying out ultrasonic oscillation reaction for 4 minutes in a water bath at the temperature of 37 ℃; respectively sucking 100 μ L of the above 9 samples, mixing in a centrifuge tube, adding 200 μ L of sodium borate buffer solution (pH 9) and 8mg of virtual magnetic surface molecular imprinting material, shaking, sealing, shaking at room temperature for 15min, separating with external magnet, adding 200 μ L of 0.1% formic acid methanol/H2Eluting with O (v/v,1:1) solution for 3 min; filtering the eluent with a filter membrane, metering to 200 mu L, and carrying out UHPLC-MS/MS (MRM) analysis and detection. The detected lyso-Gb3 content (n ═ 3) in normal human plasma was: four normal male plasma: 0.52nM, 0.60nM, 0.73nM, 0.58 nM; four normal female plasma: 0.34nM, 0.37 nM, 0.41nM, 0.50 nM.
Example 3
The detection and analysis of lyso-Gb3 in the plasma of a patient simulating Fabry disease comprises the following operation steps:
twenty random plasma samples (male: 10, female: 10; different age stages) were collected from a local hospital, the plasma samples were mixed into two portions by sex, plasma was added to a centrifuge tube containing methanol at a volume ratio of 1:4, insoluble proteins were removed by centrifugation after vortexing for two minutes, and the supernatant was taken for use. Preparing Lyso-Gb3 with 50% acetonitrile/water/0.1% formic acid solution to obtain Lyso-Gb3 standard stock solution with concentration of 2000nM, diluting the stock solution to obtain Lyso-Gb3 standard solution with different concentrations (40nM, 60nM, 150nM, 250nM, 600nM, 1500nM), and adding 10. mu.L of the Lyso-Gb3 standard solution with different concentrations to 8 aliquots of 50. mu.L plasma supernatant to obtain spiked plasma with different concentrationsSamples, at different spiking concentrations, were used to simulate different stages of fabry disease. Taking 50 μ L of 5nM lyso-Gb3 standard solution, 6 parts of each 50 μ L of simulated Fabry disease plasma supernatant and 2 parts of each 50 μ L of normal human mixed plasma supernatant, adding into a centrifuge tube containing 200 μ L of sodium borate buffer solution (pH 9), and respectively injecting 200 μ L of CH with a molar concentration of 65 μ M3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Label derivatization with-LFC-Cl, wherein CH3-LFC-Cl labeling of the derivative obtained from the lyso-Gb3 standard solution as an internal standard. Shaking up, sealing, and carrying out ultrasonic oscillation reaction in a water bath at the temperature of 37 ℃ for 3 minutes; respectively sucking 100 μ L of the above 9 samples, mixing in a centrifuge tube, adding 200 μ L of sodium borate buffer solution (pH 8) and 15mg of virtual magnetic surface molecular imprinting material, shaking, sealing, shaking at room temperature for 20 min, separating with external magnet, adding 200 μ L of 0.1% formic acid methanol/H2Eluting with O (v/v,1:1) solution for 4 min; filtering the eluent with a filter membrane, metering to 200 mu L, and carrying out UHPLC-MS/MS (MRM) analysis and detection. The detected lyso-Gb3 content (n-3) in the plasma of mock fabry patients was: normal males: 0.63nM, normal women: 0.40nM, pre-treatment fabry male (min): 101.43nM, pre-treatment fabry male (max): 241.26nM, pre-treatment Fabry female (min): 10.07nM, pre-treatment fabry female (max): 25.94nM, fabry male after treatment: 40.08nM, fabry female after treatment: 6.73nM, FIG. 4 is a mass spectrometric image of this sample.
