Composite marker and method for detecting biomarkers related to lysosomal storage diseases based on UPLC-MS / MS (Ultra Performance Liquid Chromatography-Mass Spectrometry / Mass Spectrometry)

Abstract

The invention belongs to the field of inspection, and particularly relates to a composite marker for detecting biomarkers related to lysosomal storage diseases based on UPLC-MS / MS. The composite marker comprises a marker and an isotope internal standard substance, wherein the marker comprises glucosyl sphingosine, spherical glycosyl sphingosine, hemolytic sphingomyelin, N-palmitoyl-O-phosphorylcholine-serine, 7-ketocholesterol, cholestane-3, 5, 6-triol, galactosyl sphingosine, C26-ceramide, hemolytic GM1 ganglioside and hemolytic GM2 ganglioside, and the molecular weight of the marker is 8000-9000. The isotope internal standard substance is prepared from < 13 > C < 6 >-glucosyl sphingosine, D7-spherical glycosyl sphingosine, D9-lysosphingomyelin, D9-N-palmitoyl-O-phosphorylcholine-serine, D7-7-ketocholesterol, D7-cholestane-3, 5, 6-triol and D5-galactosyl sphingosine. The invention also comprises a UPLC-MS / MS method for detecting the composite marker. According to the invention, simultaneous detection of multiple biomarkers related to lysosomal storage diseases is realized, and the method is simple and convenient to operate, high in flux and low in cost.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a liquid phase mass spectrometry method for detecting the content of lysosomal storage disease-related biomarkers in body fluids. Background Art

[0002] Lysosomal storage disorders (LSDs) are a group of inherited metabolic disorders caused by genetic mutations that result in defects in acid hydrolases in lysosomes. This leads to the inability to properly degrade biomacromolecules, resulting in their accumulation in lysosomes and causing dysfunction in cells, tissues, and organs. Gaucher disease (GD) is an autosomal recessive LSD caused by mutations in the GBA1 gene, which encodes the glucosylceramide-degrading enzyme β-glucocerebrosidase. GBA1 deficiency leads to the progressive accumulation of the substrate glucosylceramide (Gb1). Glucosylsphingosine (Lyso-Gb1), a direct metabolite of Gb1, is pathogenic to cells. Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by pathogenic variants in the GLA gene, which encodes α-galactosidase A (α-Gal A). Deficiency or loss of α-Gal A and the subsequent accumulation of glycosphingolipids, including globulin (lyso-GL3), in various tissues and cells leads to progressive damage to affected organs, life-threatening complications, and an increased risk of premature death. Niemann-Pick disease (NP) encompasses a heterogeneous group of lysosomal lipid accumulation disorders with distinct clinical, biochemical, and molecular features. Types A and B result from mutations in the acid sphingomyelinase (ASM)-sphingomyelin phosphodiesterase-1 gene, leading to accumulation of sphingomyelin in various organs and tissues. Types C and D result from dysfunction of either NPC1 or NPC2, a lysosomal protein, leading to cholesterol accumulation in lysosomes, resulting in damage to multiple organs and characterized by progressive neurological deterioration. Relevant biomarkers include plasma oxysterols—cholestane-3β,5α,6β-triol (C-Triol) and 7-ketocholesterol (7-KC)—and lysosphingolipids—lyso-sphingosine-sphingomyelin (Lyso-SM) and lyso-sphingomyelin-509 (Lyso-SM-509). Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by a deficiency of the lysosomal enzyme galactocerebrosidase (GALC), resulting in elevated levels of the substrate galactosylceramide in patients with clinical signs and symptoms of the disease. Farber disease (FD) is a rare lysosomal storage disorder caused by a deficiency of acid ceramidase and subsequent accumulation of ceramide. GM1 gangliosidosis, also known as systemic gangliosidosis, is an inherited lysosomal disorder caused by a deficiency of β-galactosidase and is inherited in an autosomal recessive manner. Clinical features include progressive central nervous system disorders and skeletal abnormalities similar to mucopolysaccharidosis type I. GM2 gangliosidosis is a rare autosomal recessive genetic disorder, also known as GM2 gangliosidosis, caused by a deficiency of the enzyme lysosomal β-hexosaminidase (Hex), which leads to excessive accumulation of GM2 gangliosides in the brain and peripheral organs.

