CN120703287A - Composite markers and methods for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS - Google Patents

Abstract

The present invention belongs to the field of inspection and specifically relates to a composite marker for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS, including a marker and an isotope internal standard; wherein the marker includes glucosylsphingosine, globular glycosylsphingosine, lyso-sphingomyelin, N-palmitoyl-O-phosphocholine-serine, 7-ketocholesterol, cholestane-3,5,6-triol, galactosylsphingosine, C26-ceramide, lyso-GM1 ganglioside, and lyso-GM2 ganglioside; and the isotope internal standard includes 13C6-glucosylsphingosine, D7-globular glycosylsphingosine, D9-lyso-sphingomyelin, D9-N-palmitoyl-O-phosphocholine-serine, D7-7-ketocholesterol, D7-cholestane-3,5,6-triol, and D5-galactosylsphingosine. The present invention also includes a UPLC-MS/MS method for detecting the composite marker. The present invention realizes the simultaneous detection of multiple lysosomal storage disease-related biomarkers, and the method is simple to operate, high in throughput and low in cost.

Description

Complex markers and methods for detection of lysosomal storage disease associated biomarkers based on UPLC-MS/MS

Technical Field

The invention relates to the technical field of biology, in particular to a liquid phase mass spectrometry method for detecting the content of a biomarker related to a lysosomal storage disease in body fluid.

Background

Lysosomal Storage Disorders (LSDs) are a group of inherited metabolic diseases, which are caused by the failure of the corresponding biomacromolecule in the body to degrade normally due to the related acid hydrolase deficiency in the lysosome caused by the mutation of the gene, and cause dysfunction of the cell tissue organ. Gaucher Disease (GD) is an autosomal recessive LSD, caused by mutations in the GBA1 gene encoding the enzyme glucosylceramide degrading enzyme β -glucocerebrosidase, GBA1 deficiency leading to progressive accumulation of the substrate glucosylceramide (Gb 1), glucosylceramide (Lyso-Gb 1) being a direct metabolite of Gb1, pathogenic to cells. Fabry Disease (FD) is a rare X-linked lysosomal storage disorder caused by pathogenic variation of the GLA gene encoding α -galactosidase a (α -Gal a). The alpha-Gal A deficiency or absence and the subsequent accumulation of glycosphingolipids (sphinganine (lyso-GL 3)) in various tissues and cells can lead to an increased risk of progressive injury, life threatening complications and premature death of the affected organ. Niemann-pick disease (NP) is a heterogeneous lysosomal lipid accumulation disease comprising a group of clinical, biochemical and molecular characteristics, type a and type B being the accumulation of sphingomyelin in different organs and tissues due to Acid Sphingomyelinase (ASM) -sphingomyelin phosphodiesterase-1 gene mutation, type C and type D being the accumulation of cholesterol in lysosomes due to NPC1 or NPC2 dysfunction of one lysosomal protein, thereby damaging multiple organs, characterized by progressive deterioration of neurological function. Related biomarkers include plasma oxysterols-cholestane-3 beta, 5 alpha, 6 beta-triol (C-Triol) and 7-ketocholesterol (7-KC) and lysosphingolipids-lysosphingosine-sphingomyelin (Lyso-SM) and lysosphingomyelin-509 (Lyso-SM-509). Kla Bei Bing (KD) is an autosomal recessive neurodegenerative disease caused by a deficiency in the lysosomal enzyme Galactocerebrosidase (GALC), resulting in an elevation of the substrate galactosylceramide in patients with clinical signs and symptoms of the disease. Fabry Disease (FD) is a rare lysosomal storage disorder caused by acid ceramidase deficiency and subsequent ceramide accumulation. GM1 gangliosidosis, also known as systemic gangliosidosis, is a hereditary lysosomal disease caused by a deficiency of β -galactosidase in a manner that is inherited by autosomal recessive inheritance. Clinical features are progressive central nervous system disorders and skeletal abnormalities like mucopolysaccharidosis type I. GM2 ganglioside deposition (GM 2 Gangliosidosis) is a rare autosomal recessive genetic disorder, also known as GM2 ganglioside storage disorder, which is caused by the deposition of excess GM2 ganglioside in the brain and surrounding organs due to lysosomal β -hexosaminidase (Hex) deficiency.