Example 4
The detection and analysis of lyso-Gb3 in urine comprises the following operation steps:
twenty normal human urine samples (male: 10, female: 10) were randomly collected, the urine samples were mixed into two portions by sex, the urine was added to a centrifuge tube containing methanol at a volume ratio of 1.5:1, vortexed for 5 minutes, centrifuged, and the supernatant was taken for use. Lyso-Gb3 was prepared from 50% acetonitrile/water/0.1% formic acid solutionThe stock solution of lyso-Gb3 standard with a concentration of 2000nM was diluted to obtain lyso-Gb3 standard solutions with different concentrations (960pM, 3000pM, 12000pM, 24000pM), and 10. mu.L of the lyso-Gb3 standard solutions with different concentrations were added to 8 aliquots of each 50. mu.L urine supernatant to obtain spiked urine samples with different concentrations (160pM, 500pM, 2000pM, 4000 pM). 50. mu.L of a 5nM lyso-Gb3 standard solution and 8 aliquots of each 50. mu.L spiked urine were added to a centrifuge tube containing 200. mu.L of sodium borate buffer solution (pH 8.5), and 200. mu.L of 55. mu.M CH was injected3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Label derivatization with-LFC-Cl, wherein CH3-LFC-Cl labeling of the derivative obtained from the lyso-Gb3 standard solution as an internal standard. Shaking up, sealing, and carrying out ultrasonic oscillation reaction in a water bath at the temperature of 40 ℃ for 2 minutes; sucking 100 μ L of each of the 9 samples, mixing in a centrifuge tube, adding 200 μ L of sodium borate buffer (pH 7.5) and 10mg of virtual magnetic surface molecularly imprinted material, shaking, sealing, shaking at room temperature for 10 min, separating with external magnet, adding 200 μ L of 0.1% formic acid in methanol/H2Eluting with O (v/v,1:1) solution for 2 min; filtering the eluent with a filter membrane, metering to 200 mu L, and carrying out UHPLC-MS/MS (MRM) analysis and detection. The detected lyso-Gb3 content (n-3) in the spiked urine samples was: concentration of analyte in four male spiked urine samples: 162.4pM, 489pM, 1978pM, 4080pM, concentration of analyte in four female spiked urine samples: 154.9pM, 490.5pM, 2038pM, 4124 pM.
Comparative example 1
This comparative example procedure was the same as example 3, and lyso-Gb3 was detected in plasma. The difference lies in that: in the sample pretreatment process, firstly, lyso-Gb3 in plasma is extracted and then is subjected to derivatization, a derivatization reagent adopts o-phthalaldehyde disclosed by Breemen et al (Biochimica Biophysica Acta,2011,1812:70-76), and the obtained derivative is directly subjected to high performance liquid chromatography-fluorescence detection; no magnetic dispersion solid phase extraction was performed after derivatization.
Comparative example 2
This comparative example procedure was the same as examples 3, 4, and plasma and urine were assayed for lyso-Gb 3. The difference lies in that: in the sample pretreatment process, Gold et al (Clinical Chemistry,2013,59:547-556) disclose that the sample is not derivatized with a derivatization reagent and subjected to magnetic dispersion solid phase extraction after derivatization, lyso-Gb3 in plasma and urine is extracted first, and the sample is self-synthesized13C5lysoGb3 was used as an internal standard for detection in combination with an ultra performance liquid chromatography tandem mass spectrometry system.
Comparative example 3
The procedure of this comparative example is the same as in example 4, and the detection of lyso-Gb3 in urine is different in that during the pretreatment of the sample, the solid phase extraction method is used to extract lyso-Gb3 in urine, and then the liquid-mass spectrometry detection is performed using 1- β -D-glucosylceramide as an internal standard, using derivatization-free derivatization reagent disclosed by Auray-Blais et al (clinical Chimica Acta,2010,411: 1906-.
Table 2 below shows the results of experiments in examples 3 and 4 in comparison with comparative examples 1 to 3.
TABLE 2
As can be seen from Table 2, the present invention utilizes CH in comparison with the related reports3The derivatization method has the advantages that the derivatization reagent is labeled and derivatized by multi-channel mass spectrometry such as-LFC-Cl and the like, the derivatization condition is mild and rapid, the sensitivity is high, the detection limit of the derivatization method is about 1-300 times lower than the comparison ratio, the detection limit is the same as that of the comparison example 3, but the method also has the obvious advantage of high flux, and the analysis time of the sample is greatly shortened. The recovery rate of the virtual magnetic surface molecular imprinting extraction technology is good, and the accuracy of the analysis method is guaranteed.
To verify the applicability of the established assay, the accuracy, precision of example 1, and recovery and precision in the simulated fabry disease plasma sample of example 3 were examined in detail and the results are shown in table 3.