[0003] Screening for LSDs typically involves direct measurement of the activity of disease-associated enzymes for initial diagnosis. Enzyme activity quantification is widely used in newborn screening and is considered the gold standard for primary screening in newborns. Enzyme activity is often measured using a PE-based assay, the NeoLSD™ MSMS Kit, which detects the levels of six enzymes in blood spots, covering all six LSD disorders. However, while some enzyme activities may be affected by sample processing or experimental conditions, enzyme activity assays are limited in throughput and subject to numerous experimental constraints. Another advantage of using biomarkers over enzyme activity assays is the risk of false positives and false negatives. Fabry disease (FD) is a rare X-linked lysosomal storage disorder. The diagnosis of FD in males is confirmed by demonstrating α-Gal A activity deficiency through enzyme testing. In heterozygous females, the non-pathogenic allele may be absent, making enzyme testing unreliable and requiring diagnosis based on gene sequencing. Biomarker testing often supports the diagnosis and is particularly useful for patients with a diminished phenotype. Plasma biomarkers have a higher sensitivity for diagnosing FD in females than α-Gal A enzyme activity assays. Secondary testing of screen-positive samples through quantification of biomarkers can address the high false-positive rate associated with low enzyme activity due to pseudo-deficiency.

[0004] Furthermore, while most LSDs are caused by enzyme deficiencies, some are caused by pathogenic alterations in lysosomal proteins that lack enzymatic activity. For example, NPC, which is caused by the NPC1 or NPC2 genes, can only be diagnosed by evaluating multiple biomarkers and confirmatory genetic analysis.

[0005] Conventional treatments for LSDs include enzyme replacement therapy (ERT), substrate reduction therapy (SRT), chaperone therapy, or hematopoietic stem cell transplantation. However, enzyme activity is less useful for monitoring disease progression once ERT has been initiated, compared to diagnostics using enzyme activity. For example, Pompe uses a more effective biomarker, the tetrasaccharide glucose (Glc4), derived from the digestion of glycogen and other branched glucose polymers. Biomarker analysis can be used to assess disease severity, serve as an indicator of disease progression, and monitor patient adherence to treatment regimens.

[0006] There are also a few reports in the prior art that use liquid chromatography-mass spectrometry (LC-MS) to detect biomarkers for the diagnosis of some LSDs. For example, patent publication number WO2023232942A1 discloses a biomarker and method for detecting lysosomal storage diseases, specifically describing the diagnosis of LSDs through the detection of Lyso-Gb1, Lyso-Gb3, and Lyso-Gb3-IS. Patent publication number US10983112B2 discloses a bile acid biomarker for Niemann-Pick disease, its method, and use, specifically describing the detection of 3B,5A,6B-trihydroxycholestanoic acid for the diagnosis of Niemann-Pick disease.

[0007] In summary, although there are some reports on biomarker diagnosis in the existing technology, the diagnostic scope of existing methods is limited, and the breadth, stability and specificity of detection need to be improved. Summary of the Invention

[0008] In response to the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a composite marker based on UPLC-MS / MS for detecting lysosomal storage disease-related biomarkers, aiming to improve the broad spectrum, sensitivity and accuracy of LSDs detection through the identification of the composite marker.

[0009] The second purpose of the present invention is to provide a method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS / MS, aiming to provide a method that can effectively detect lysosomal storage disease-related biomarkers.

[0010] The current diagnostic scope of LSDs is relatively narrow, making it difficult to achieve broad-spectrum and accurate diagnosis and identification of LSD-related diseases. To address this problem, the present invention provides the following improvements:

[0011] A composite marker for detecting lysosomal storage disease-related biomarkers based on UPLC-MS / MS, comprising a marker and an isotope internal standard;

[0012] 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;

[0013] 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.

[0014] The innovative research of the present invention shows that the innovative use of the combination of multiple biomarkers, combined with the use of targeted multiple isotope internal standards, offsets the influence of the matrix effect in the ionization process, thereby achieving synergy and enabling the broad-spectrum, accurate and sensitive identification of LSDs, thereby meeting the detection requirements of LSDs scientific research and diagnosis.

[0015] Lysosomal storage diseases include Gaucher disease (GD), Fabry disease (FD), Niemann-Pick disease types A, B, and C / D (NPA / B, C / D), Krabbe disease (KD), Farber disease (FD), GM1 gangliosidosis, and GM2 gangliosidosis.