Screening of LSDs is often performed by direct measurement of the activity of disease-related enzymes for primary diagnosis, and enzyme activity quantification techniques are widely used in neonatal screening, considered as the gold standard for primary screening of NBS. PE kit-NeoLSDTM MSMS Kit is adopted for detecting enzyme activity, 6 enzyme contents in blood spots are detected, and 6 LSD diseases are covered, wherein on one hand, partial enzyme activity can be affected by sample treatment or experimental conditions, and on the other hand, the detection flux of enzyme activity is limited, and the condition change limit is more. Another advantage of using biomarkers compared to enzyme activity assays is that the enzyme activity assay is at risk of false positives or false negatives, fabry Disease (FD) is a rare X-linked genetic lysosomal storage disorder, and male fabry disease diagnosis is determined by enzyme assays that demonstrate a-Gal a activity deficiency. In heterozygotes, there may be no non-pathogenic allele, and the enzyme detection is unreliable, and the diagnosis is based on gene sequencing. Biomarker detection generally supports diagnosis, and is particularly useful for patients with reduced phenotype. The sensitivity of the plasma biomarker to diagnosis of female Fabry disease is higher than that of the alpha-GalA enzyme activity detection. And the secondary test analysis is carried out on the screened positive sample through the quantification of the biomarker, so that the high false positive rate related to low enzyme activity caused by the false defect can be solved.

Furthermore, while most LSDs are caused by enzyme deficiency, there are also diseases caused by pathogenic changes in lysosomal associated proteins that have no enzymatic activity. For example, NPC, which is caused by NPC1 or NPC2 genes, can only be assessed for a variety of biomarkers and confirmed genetic analysis.

Conventional therapies for LSDs include Enzyme Replacement Therapy (ERT), substrate Reduction Therapy (SRT), chaperone therapy, or hematopoietic stem cell transplantation, etc., and once ERT is administered, enzyme activity is not useful for monitoring disease progression, as compared to enzyme activity diagnosis. For example, pompe, is more effective using the biomarker produced by glycogen digestion and other bifurcated glucose polymers, i.e., the tetraose glucose (Glc 4). Biomarker analysis can be used to assess disease severity as an indicator of disease progression, monitoring compliance with patient treatment regimens.

There are also few reports in the prior art of partial LSDs disease diagnosis by detecting biomarkers by liquid chromatography-mass spectrometry, for example, the patent document published under the number WO2023232942A1 discloses a biomarker and a method for detecting lysosomal storage diseases, specifically describing LSDs disease diagnosis by detecting Lyso-Gb1 and Lyso-Gb3& Lyso-Gb 3-IS. Patent document publication No. US10983112B2 discloses a bile acid biomarker for niemann pick disease, a method and use thereof, and specifically reports on the diagnosis of niemann pick disease by detecting 3-beta, 5-alpha, 6-BETA-trihydroxy cholanic acid.

In summary, although some biomarker diagnoses are reported in the prior art, the diagnostic scope of the existing methods is limited, and the broad spectrum, stability and specificity of the detection need to be improved.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention aims to provide a composite marker for detecting the lysosomal storage disease related biomarker based on UPLC-MS/MS, and aims to improve the detection broad spectrum, sensitivity and accuracy of LSDs through the identification of the composite marker.

A second object of the present invention is to provide a method for detecting a lysosomal storage disease related biomarker based on UPLC-MS/MS, aiming at providing a method capable of effectively detecting a lysosomal storage disease related biomarker.

The existing LSDs have relatively narrow diagnosis range, are difficult to realize the broad spectrum and accurate diagnosis and identification of LSDs diseases, and aiming at the problems, the invention provides the following improvement scheme:

A complex label for detecting a lysosomal storage disease related biomarker based on UPLC-MS/MS, comprising a label and an isotopic internal standard;

Wherein the marker comprises glucosylceramine, globosulferamine, lysosphingomyelin, N-palmitoyl-O-phosphorylcholine-serine, 7-ketocholesterol, cholestane-3, 5, 6-triol, galactosylsphingosine, C26-ceramide, lysoGM 1 ganglioside, and lysoGM 2 ganglioside;

Isotopic internal standards include 13C 6-glucosylceramide, D7-globular glycosylsphingosine, D9-lysosphingomyelin, D9-N-palmitoyl-O-phosphocholine-serine, D7-7-ketocholesterol, D7-cholestane-3, 5, 6-triol, and D5-galactosylsphingosine.

The innovative research of the invention shows that the combination of multiple biomarkers is innovatively adopted, and the use of targeted multiple isotope internal standard substances is matched, so that the influence of matrix effect in the ionization process is counteracted, thus the synergy can be realized, the broad-spectrum, accurate and sensitive identification of LSDs can be realized, and the detection requirements of LSDs scientific research and diagnosis can be realized.

Lysosomal storage diseases include Gaucher Disease (GD), fabry Disease (FD), niemann-pick disease type a, B and C/D (NPA/B, C/D), clara Bei Bing (KD), fabry Disease (FD), GM1 gangliosidosis, GM2 gangliosidosis.