TABLE 3
As can be seen from tables 2 and 3, for lyso-Gb3 in the plasma samples, the linear range was 0.05-300nM, the detection limit was 0.01nM, and the quantification limit was 0.05 nM. The result shows that the established analysis method has high sensitivity and good recovery rate, effectively reduces matrix interference, and can be well applied to detecting the content of lyso-Gb3 in plasma and urine.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (10)
1. A multichannel mass spectrometry derivation reagent based on levofloxacin, wherein the structural formula of the reagent is as follows:
r is CH3、CH2D、CHD2、CD3、13CD3、C2H5、C2H3D2、C2H2D3Or C2D5。
2. A method of preparing a multichannel mass spectrometry derivatisation reagent according to claim 1, comprising the steps of:
a. dissolving 1.5g levofloxacin in 100mL chromatographic acetonitrile, performing ultrasonic treatment for 2 minutes, adding 10.038g ammonium bicarbonate and 48.6mmol CH3I、CH2DI、CHD2I、CD3I、13CD3I、C2H5Br、C2H3D2I、C2H2D3I or C2D5I, putting in magnetons, sealing, and stirring at room temperature for 150 hours;
drying the solid obtained by suction filtration in a vacuum oven at 50 ℃ for 7-8h to obtain a white solid;
b. dissolving 0.5g white solid in 5mL freshly distilled thionyl chloride, magnetically stirring, heating to 80 ℃, refluxing for 4h, distilling under reduced pressure to remove thionyl chloride, and cooling to room temperature to obtain a red solid which is CH3-LFC-Cl、CH2D-LFC-Cl、CHD2-LFC-Cl、CD3-LFC-Cl、13CD3-LFC-Cl、C2H5-LFC-Cl、C2H3D2-LFC-Cl、C2H2D3-LFC-Cl or C2D5-LFC-Cl；
C3H7The synthesis of-LFC-Cl was similar to that of the 9 mass spectrum derivation reagents described above, except that C was used in step a3H7Br。
3. A method for detecting ceramide hexoside by using the levofloxacin-based multichannel mass spectrometry derived reagent of claim 1 or 2, which comprises the following steps: carrying out derivatization reaction on a target by 9 levofloxacin-based multichannel mass spectrum derivatization reagents: wherein CH3-derivative of LFC-Cl and ceramide hexatriglycoside (lyso-Gb3) as internal standard substance for mass spectrum quantification, and the remaining 8 derivative reagents CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5LFC-Cl labels 8 actual samples, respectively; c3H7The synthetic product of-LFC-Cl and lyso-Gb3 is used as a virtual template for synthesizing a magnetic surface molecularly imprinted material, the virtual magnetic surface molecularly imprinted material is used for extracting the 9 derivatives, and eluent is filtered by a filter membrane and then is combined with an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system for synthesisAnd (5) analyzing and detecting.
4. The method according to claim 3, characterized in that it comprises in particular the steps of:
a. and (3) a labeling and derivatization process: taking 50 muL of standard substance solution or sample to be detected, adding the 50 muL of standard substance solution or sample to be detected into a centrifuge tube containing 200 muL of pH 8.5-10.5 sodium borate buffer solution, and respectively injecting 200 muL of 55-70 muM CH3/CH2D/CHD2/CD3/13CD3/C2H5/C2H3D2/C2H2D3/C2D5Label derivatization with-LFC-Cl, wherein CH3taking-LFC-Cl and a derivative of lyso-Gb3 as internal standard substances, shaking up, sealing, and carrying out ultrasonic oscillation reaction for 1-4 minutes in a water bath at the temperature of 37-40 ℃;
b. the extraction process comprises the following steps: absorbing 100 muL of solution from the 9 samples, uniformly mixing the solution in a centrifuge tube, adding 200 mug LpH 7.5, 7.5-9 sodium borate buffer solution and 8-15mg of virtual magnetic surface molecular imprinting material, shaking uniformly, sealing, violently shaking for 10-20 minutes at room temperature, finally realizing separation by using an external magnet, and adding 200 muL of desorption solution for elution for 2-4 minutes;
c. and c, filtering the eluent in the step b by using a filter membrane, fixing the volume to 200 mu L, and performing quantitative analysis and detection by using an ultra-high performance liquid chromatography triple quadrupole mass spectrometry system.