[0016] Related disease biomarkers include glucosylsphingosine (Lyso-Gb1) in Gaucher disease (GD), globular glycosylsphingosine (Lyso-Gb3) in Fabry disease (FD), lyso-sphingomyelin (Lyso SM) in Niemann-Pick disease, N-palmitoyl-O-phosphocholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3,5,6-triol (C-triol), galactosylsphingosine (Psychosine / PSY) in Krabbe disease (KD), C26-ceramide (C26-Ceramide / C26-C) in FD, lysoGM1 ganglioside (lysoGM1) in GM1 gangliosidosis, and lysoGM2 ganglioside (lysoGM2) in GM2 gangliosidosis.

[0017] The corresponding isotope internal standards include 13C6-glucosylsphingosine (13C6-Lyso-Gb1), D7-globulosylsphingosine (D7-Lyso-Gb3), D9-lyso-sphingomyelin (D9-Lyso SM), D9-N-palmitoyl-O-phosphocholine-serine (D9-PPCS), D7-7-ketocholesterol (D7-7-KC), D7-cholestane-3,5,6-triol (D7-C-triol) and D5-galactosylsphingosine (D5-PSY), which correspond to the biomarkers glucosylsphingosine (Lyso-Gb1), globular glycosylsphingosine (Lyso-Gb3), lyso-sphingomyelin (Lyso SM), D9-N-palmitoyl-O-phosphocholine-serine (D9-PPCS), D7-7-ketocholesterol (D7-7-KC), D7-cholestane-3,5,6-triol (D7-C-triol) and D5-galactosylsphingosine (D5-PSY). SM), N-palmitoyl-O-phosphocholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3,5,6-triol (C-triol), and galactosylsphingosine (PSY).

[0018] The present invention also provides an application of the composite marker in preparing a detection reagent for determining LSDs, specifically:

[0019] A method for detecting lysosomal storage disease-related biomarkers based on UPLC-MS / MS, wherein each component of the composite marker is measured using the UPLC-MS / MS method, and a standard curve for each marker is drawn;

[0020] 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;

[0021] The UPLC test conditions for UPLC-MS / MS are:

[0022] 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;

[0023] Gradient elution conditions: 0 - 0.2 min, 0 - 16.5% B;

[0024] 0.2 - 3.25min, 16.5 - 16.8%B;

[0025] 3.25-3.75min, 16.8-33.8%B;

[0026] 3.75-3.8min, 33.8-34%B;

[0027] 3.8 - 10.25min, 34 - 34.5%B;

[0028] 10.25-10.3min, 34.5-34.5%B;

[0029] 10.3-10.85min, 34.5-40%B;

[0030] 10.85-11min, 40-62.5%B;

[0031] 11 - 12 min, 62.5 - 64% B;

[0032] 12 - 12.35min, 64 - 64%B;

[0033] 12.35-13.55min, 64-100%B;

[0034] 13.55-14min, 100-100%B;

[0035] 14 - 14.01min, 100 - 0%B;

[0036] 14.01 - 15min, 0 - 0%B.

[0037] The present invention proposes for the first time the idea of ​​diagnosing LSDs by detecting the above-mentioned composite markers. However, early research and development showed that the physicochemical properties of composite markers vary greatly. How to achieve highly selective separation of numerous markers under a set of detection conditions and achieve highly sensitive responses to markers of different properties is the main difficulty in implementing this technical idea. In response to the difficulties in implementing this technical idea, the present invention has shown through in-depth research that special control of the chromatographic column type, eluent, and elution mechanism during the UPLC test process can achieve synergy, achieve highly selective separation of the above-mentioned composite markers, and achieve highly sensitive and linear peak extraction. The method of the present invention can identify LSDs diseases broadly, and has excellent identification accuracy and sensitivity, which can effectively reduce the problems of false positives and false negatives in detection. The method of the present invention can realize in vitro detection of the composite markers. The method of the present invention can be used in scientific research related to LSDs.

[0038] In the present invention, lysosomal storage diseases include at least one of Gaucher disease (GD), Fabry disease (FD), Niemann-Pick disease type A, type B, and type C / D (NPA / B, C / D), Krabbe disease (KD), Farber disease (FD), GM1 gangliosidosis, and GM2 gangliosidosis.