Relevant disease biomarkers include glucosylceramine (Lyso-Gb 1) of Gaucher Disease (GD), globular glycosylsphingosine (Lyso-Gb 3) of Fabry Disease (FD), lysosphingomyelin (Lyso SM) of Niman-Picker disease, N-palmitoyl-O-phosphorylcholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3, 5, 6-triol (C-triol), galactosylsphingosine (Psychosine/PSY) of Crar Bei Bing (KD), C26-Ceramide (C26-Ceramide/C26-C) of Fabry Disease (FD), lysoGM1 ganglioside (lysoGM 1) of GM1 gangliosidosis and lysoGM2 ganglioside (lysoGM) of GM2 gangliosidosis;

Corresponding isotopic internal standards include 13C 6-glucosylceramine (13C 6-Lyso-Gb 1), D7-globular glycosylsphingosine (D7-Lyso-Gb 3), D9-lysosphingomyelin (D9-Lyso SM), D9-N-palmitoyl-O-phosphorylcholine-serine (D9-PPCS), D7-7-ketocholesterol (D7-7-KC), D7-cholestane-3, 5, 6-triol (D7-C-triol) and D5-galactosyl sphingosine (D5-PSY), corresponding in sequence to the biomarkers glucosylceramine (Lyso-Gb 1), globular glycosylsphingosine (Lyso-Gb 3), lysosphingomyelin (Lyso SM), N-palmitoyl-O-phosphorylcholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3, 5, 6-triol (C-triol) and galactosyl sphingosine (PSY).

The invention also provides an application of the composite marker in preparing a detection reagent for determining LSDs, which is specifically characterized in that:

a method for detecting a lysosomal storage disease related biomarker based on UPLC-MS/MS, which adopts the UPLC-MS/MS method to measure each component in the composite marker and draws a standard curve of each marker;

Preparing a sample to be tested to obtain a solution to be tested, and adopting the UPLC-MS/MS to measure so as to perform qualitative analysis or perform quantitative analysis based on a drawn standard curve;

UPLC test conditions of UPLC-MS/MS are:

The chromatographic column is a phenyl column, the mobile phase A is an aqueous solution containing 0.005% -0.01% of ammonia water plus 10+/-1% of acetonitrile, and the mobile phase B is an acetonitrile solution containing 0.005% -0.01% of ammonia water plus 10+/-1% of isopropanol;

Gradient elution condition is 0-0.2min,0-16.5% B;

0.2 - 3.25min,16.5 - 16.8%B;

3.25 - 3.75min,16.8 - 33.8%B;

3.75 - 3.8min,33.8 - 34%B;

3.8 - 10.25min,34 - 34.5%B;

10.25 - 10.3min,34.5 - 34.5%B;

10.3 - 10.85min,34.5 - 40%B;

10.85 - 11min,40 - 62.5%B;

11 - 12min,62.5 - 64%B;

12 - 12.35min,64 - 64%B;

12.35 - 13.55min,64 - 100%B;

13.55 - 14min,100 - 100%B;

14 - 14.01min,100 - 0%B;

14.01 - 15min,0 - 0%B。

The invention provides the thought of realizing LSDs diagnosis by detecting the composite markers for the first time, however, early research and development show that how to realize high-selectivity separation of a plurality of markers under a set of detection conditions and realize high-sensitivity response of the markers with different properties is a main difficulty in realizing the technical thought. Aiming at the difficulty of implementing the technical thought, the invention shows that the special control of chromatographic column type, eluent and elution mechanism in the UPLC test process can realize the synergy, can realize the high-selectivity separation of the composite marker, and can realize the high-sensitivity and high-linearity peak. The method can identify LSDs disease in a broad spectrum, has excellent identification accuracy and sensitivity, and can effectively reduce the problems of false positive and false negative of detection. The method can realize in vitro detection of the composite marker. The method can be used for LSDs related scientific research.

In the present invention, the lysosomal storage disorder includes at least one of Gaucher Disease (GD), fabry Disease (FD), niemann-pick disease type a, type B and type C/D (NPA/B, type C/D), clara Bei Bing (KD), fabry Disease (FD), GM1 gangliosidosis, GM2 gangliosidosis.

In the present invention, the standard curve may be drawn based on known principles and means. For example, the standard substance of each marker and the corresponding isotope internal standard substance are configured into 5-10 concentration gradients, then the concentration gradients are respectively measured, the ratio of the standard substance to the internal standard peak area is the Y axis by taking the concentration of the standard substance relative to the internal standard solution as the X axis, and the standard curve of each marker is obtained by linear regression analysis.

In the invention, the steps of preparing the solution to be measured from the sample to be measured are as follows:

Step (1):

transferring a sample to be measured, placing the sample into a container, sequentially adding an internal standard working solution and a formic acid-containing precipitant, uniformly mixing, and centrifuging to obtain a supernatant;

Step (2):

Drying the supernatant in the step (1), re-dissolving the protein precipitate by using a re-dissolving solution, and then centrifuging, wherein the obtained supernatant is used as a solution to be tested.

In the invention, the sample to be tested comprises at least one of serum, plasma, urine, amniotic fluid and cerebrospinal fluid.