5. The method according to claim 4, wherein the virtual magnetic surface molecularly imprinted material is prepared by the following method:
a.1g ferric chloride hexahydrate, 2g anhydrous sodium acetate and 6.5g 1, 6-hexanediamine are dispersed in 30mL ethylene glycol in turn under vigorous stirring, and are continuously stirred for 30 minutes at room temperature;
transferring the solution into a polytetrafluoroethylene high-pressure kettle, and reacting for 24 hours at 200 ℃;
washing the obtained product with water and ethanol for three times respectively, and vacuum drying at 60 ℃ for 24 hours to obtain a black solid Fe3O4@NH2；
b. 100 mg Fe3O4@NH2200mg of 4-formylphenylboronic acid and 250mg of sodium cyanoborohydride are dispersed in 25mL of anhydrous methanol, ultrasonic treatment is carried out for 20 minutes, reflux is carried out for 24 hours at 65 ℃, water and methanol are respectively washed for three times, vacuum drying is carried out for 24 hours at 60 ℃, and the obtained black solid is Fe3O4@NH2@FPBA；
c.0.4mg of lyso-Gb3, 10mL of dichloromethane and 20. mu.L of pyridine were mixed well in an ice bath, 0.3587 mgC3H7-LFC-Cl is dissolved in 10mL dichloromethane, poured into a constant pressure funnel, slowly added dropwise to the reaction flask, after 2 hours the ice bath is removed, stirring is continued for 2 hours at room temperature, 5mL sodium carbonate buffer pH 9.5 is added for reaction for 0.5 hours, filtered to obtain the solid product: c3H7-LFC-Cl-lyso-Gb3；
d. 0.6 mg C3H7-LFC-Cl-lyso-Gb3、50 mg Fe3O4@NH2@ FPBA was dissolved in 20mL of 20mM phosphate buffer pH 8;
stirring for 1 hour at 25 ℃, rinsing twice with phosphate buffer solution, washing twice with water and ethanol, and vacuum drying for 24 hours at 60 ℃;
dissolving the product with 10mL of acetonitrile as a solvent, adding 0.1422mg of acrylamide, carrying out ultrasonic treatment for 15min, and carrying out dark treatment for 12 h;
1.9822mg of ethylene glycol dimethacrylate and 0.1mg of azobisisobutyronitrile are uniformly dispersed in the prepolymerization solution, ultrasonic treatment is carried out for 15min, 70mL of acetonitrile is added, nitrogen purification is carried out, reaction is carried out for 8h at 60 ℃ under mechanical stirring, reaction is carried out for 2h at 75-80 ℃, separation is carried out under the action of an external magnet, water and ethanol are respectively washed for three times, vacuum drying is carried out for 24h at 60 ℃, soxhlet extraction is carried out for 24h at 80 ℃, water and ethanol are respectively washed for three times, vacuum drying is carried out for 24h at 60 ℃, and the obtained solid powder is the virtual magnetic surface molecularly imprinted polymer.
6. The method according to claim 4 or 5, characterized in that the desorption solution is acetonitrile, methanol, ethanol, acetone, methanol/H of 0.01-0.20% formic acid2O (v/v1:1) solution.
7. The method according to claim 6, wherein the desorption solution is 0.1% formic acid in methanol/H2O(v/v1:1) solution.
8. The method according to any one of claims 3 to 7, wherein the ultra performance liquid chromatography triple quadrupole tandem mass spectrometry system is used for chromatographic separation with an Agilent SB C18 column: 2.1mm multiplied by 50mm, 1.8 mu m, 2 mu L of sample introduction volume, 30 ℃ of column temperature and a gradient elution method.
9. The process of claim 8, wherein the gradient elution is performed over a period of 2.0min at a flow rate of 0.2mL/min, mobile phase A is a 5% aqueous acetonitrile solution containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, 0min mobile phase composition is 40% A +60% B, 0.5min 15% A +85% B, 1.5min 8% A +92% B, 1.6min 2% A +98% B, 2.0min 0% A +100% B; each fraction in the mobile phase is a volume fraction.
10. The method according to claim 8 or 9, wherein the conditions of the mass spectrum of the ultra performance liquid chromatography triple quadrupole tandem mass spectrometry system in the analysis detection are as follows: the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the air pressure of the sprayer is 40psi, the temperature of the sheath gas is 280 ℃, the flow rate is 11L/min, and the voltage of the capillary tube is 3.5 kV.
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