[0039] In the present invention, the standard curve can be drawn based on known principles and methods. For example, each marker standard and the corresponding isotope internal standard can be prepared into 5-10 concentration gradients, and then measured separately. The standard curve of each marker can be obtained by performing linear regression analysis with the concentration of the standard relative to the internal standard solution as the X-axis and the ratio of the standard and internal standard peak areas as the Y-axis.

[0040] In the present invention, the steps of preparing the test solution from the test sample are:

[0041] Step (1):

[0042] 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;

[0043] Step (2):

[0044] The supernatant of step (1) was blown dry, and the protein precipitate was redissolved with a redissolving solution; the mixture was then centrifuged, and the resulting supernatant was used as the test solution.

[0045] In the present invention, the sample to be tested includes at least one of serum, plasma, urine, amniotic fluid, and cerebrospinal fluid.

[0046] In the present 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, wherein the methanol content in the organic solvent is 30-60% by volume. Studies of the present invention have shown that the use of this precipitation process helps to further improve the precipitation effect and recovery rate of the marker, enhance the response of the detection index, reduce interfering substances, and help reduce false negatives in the determination.

[0047] In the present invention, the reconstitution solution is a methanol-water solution containing 0.1-0.2% formic acid, wherein the methanol content in the methanol-water solution is 80-90% by volume. Studies in the present invention have shown that adopting this reconstitution process helps to further improve the reconstitution effect, while reducing the influence of solvent effects, ensuring the separation of isomers and interfering ion pairs, and improving the detection sensitivity of multiple indicators.

[0048] In the present invention, the UPLC-MS / MS assay process, specifically the combined control of column type, eluent, and elution mechanism, is key to achieving selective, highly sensitive, and linear separation of the markers. Preferably, the phenyl column is an ACQUITY UPLC BEH Phenyl 2.1*50mm. Studies have shown that using this UPLC column, combined with the elution process described herein, further improves the separation selectivity of complex markers.

[0049] In the present invention, the specific mass spectrometry conditions are as follows: 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. Hr refers to hours.

[0050] Beneficial effects:

[0051] The present invention innovatively adopts the composite marker for the detection of LSDs, which has excellent measurement broad spectrum, stability and accuracy, and can promote the early diagnosis and treatment of LSDs as well as scientific research.

[0052] The present invention jointly optimizes and controls the conditions of the biological sample's protein precipitant, resolvent, chromatographic column, and elution mechanism, thereby achieving highly sensitive, highly selective, and highly linear separation of the composite markers, and enabling efficient diagnosis and scientific research of LSDs. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 - A standard calibration curve of Lyso-Gb1 obtained by the method of the present invention.

[0054] Figure 2 - TIC diagrams obtained by the method of the present invention for the determination of 10 biomarkers and their corresponding isotope internal standards.

[0055] Figure 3 - Chromatograms of two isomers of Lyso-Gb1 and PSY and the corresponding isotope internal standards determined by the method of the present invention.

[0056] Figure 4 -Comparison of TICs obtained from negative samples and Niemann-Pick disease-positive samples using this assay.

[0057] Figure 5 -Comparison of TICs obtained from negative samples and Krabbe disease-positive samples using this assay.

[0058] Figure 6 -Comparison of TICs obtained from negative samples and GM1 gangliosidosis-positive samples obtained through this experiment.

[0059] Figure 7 -Comparison of TICs obtained from negative samples and GM2 gangliosidosis-positive samples obtained through this experiment.

[0060] Figure 8 Comparison of TICs collected using different chromatographic columns.

[0061] Figure 9 Comparison of TICs collected for different mobile phase compositions and different additive ratios.

[0062] Figure 10 Comparison of TICs collected at different liquid gradients. DETAILED DESCRIPTION

[0063] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0064] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0065] In order to achieve the above object, the present invention adopts the following technical solutions:

[0066] A liquid chromatography-mass spectrometry method for detecting the content of lysosomal storage disease-related biomarkers in body fluids, wherein applicable matrix types include but are not limited to: serum, plasma, urine, amniotic fluid, and cerebrospinal fluid.