In the invention, the precipitant is an organic solvent containing 0.05-0.5% v/v formic acid, wherein the organic solvent is a methanol-acetonitrile mixed solvent or a methanol-acetone mixed solvent, and the content of methanol in the organic solvent is 30-60% v. The research of the invention shows that the adoption of the precipitation process is beneficial to further improving the precipitation effect and recovery rate of the marker, improving the response of the detection index, reducing the interference substances and reducing the false negative of the detection.

In the invention, the complex solution is a methanol aqueous solution containing 0.1-0.2% formic acid, wherein the content of methanol in the methanol aqueous solution is 80-90% by volume. The research of the invention shows that the adoption of the re-dissolution process is beneficial to further improving the re-dissolution effect, reducing the influence of the solvent effect, guaranteeing the separation of isomerides and interfering ion pairs, and improving the detection sensitivity of multiple indexes.

In the present invention, the UPLC-MSMS assay process, especially the combined control of chromatographic column type, eluent and elution mechanism, achieves the key of the marker selectivity, high sensitivity and high linearity separation, preferably the phenyl column is ACQUITY UPLC BEH Phenyl 2.1.1 x 50mm. Research shows that the UPLC chromatographic column with the model is matched with the elution process disclosed by the invention, and is favorable for further improving the separation selectivity of the composite marker.

The mass spectrum of the invention has the specific conditions that a multi-ion reaction monitoring mode of positive ion electrospray ionization is adopted, the capillary voltage is 2-3 Kv, the ion source temperature is 150+/-10 ℃, the desolvation gas temperature is 600+/-20 ℃, the desolvation gas flow rate is 800+/-50L/Hr, and the back blowing gas flow rate is 150+/-10L/Hr. Hr means hours.

The beneficial effects are that:

The composite marker is innovatively used for detecting LSDs, has excellent broad-spectrum measurement, stability and accuracy, and can promote LSDs early diagnosis and early treatment and scientific research.

The invention performs joint optimization control on conditions such as a protein precipitator, a complex solvent, a chromatographic column, an elution mechanism and the like of a biological sample, can realize high-sensitivity, high-selectivity and high-linearity separation of the composite marker, and can realize high-efficiency diagnosis and scientific research of LSDs.

Drawings

FIG. 1-A standard calibration curve of Lyso-Gb1 obtained by the method of the invention.

FIG. 2-TIC diagram of the determination of 10 biomarkers and their corresponding isotopic internal standard obtained by the method of the invention.

FIG. 3-chromatograms of Lyso-Gb1 and PSY 2 isomers and corresponding isotopic internal standards determined by the method of the present invention.

FIG. 4-TIC comparison of negative samples and NIMANPIKE positive by this experiment.

FIG. 5-comparative plot of negative samples and Krabeliant positive TIC obtained by this experiment.

FIG. 6-TIC comparison of negative samples obtained by this experiment and positive for GM1 gangliosidosis.

FIG. 7-TIC comparison of negative samples obtained by this experiment and positive for GM2 gangliosidosis.

Fig. 8 is a comparative view of TICs collected using different chromatographic columns.

Fig. 9 is a comparative view of TICs collected for different mobile phase compositions and different proportions of additives.

FIG. 10 is a comparative TIC plot of different liquid phase gradient acquisitions.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present application. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

A liquid phase mass spectrometry method for detecting the content of a biomarker related to lysosomal storage disease in body fluid is applicable to matrix types including, but not limited to, serum, plasma, urine, amniotic fluid and cerebrospinal fluid.

The method comprises the steps of carrying out qualitative and quantitative detection on 10 relevant biomarkers and corresponding isotope internal standards in body fluid by adopting a UPLC-MS/MS method, wherein a mobile phase A of liquid chromatography is an aqueous solution containing ammonia water and acetonitrile, a mobile phase B of liquid chromatography is an acetonitrile solution containing ammonia water and isopropanol, gradient elution is carried out, and mass spectrometry adopts a multi-ion reaction monitoring mode of positive ion electrospray ionization.

The 10 related biomarkers are respectively glucosylceramine (Lyso-Gb 1), globular glycosylsphingosine (Lyso-Gb 3), lysosphingomyelin (Lyso SM), N-palmitoyl-O-phosphorylcholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3, 5, 6-triol (C-triol), galactosylsphingosine (Psychosine/PSY), C26-Ceramide (C26-Ceramide/C26-C), lysoGM1 ganglioside (lysoGM 1), and lysoGM2 ganglioside (lysoGM 2).

The corresponding isotope internal standard of the biomarker is 13C 6-glucosylceramine (13C 6-Lyso-Gb 1), D7-globular glycosylsphingosine (D7-Lyso-Gb 3), D9-lysosphingomyelin (D9-Lyso SM), D9-N-palmitoyl-O-phosphorylcholine-serine (D9-PPCS), D7-7-ketocholesterol (D7-7-KC), D7-cholestane-3, 5, 6-triol (D7-C-triol) and D5-galactosyl sphingosine (D5-PSY) respectively.