[0067] UPLC-MS / MS was used for qualitative and quantitative detection of 10 relevant biomarkers and their corresponding isotopic internal standards in body fluids. The mobile phase A of the liquid chromatography was an aqueous solution containing ammonia and acetonitrile, and the mobile phase B was an acetonitrile solution containing ammonia and isopropanol, with gradient elution. The mass spectrometry used positive ion electrospray ionization in multiple ion reaction monitoring mode.

[0068] The 10 related biomarkers are: glucosylsphingosine (Lyso-Gb1), globular glycosylsphingosine (Lyso-Gb3), lyso-sphingomyelin (Lyso SM), N-palmitoyl-O-phosphocholine-serine (PPCS), 7-ketocholesterol (7-KC), cholestane-3,5,6-triol (C-triol), galactosylsphingosine (Psychosine / PSY), C26-ceramide (C26-Ceramide / C26-C), lyso-GM1 ganglioside (lysoGM1), and lyso-GM2 ganglioside (lysoGM2).

[0069] The isotope internal standards corresponding to the biomarkers are: 13C6-glucosylsphingosine (13C6-Lyso-Gb1), D7-globulosylglycosphingosine (D7-Lyso-Gb3), D9-lyso-sphingomyelin (D9-Lyso SM), D9-N-palmitoyl-O-phosphocholine-serine (D9-PPCS), D7-7-ketocholesterol (D7-7-KC), D7-cholestane-3,5,6-triol (D7-C-triol), and D5-galactosylsphingosine (D5-PSY).

[0070] The determination of biomarkers and corresponding isotopic internal standards includes the separation of isomers - Lyso-Gb1 and PSY. The effective separation of this group of isomers can ensure the sensitivity and specificity of the detection of Gaucher disease (GD) and Krabbe disease (KD).

[0071] In the present invention, MeOH refers to methanol, ACE refers to acetonitrile, and FA refers to formic acid.

[0072] Preferably, the specific liquid chromatography conditions are: chromatographic column: ACQUITY UPLC BEH Phenyl 2.1×50mm1.7μM, column temperature: 40°C; mobile phase A: water containing 0.005~0.01% NH3H2O ​​+ 10% acetonitrile, mobile phase B: acetonitrile solution containing 0.005~0.01% NH3H2O ​​+ 10% IPA (isopropanol); gradient elution conditions (as shown 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; flow rate 0.4mL / min; injection volume: 7.5μL.

[0073]

[0074] Preferably, the specific conditions of mass spectrometry are: using positive ion electrospray ionization in multiple ion reaction monitoring mode, capillary voltage: 2.5Kv; ion source temperature: 150°C; desolvation gas temperature: 600°C; desolvation gas flow: 800L / Hr; backflush gas flow: 150L / Hr.

[0075]

[0076] Preferably, the above method further comprises the step of pre-treating samples such as serum, plasma, urine, amniotic fluid, and cerebrospinal fluid.

[0077] Further preferably, the pretreatment step is:

[0078] (1) Protein precipitation

[0079] Transfer serum / plasma sample to a 1.5 ml centrifuge tube, add 5 μL of internal standard working solution and 300 μL of formic acid-containing precipitant, vortex thoroughly to mix, and centrifuge at 4°C and 14,000 rpm for 10 min.

[0080] (2) Nitrogen blowing and re-dissolution

[0081] Transfer the supernatant to a clean 1.5 ml centrifuge tube and blow dry with nitrogen at 40 °C. Reconstitute with 100 μL of reconstitution solution, vortex thoroughly, and centrifuge at 10 °C and 14,000 rpm for 10 min.

[0082] (3) Loading:

[0083] After centrifugation, the supernatant was taken to obtain the sample to be tested.

[0084] Preferably, the concentrations of the biomarkers Lyso-Gb1, Lyso-Gb3, Lyso SM, 7-KC, PSY, C26-C, GM1, GM2, PPCS, and C-triol are 250, 250, 500, 2500, 100, 2500, 500, 500, 2000, and 10000 ng / ml, respectively. The preparation scheme of the mixed standard working solution is shown in Table 3.

[0085]

[0086] The mixed standard working solution is prepared by diluting the 10-fold working solution 10 times.

[0087] Preferably, the mixed internal standard is prepared into a mixed internal standard solution by methanol-water solution, and the preparation scheme of the mixed internal standard solution is shown in Table 4.