The determination of the biomarker and the corresponding isotope internal standard comprises the separation of the isomers-Lyso-Gb 1 and PSY, and the effective separation of the isomers can ensure the sensitivity and the specificity of the detection of two diseases, namely Gaucher Disease (GD) and Crab Bei Bing (KD).

In the present invention, meOH refers to methanol. ACE refers to acetonitrile. FA refers to formic acid.

Preferably, specific liquid chromatography conditions include a chromatographic column of ACQUITY UPLC BEH Phenyl 2.1.1X105 mm 1.7. Mu.M, a column temperature of 40 ℃, a mobile phase A of water containing 0.005-0.01% NH ₃H₂ O+10% acetonitrile, a mobile phase B of acetonitrile solution containing 0.005-0.01% NH ₃H₂ O+10% IPA (isopropyl alcohol), and gradient elution conditions (such as a flow rate of 0.4mL/min in Table 1)：0 - 0.2min,0 - 16.5%B;0.2 - 3.25min,16.5 - 16.8%B;3.25 - 3.75min,16.8 - 33.8%B;3.75 - 3.8min,33.8 - 34%B;3.8 - 10.25min,34 - 34.5%B;10.25 - 10.3min,34.5 - 34.5%B;10.3 - 10.85min,34.5 - 40%B;10.85 - 11min,40 - 62.5%B;11 - 12min,62.5 - 64%B;12 - 12.35min,64 - 64%B;12.35 - 13.55min,64 - 100%B;13.55 - 14min,100 - 100%B;14 - 14.01min,100 - 0%B;14.01 - 15min,0 - 0%B;, and a sample injection amount of 7.5. Mu.L).

Preferably, the specific conditions of the mass spectrum are that a multi-ion reaction monitoring mode of positive ion electrospray ionization is adopted, the capillary voltage is 2.5Kv, the ion source temperature is 150 ℃, the desolvation gas temperature is 600 ℃, the desolvation gas flow rate is 800L/Hr, and the back blowing gas flow rate is 150L/Hr.

Preferably, the method further comprises the step of pre-treating the sample of serum, plasma, urine, amniotic fluid, cerebrospinal fluid or the like.

Further preferably, the pretreatment step is as follows:

(1) Protein precipitation

The serum/plasma samples were removed and placed in a 1.5ml centrifuge tube, 5. Mu.L of internal standard working solution and 300. Mu.L of formic acid-containing precipitant were added in sequence, thoroughly vortexed and mixed, and centrifuged at 4℃and 14000rpm for 10min. .

(2) Blowing and redissolving nitrogen

Transferring the supernatant into a clean 1.5ml centrifuge tube, drying with nitrogen at 40 ℃, re-dissolving with 100 mu L of re-solution, fully vortex-dissolving, and centrifuging for 10min at 10 ℃ and 14000 rpm.

(3) Loading:

and after the centrifugation is finished, taking a supernatant to obtain a sample to be tested.

Preferably, the concentration of the biomarkers Lyso-Gb1, lyso-Gb3, lyso SM, 7-KC, PSY, C26-C, GM1, GM2, PPCS, C-triol is 250, 500, 2500, 100, 2500, 500, 2000, 10000 ng/ml, respectively. The formulation scheme of the mixed standard working solution is shown in table 3.

And diluting the working solution 10 times by 10 times to obtain the mixed standard working solution.

Preferably, the mixed internal standard is prepared into a mixed internal standard solution by using a methanol aqueous solution, and the preparation scheme of the mixed internal standard solution is shown in table 4.

The calibration line is prepared by preparing a standard curve from seven-concentration mixed standard substance solutions, wherein the seven-concentration mixed standard substance solution is prepared by adding a compound solution into a mixed standard working solution for dilution, and the concentration of the calibration line is shown in table 5. Performing linear regression analysis to obtain a regression equation, substituting the peak area of the corresponding biomarker into the regression equation, and calculating the concentration of the biomarker in the sample;

Preferably, the controlled matrix is selected from serum, plasma, urine, amniotic fluid, cerebrospinal fluid, and the like.

The method comprises a quality control product, wherein the quality control product comprises three (low, medium and high) concentration levels of quality control solution, the quality control product is prepared by adding mixed biomarker working solution into treated or commercialized serum, plasma, urine, amniotic fluid, cerebrospinal fluid and the like, the concentration of each biomarker of the low, medium and high quality control is distributed in seven gradient concentrations of mixed standard solution, and the target value is determined by detection.

Example 1:

Preparing a sample to be tested:

for obtained body fluid samples, such as serum, plasma, urine, amniotic fluid, cerebrospinal fluid, etc., the pretreatment reference steps are as follows:

① Protein precipitation

Transfer 50. Mu.L serum/plasma sample into 1.5ml centrifuge tube, sequentially add 5. Mu.L internal standard working solution and 300. Mu.L formic acid-containing precipitant, vortex well, centrifuge at 4 deg.C and 14000rpm for 10min to obtain supernatant.