[0088]

[0089] The calibration line was prepared using a standard curve with seven concentrations of mixed standard solution, which was prepared by diluting the mixed standard working solution with the reconstituted solution. The calibration line concentrations are shown in Table 5. The concentration of the standard relative to the internal standard solution was plotted on the X-axis, and the ratio of the peak area of ​​the standard to the internal standard was plotted on the Y-axis. Linear regression analysis was performed to obtain a regression equation, and the corresponding biomarker peak area was substituted into the regression equation to calculate the concentration of the biomarker in the sample.

[0090]

[0091] Preferably, the matrix for quality control is serum, plasma, urine, amniotic fluid, cerebrospinal fluid, etc.

[0092] The above method includes quality control products: quality control solutions containing three concentration levels (low, medium, and high). The quality control products are prepared by adding mixed biomarker working solutions to processed or commercialized serum, plasma, urine, amniotic fluid, cerebrospinal fluid, etc. The concentrations of each biomarker in the low, medium, and high quality control are distributed in seven gradient concentrations of the mixed standard solution, and the target value is determined by detection.

[0093] Example 1:

[0094] Prepare the sample to be tested:

[0095] For body fluid samples obtained, such as serum, plasma, urine, amniotic fluid, cerebrospinal fluid, etc., the pre-processing reference steps are:

[0096] ① Protein precipitation

[0097] Pipette 50 μL of serum / plasma sample into a 1.5 ml centrifuge tube, add 5 μL of internal standard working solution and 300 μL of formic acid-containing precipitant in sequence, vortex mix thoroughly, and centrifuge at 4°C and 14,000 rpm for 10 min to obtain the supernatant.

[0098] ②Nitrogen blowing and re-dissolution

[0099] Transfer the supernatant to a clean 1.5 ml centrifuge tube and blow dry with nitrogen at 40 °C. Then reconstitute with 100 μL of reconstitution solution, vortex thoroughly, and centrifuge at 10 °C and 14,000 rpm for 10 min.

[0100] ③ Loading:

[0101] After centrifugation, the sample to be tested is obtained and ready for testing on the machine.

[0102] (2) Ultra-high performance liquid chromatography-mass spectrometry detection

[0103] The sample to be tested enters the chromatographic column for separation through gradient elution mode. The reference conditions of liquid chromatography are:

[0104] Column: ACQUITY UPLC BEH Phenyl 2.1*50mm 1.7μM

[0105] Mobile phase A: 0.01% NH3H2O ​​+10% acetonitrile +90% water

[0106] Mobile phase B: 0.01% NH3H2O ​​+ 10% IPA + 90% acetonitrile

[0107] Column temperature: 40°C

[0108] Injection volume: 7.5 μL

[0109] Elution gradient (see Table 1):

[0110] Mass spectrometry parameter reference conditions:

[0111] Positive electrospray ionization was used in multiple ion reaction monitoring mode with a capillary voltage of 2.5 kV, an ion source temperature of 150°C, a desolvation temperature of 600°C, a desolvation gas flow rate of 800 L / Hr, and a backflush gas flow rate of 150 L / Hr. MRM mass spectrometry parameters are shown in Table 2.

[0112] (3) Calculation results

[0113] Preparation of standard working solutions: The concentrations of Lyso-Gb1, Lyso-Gb3, Lyso SM, 7-KC, PSY, C26-C, GM1, GM2, PPCS, and C-triol were 250, 250, 500, 2500, 100, 2500, 500, 500, 2000, and 10,000 ng / mL, respectively. The preparation of the mixed standard working solution is shown in Table 3: The mixed standard working solution is prepared by diluting the 10x working solution 10-fold.

[0114] The mixed internal standard was prepared into a mixed internal standard solution by methanol-water solution. The preparation scheme of the mixed internal standard solution is shown in Table 4.

[0115] The calibration line was prepared using a standard curve using seven concentrations of mixed standard solution, prepared by diluting the mixed standard working solution with the reconstituted solution. The calibration line concentrations are shown in Table 5. The concentration of the standard relative to the internal standard solution was plotted on the X-axis, and the ratio of the standard to internal standard peak areas was plotted on the Y-axis. Linear regression analysis was performed to determine the regression equation, and the corresponding biomarker peak areas were substituted into the regression equation to calculate the biomarker concentrations in the sample. The calibration line concentrations were designed as shown in Table 5.