② Blowing and redissolving nitrogen

The supernatant was transferred to a clean 1.5ml centrifuge tube and dried under nitrogen at 40 ℃ and then reconstituted with 100 μl of reconstituted solution and vortexed well and centrifuged at 10 ℃ and 14000rpm for 10min.

③ Loading:

after the centrifugation is completed, a sample to be detected is obtained, and the sample is prepared for on-machine detection.

(2) Ultra-high performance liquid chromatography-mass spectrometry detection

The sample to be tested enters a chromatographic column for separation through a gradient elution mode, and the liquid chromatography reference conditions are as follows:

ACQUITY UPLC BEH Phenyl 2.1.1 x 50mm 1.7. Mu.M column

Mobile phase A, 0.01% NH ₃H₂ O +10% acetonitrile +90% water

Mobile phase B, 0.01% NH ₃H₂ O+10% IPA+90% acetonitrile

Column temperature of 40 DEG C

Sample volume 7.5. Mu.L

Elution gradient (see table 1):

Mass spectrometry parameter reference conditions:

The multi-ion reaction monitoring mode of positive ion electrospray ionization is adopted, wherein the capillary voltage is 2.5Kv, the ion source temperature is 150 ℃, the desolvation gas temperature is 600 ℃, the desolvation gas flow rate is 800L/Hr, and the back-blowing gas flow rate is 150L/Hr. The MRM mass spectral parameters are shown in table 2.

(3) Calculation result

The standard working solution is prepared by the concentration of Lyso-Gb1, lyso-Gb3, lyso SM, 7-KC, PSY, C26-C, GM1, GM2, PPCS and C-triol of 250, 500, 2500, 100, 2500, 500, 2000 and 10000 ng/ml respectively. The preparation scheme of the mixed standard working solution is shown in the table 3, wherein the mixed standard working solution is obtained by diluting the working solution 10 times.

The mixed internal standard is prepared into a mixed internal standard solution by methanol aqueous solution, and the preparation scheme of the mixed internal standard solution is shown in table 4.

The calibration line is prepared by preparing a standard curve from seven-concentration mixed standard substance solutions, wherein the seven-concentration mixed standard substance solution is prepared by adding a compound solution into a mixed standard working solution for dilution, and the concentration of the calibration line is shown in table 5. The concentration of the standard substance relative to the internal standard solution is taken as an X axis, the ratio of the standard substance to the internal standard peak area is taken as a Y axis, a regression equation is obtained by linear regression analysis, the peak area of the corresponding biomarker is substituted into the regression equation, the concentration of the biomarker in the sample is calculated, and the concentration design of the correction line is shown in Table 5.

Analysis of data the linearity measured with the method of the invention is shown in Table 6 below:

the correlation coefficient r2 is larger than 0.995, the method has excellent linearity, and the addition concentration and the measured concentration have good correlation.

The method comprises a quality control product, wherein the quality control product comprises three (low, medium and high) concentration levels of quality control solution, the quality control product is prepared by adding mixed biomarker working solution into treated or commercialized serum, plasma, urine, amniotic fluid, cerebrospinal fluid and the like, the concentration of each biomarker of the low, medium and high quality control is distributed in seven gradient concentrations of mixed standard solution, and the target value is determined by detection.

Example 2:

According to the method of example 1, the accurate magnitude of each biomarker of normal samples and LSDs positive samples of different diseases is obtained by the method of the invention, the magnitude is shown in the following table 7, and typical spectrograms are shown in fig. 2-7:

TABLE 7

As shown in the above table, we performed sample validation on 4 different LSD disease samples for niemann pick disease, cla Bei Bing, GM1 gangliosidosis, GM2 gangliosidosis. For different LSD diseases, the method provided by the invention sensitively detects the increase of the specific biomarker on the disease sample, and quantifies the increase through a standard substance and an isotope internal standard.

The method detects the magnitude of significant elevation of specific biomarkers PPCS and LysoSM relative to negative samples in niemann pick disease;

The method detects the magnitude of the obvious increase of the specific biomarker PSY relative to a negative sample in the Crabbe disease;

The method detects the specific biomarker in the GM1 gangliosidosis, namely the magnitude of the significant increase of GM1 relative to the negative sample;

The method detects the specific biomarker in the GM2 gangliosidosis, namely the magnitude of the significant increase of GM2 relative to a negative sample;

Example 3:

The only difference compared to example 1 is that the chromatographic Column 1 was ACQUITY UPLC BEH Amide Column,10cm, the chromatographic Column 2 was ACQUITY UPLC BEH C, 18 Column,5cm, and the chromatographic Column 3 was ACQUITY UPLC BEH Phenyl,5cm were optimized and compared to the mobile phase. The TIC spectrum is shown in FIG. 8.

Column 1 is mainly characterized by the absence of several indicators of C26-C, 7-KC and C-triol. Meanwhile, the composition and distribution of the mobile phase are optimized and compared for the chromatographic column, and the composition and distribution of the mobile phase are not obviously improved. The chromatographic column 2 was not completely separated from the baseline of lyso-Gb1 and PSY by adjustment of the elution gradient, nor was the elution of the strong retention of C26-C good. The chromatographic column 3 can not separate the lyso-Gb1 from the PSY under the acidic pH mobile phase, and the NH ₃H₂ O is more than or equal to 0.05% under the alkaline pH mobile phase, the index lyso-Gb1 and the PSY can not be separated, and the NH ₃H₂ O can be separated within the range of 0.005% -0.01%, as shown in the attached figures 2 and 3. Preferably, the chromatographic column has the best eluting effect of 3-ACQUITY UPLC BEH Phenyl cm.

Example 4:

The only difference compared to example 1 is that the pre-treated precipitant components, pH and amounts are all optimized and compared, the parameters and structures are shown in table 8;

The best precipitating agent was determined to be 300. Mu.L 0.2% FA methanol/acetone (1:1).

Example 5:

the only difference compared to example 1 is that the pre-treatment complex solution components, pH and amounts were all optimized and compared, the parameters and structures are shown in table 9;

the formic acid ratio in the complex solution collected peak area data are shown in table 10:

Table 10

The solvent which determined the best effect of the multiple solution was 100. Mu.L of 0.1% FA 85% methanol.

Example 6:

the only difference compared to example 1 is the group:

the acetonitrile in the mobile phases A and B is replaced by methanol;

Adopting formic acid as an additive to replace ammonia water in the mobile phases A and B in an equal volume;

Changing the addition amount (0.005%, 0.01% or 0.02%) of the ammonia water in the mobile phases A and B;

the TIC spectrum is shown in FIG. 9.

The mobile phase components, additives and elution gradients are all optimized and compared, and compared with different mobile phase components, the lyso-Gb1 and PSY cannot be separated after the MEOH peak time. Compared with different mobile phase additives, the lyso-Gb1 and the PSY cannot be separated under the acidic PH, NH ₃H₂ O is more than or equal to 0.02% under the alkaline PH, the index lyso-Gb1 and the PSY cannot be separated, NH ₃H₂ O can be completely separated within the range of 0.005% -0.01%, and the separation degree is good.

Gradient screening TIC spectra comparison is shown in fig. 10:

in gradient elution, the structures of lyso-Gb1 and PSY are very similar and are mutually optical isomers, the method comprises 3.8-10.25min,34-34.5% of B and 0.5 mL/min, 10.25-10.3min,34.5-34.5% of B and 0.5-0.4mL/min are combined with the nearly equal A1B1 component and the variable flow rate, the separation of lyso-Gb1 and PSY is realized, and the effective separation of the isomers of the components can ensure the sensitivity and the specificity of the detection of two diseases of Gaucher Disease (GD) and Crabbe Bei Bing (KD). In addition, GM1 and GM2 interfere with each other, and separation of GM1 and GM2 is achieved by modulation of 0 - 0.2min,0 - 16.5%B;0.2 - 3.25min,16.5 - 16.8%B;3.25 - 3.75min,16.8 - 33.8%B;3.75 - 3.8min,33.8 - 34%B gradients.

An optimal liquid phase method is determined, namely a mobile phase A is water containing 0.005% -0.01% of NH ₃H₂ O+10% of acetonitrile, a mobile phase B is acetonitrile solution containing 0.005% -0.01% of NH ₃H₂ O+10% of IPA, and the gradient elution condition ：0 - 0.2min,0 - 16.5%B;0.2 - 3.25min,16.5 - 16.8%B;3.25 - 3.75min,16.8 - 33.8%B;3.75 - 3.8min,33.8 - 34%B;3.8 - 10.25min,34 - 34.5%B;10.25 - 10.3min,34.5 - 34.5%B;10.3 - 10.85min,34.5 - 40%B;10.85 - 11min,40 - 62.5%B;11 - 12min,62.5 - 64%B;12 - 12.35min,64 - 64%B;12.35 - 13.55min,64 - 100%B;13.55 - 14min,100 - 100%B;14 - 14.01min,100 - 0%B;14.01 - 15min,0 - 0%B. does not adopt the elution mechanism and the elution gradient of the invention, so that the ideal separation of the composite marker is difficult to realize.