[0116] Data analysis: The linearity measured by the method of the present invention is shown in Table 6 below:

[0117]

[0118] The correlation coefficients r^2 were all greater than 0.995, indicating that the method had excellent linearity and good correlation between the added concentration and the measured concentration.

[0119] The above method includes quality control products: quality control solutions containing three concentration levels (low, medium, and high). The quality control products are prepared by adding mixed biomarker working solutions to processed or commercialized serum, plasma, urine, amniotic fluid, cerebrospinal fluid, etc. The concentrations of each biomarker in the low, medium, and high quality control are distributed in seven gradient concentrations of the mixed standard solution, and the target value is determined by detection.

[0120] Example 2:

[0121] According to the method of Example 1, the accurate values ​​of each biomarker of normal samples and LSDs positive samples of different diseases were obtained by the method of the present invention. The values ​​are shown in Table 7 below, and the typical spectra are shown in Table 7 below. Figures 2 to 7 :

[0122] Table 7

[0123]

[0124] As shown in the table above, we validated this method in samples from four different LSD disorders: Niemann-Pick disease, Krabbe disease, GM1 gangliosidosis, and GM2 gangliosidosis. For each LSD disorder, the method sensitively detected elevated levels of specific biomarkers in disease samples and quantified them using standards and isotope internal standards.

[0125] The method of the present invention detected significantly increased values ​​of specific biomarkers, PPCS and LysoSM, in Niemann-Pick disease compared with negative samples;

[0126] The method of the present invention detected a specific biomarker, PSY, in Krabbe disease, with a significantly increased value relative to negative samples;

[0127] The method of the present invention detects a specific biomarker in GM1 gangliosidosis, the value of which is significantly increased compared with the negative sample;

[0128] The method of the present invention detects a specific biomarker in GM2 gangliosidosis, the value of which is significantly increased compared with the negative sample;

[0129] Example 3:

[0130] Compared with Example 1, the only difference is that the chromatographic columns and mobile phases were optimized and compared. Among them, chromatographic column 1 is ACQUITY UPLC BEH Amide Column, 10 cm; chromatographic column 2 is ACQUITY UPLC BEH C18 Column, 5 cm; chromatographic column 3 is ACQUITY UPLC BEH Phenyl, 5 cm. TIC spectrum is shown in Figure 8 .

[0131] Column 1 mainly showed the lack of retention of several indicators, including C26-C, 7-KC, and C-triol. The composition and distribution of the mobile phase were also optimized and compared for this column, but no significant improvement was found. By adjusting the elution gradient of column 2, the baselines of lyso-Gb1 and PSY could not be completely separated, and the elution of the strongly retained C26-C was also poor. Column 3 could not separate lyso-Gb1 and PSY under acidic pH mobile phase; under alkaline pH mobile phase, NH3H2O ​​≥ 0.05%, the indicators lyso-Gb1 and PSY were inseparable, but they could be separated within the range of 0.005%-0.01% NH3H2O, see attached. Figure 2 and attached Figure 3 Preferably, the chromatographic column with the best elution effect is chromatographic column 3 - ACQUITY UPLC BEH Phenyl, 5 cm.

[0132] Example 4:

[0133] Compared with Example 1, the only difference is that the components, pH and amount of the pretreatment precipitant are optimized and compared. The parameters and structure are shown in Table 8;

[0134]

[0135] It was determined that the precipitant with the best precipitation effect was 300 μL 0.2% FA methanol / acetone (1:1).

[0136] Example 5:

[0137] Compared with Example 1, the only difference is that the components, pH and amount of the pre-treated reconstituted solution are optimized and compared. The parameters and structure are shown in Table 9;

[0138]

[0139] The peak area data of formic acid ratio in the reconstituted solution are shown in Table 10:

[0140] Table 10

[0141]

[0142] It was determined that the solvent with the best reconstitution effect was 100 μL 0.1% FA 85% methanol.

[0143] Example 6:

[0144] Compared with Example 1, the only difference is that the groups are:

[0145] A: Replace acetonitrile in mobile phases A and B with methanol;

[0146] B: Use formic acid as an additive to replace the ammonia in mobile phases A and B by equal volume;

[0147] C: Change the amount of ammonia added to mobile phases A and B (0.005%, 0.01% or 0.02%);

[0148] TIC spectrum see Figure 9 .