Example 7:

In the invention, the introduction of a plurality of isotope internal standards is introduced, and referring to CLSI C62-A: "Liquid Chromatography-Mass Spectrometry Methods; approved Guideline" matrix effect investigation implementation scheme-the blank blood matrix of 6 different individuals is investigated, and the matrix factors normalized by the internal standards are calculated by comparing the signals of the signal vs of the analyte added with the standard sample after extraction and the signals of the analyte added with the standard curve matrix after extraction. Formula: matrix Element (ME) =peak area response of blank matrix after extraction to test object/peak area response of test object in pure solution×100%, internal standard normalized me=test object ME/internal standard me×100%. Taking D9-PPCS as an example, the data are given in Table 11 below:

TABLE 11

The variation coefficient of the ME% of the normalized matrix factors of the 6 different individuals is 9.3% and less than 15%, and the mean value of the ME% of the matrix factors of the internal standard is 97%, which indicates that the use of the internal standard of the isotope effectively eliminates the influence of response inhibition generated among different matrixes and ensures the accurate quantification of target analytes in terms of a method.

The present application is not limited to the above-mentioned preferred embodiments, and any person who can obtain other various products under the teaching of the present application can make any changes in shape or structure, and all the technical solutions that are the same or similar to the present application fall within the scope of the present application.

The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (9)

1. A composite marker for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS, comprising a marker and an isotope internal standard; Among them, markers include glucosylsphingosine, globular glycosylsphingosine, lyso-sphingomyelin, N-palmitoyl-O-phosphocholine-serine, 7-ketocholesterol, cholestane-3,5,6-triol, galactosylsphingosine, C26-ceramide, lyso-GM1 ganglioside, and lyso-GM2 ganglioside; Isotopic internal standards included 13C6-glucosylsphingosine, D7-globulosylsphingosine, D9-lyso-sphingomyelin, D9-N-palmitoyl-O-phosphocholine-serine, D7-7-ketocholesterol, D7-cholestane-3,5,6-triol, and D5-galactosylsphingosine. 2. A method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS, characterized in that each component of the composite marker of claim 1 is measured using the UPLC-MS/MS method, and a standard curve for each marker is drawn; The sample to be tested is prepared to obtain a test solution, and the solution is measured by UPLC-MS/MS, and then a qualitative analysis is performed or a quantitative analysis is performed based on a drawn standard curve; The UPLC test conditions for UPLC-MS/MS are: Chromatographic column: phenyl column, mobile phase A: aqueous solution containing 0.005%-0.01% ammonia + 10±1% acetonitrile, mobile phase B: acetonitrile solution containing 0.005%-0.01% ammonia + 10±1% isopropanol; Gradient elution conditions: 0 - 0.2 min, 0 - 16.5% B; 0.2 - 3.25min, 16.5 - 16.8%B; 3.25-3.75min, 16.8-33.8%B; 3.75-3.8min, 33.8-34%B; 3.8 - 10.25min, 34 - 34.5%B; 10.25-10.3min, 34.5-34.5%B; 10.3-10.85min, 34.5-40%B; 10.85-11min, 40-62.5%B; 11 - 12 min, 62.5 - 64% B; 12 - 12.35min, 64 - 64%B; 12.35-13.55min, 64-100%B; 13.55-14min, 100-100%B; 14 - 14.01min, 100 - 0%B; 14.01 - 15min, 0 - 0%B. 3. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 2, wherein the lysosomal storage disease comprises at least one of Gaucher disease (GD), Fabry disease (FD), Niemann-Pick disease type A, type B, and type C/D (NPA/B, type C/D), Krabbe disease (KD), Farber disease (FD), GM1 gangliosidosis, and GM2 gangliosidosis. 4. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 2, wherein the step of preparing the test solution from the test sample comprises: Step (1): Pipette the sample to be tested into a container, add the internal standard working solution and the precipitant containing formic acid in sequence, mix well and centrifuge to obtain the supernatant; Step (2): The supernatant of step (1) was blown dry and then re-dissolved with the reconstitution solution; then centrifuged and the obtained supernatant was used as the test solution. 5. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 4, wherein the sample to be tested comprises at least one of serum, plasma, urine, amniotic fluid, and cerebrospinal fluid. 6. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 4, wherein the precipitant is an organic solvent containing 0.05-0.5% by volume of formic acid, wherein the organic solvent is a methanol-acetonitrile mixed solvent or a methanol-acetone mixed solvent, wherein the content of methanol in the organic solvent is 30-60% by volume. 7. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 4, wherein the reconstitution solution is a methanol aqueous solution containing 0.1-0.2% formic acid, wherein the methanol content in the methanol aqueous solution is 80-90% by volume. 8. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 2, wherein the phenyl column is ACQUITY UPLC BEH Phenyl 2.1×50 mm. 9. The method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS/MS according to claim 2, wherein the specific conditions of mass spectrometry are: positive ion electrospray ionization in multiple ion reaction monitoring mode, capillary voltage: 2-3 kV; ion source temperature: 150±10°C; desolvation temperature: 600±20°C; desolvation gas flow rate: 800±50 L/Hr; backflush gas flow rate: 150±10 L/Hr.