[0149] Mobile phase components, additives, and elution gradients were optimized and compared. Comparisons of different mobile phase components revealed that MEOH eluted later, making it impossible to separate lyso-Gb1 and PSY. Comparisons of different mobile phase additives revealed that lyso-Gb1 and PSY could not be separated at acidic pH. At alkaline pH, lyso-Gb1 and PSY were inseparable when NH3H2O ​​was ≥ 0.02%. However, complete separation was achieved within the 0.005%-0.01% NH3H2O ​​range, demonstrating excellent resolution.

[0150] Gradient screening TIC spectrum comparison see Figure 10 :

[0151] In gradient elution, lyso-Gb1 and PSY have extremely similar structures and are optical isomers of each other. The method uses a flow rate of 3.8-10.25 min, 34-34.5% B, 0.5 mL / min; and a flow rate of 10.25-10.3 min, 34.5-34.5% B, 0.5-0.4 mL / min. The combination of nearly isocratic A1B1 components and variable flow rate enables the separation of lyso-Gb1 and PSY. The effective separation of this group of isomers can ensure the sensitivity and specificity of detecting Gaucher disease (GD) and Krabbe disease (KD). In addition, GM1 and GM2 interfered with each other, and the separation of GM1 and GM2 was achieved by adjusting the gradient 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.

[0152] The optimal liquid phase method was determined: mobile phase A: water containing 0.005%-0.01% NH3H2O ​​+10% acetonitrile, mobile phase B: acetonitrile solution containing 0.005%-0.01% NH3H2O+10% IPA; gradient elution conditions: 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- 11 min, 40 - 62.5% B; 11 - 12 min, 62.5 - 64% B; 12 - 12.35 min, 64 - 64% B; 12.35 - 13.55 min, 64 - 100% B; 13.55 - 14 min, 100 - 100% B; 14 - 14.01 min, 100 - 0% B; 14.01 - 15 min, 0 - 0% B. Without the elution mechanism and elution gradient described in the present invention, it is difficult to achieve the ideal separation of the composite marker.

[0153] Example 7:

[0154] This method incorporates the use of multiple isotopic internal standards. Matrix effect investigation implementation plan, as described in CLSI C62-A: Liquid Chromatography - Mass Spectrometry Methods; Approved Guideline, examines blank blood matrices from six different individuals. By comparing the signal of the analyte spiked into the extracted sample with the signal of the analyte spiked into the extracted standard curve matrix, the matrix factor (ME) for each analyte (low, medium, and high quality control concentrations) and internal standard was calculated. The matrix factor normalized to the internal standard was then calculated. The formula is: Matrix Factor (ME) = Peak Area Response of Analyte in Extracted Blank Matrix / Peak Area Response of Analyte in Pure Solution × 100%; Internal Standard Normalized ME = Analyte ME / Internal Standard ME × 100%. Using D9-PPCS as an example, the resulting data are shown in Table 11:

[0155] Table 11

[0156]

[0157] The coefficient of variation of the internal standard-normalized matrix factor ME% of 6 different individuals was 9.3%, which was less than 15%, and the average internal standard-normalized matrix factor ME% was 97%, indicating that the use of isotope internal standards effectively eliminated the influence of response suppression caused by different matrices, ensuring the accurate quantification of the target analytes from a methodological perspective.

[0158] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, any technical solution that is the same or similar to that of the present application falls within the scope of protection of the present invention.

[0159] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A composite marker for detecting lysosomal storage disease-related biomarkers based on UPLC-MS / MS, characterized in that: Including markers and isotope internal standards; 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: The components of the composite marker according to claim 1 are measured by UPLC-MS / MS method, and a standard curve of 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: Lysosomal storage diseases include at least one of Gaucher disease (GD), Fabry disease (FD), Niemann-Pick disease type A, B, and C / D (NPA / B, 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 steps for preparing the test solution from the test sample are as follows: 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, characterized in that: The sample to be tested includes 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, characterized in that: The precipitant is an organic solvent containing 0.05-0.5v% 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 30-60v%.

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 content of methanol 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 were as follows: 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.

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