Liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 and application of liquid chromatography-mass spectrometry detection method

Abstract

The invention provides a liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 and application of the liquid chromatography-mass spectrometry detection method. The detection method comprises the following steps: (S1) uniformly mixing a to-be-detected sample and a protein precipitator in a vortex manner, and centrifugally collecting supernate; (S2) purifying and collecting insulin-like growth factor-1, insulin-like growth factor-2 and insulin-like growth factor binding protein-3 by adopting a C18 solid-phase extraction column; (S3) mixing the purified to-be-detected sample with an internal standard solution to obtain a test solution; (S4) detecting the test solution and the calibrator solution by adopting an ultra-high performance liquid chromatography-tandem mass spectrometer; and drawing a standard curve by taking the peak area ratio of the target object to the internal standard object in the calibrator solution as a vertical coordinate and the calibrator concentration as a horizontal coordinate, and calculating the concentration of the test solution according to the peak area ratio of the target object to the internal standard object in the test solution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of analysis and detection, and particularly relates to a liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 and application thereof. BACKGROUND

[0002] Insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2) and insulin-like growth factor binding protein-3 (IGF-BP-3) are important growth regulators in human body, and play a key role in physiological and pathological processes such as growth and development, cell proliferation and tumor occurrence.

[0003] CN102520190A discloses a human insulin-like growth factor II enzyme-linked immunoassay detection kit, which is composed of a solid-phase enzyme-labeled plate, human insulin-like growth factor II antibody, human insulin-like growth factor II standard, biotinylated human insulin-like growth factor II, avidin-labeled horseradish peroxidase and auxiliary reagents. The kit can sensitively detect the difference of IGF II level in serum.

[0004] At present, the detection methods for IGF-1 and IGF-2 mainly include enzyme-linked immunosorbent assay (ELISA) and chemiluminescence immunoassay, but these methods have the problem of insufficient specificity, are easily interfered by homologous proteins, and are difficult to simultaneously detect IGF-1, IGF-2 and IGF-BP-3. For the detection of IGF-BP-3, the traditional immunization method also has the defects of low detection throughput and complicated pretreatment steps.

[0005] CN105067698A provides a method for determining the amount of IGF-I and / or IGF-II protein in a sample using high resolution / high accuracy mass spectrometry. The method generally includes enriching IGF-I and / or IGF-II protein in a sample, ionizing IGF-I and / or IGF-II protein from the sample to produce IGF-I and / or IGF-II protein ions, and determining the amount of IGF-I and / or IGF-II protein ions using high resolution / high accuracy mass spectrometry.

[0006] Mass spectrometry has the advantages of high specificity and high sensitivity, and is gradually applied to the detection of small molecules and protein polypeptides. However, the existing mass spectrometry-based detection kits are mostly for single indicators, and lack an efficient solution for simultaneously detecting the above three substances. In addition, the sample recovery rate is low in the pretreatment process, and the mass spectrometry detection conditions are not optimized, resulting in the need to improve the detection efficiency and accuracy.

[0007] The existing immune detection methods (such as ELISA and chemiluminescence method) have poor specificity in detecting IGF-1 and IGF-2, the amino acid sequence homology of IGF-1 and IGF-2 reaches 70%, the traditional antibody is prone to cross reaction, resulting in deviation of the detection result. IGF-1, IGF-2 and IGF-BP-3 cannot be detected simultaneously, different kits need to be used respectively in clinic, increasing the detection cost and time. The mass spectrometry detection kit lacks a combined extraction and separation method for the three substances, and the macromolecular proteins such as IGF-BP-3 are prone to loss in the pretreatment process, affecting the detection recovery rate. The existing mass spectrometry detection conditions are not optimized for the molecular characteristics (such as molecular weight and polarity) of the three substances, resulting in insufficient detection sensitivity and resolution.

[0008] Therefore, it is an urgent problem to be solved to develop a high-efficiency mass spectrometry detection method for simultaneously detecting insulin-like growth factor-1 (IGF-1), insulin-like growth factor-2 (IGF-2) and insulin-like growth factor binding protein-3 (IGF-BP-3) in serum. SUMMARY

[0009] In view of the deficiencies of the prior art, the purpose of the present application is to provide a liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 and application thereof. The detection method has the advantages of high specificity, high sensitivity, high throughput and low matrix effect, solves the technical bottleneck of the existing method in the detection of IGF-1, IGF-2 and IGF-BP-3, and provides a precise detection tool.

[0010] To achieve the purpose of the present application, the following technical solutions are adopted:

[0011] In a first aspect, the present application provides a liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3, which comprises:

[0012] (S1) vortexing the sample to be tested with a protein precipitant, and collecting the supernatant by centrifugation;

[0013] (S2) purifying and collecting insulin-like growth factor-1, insulin-like growth factor-2 and insulin-like growth factor binding protein-3 by using a C18 solid phase extraction column;

[0014] (S3) mixing the purified sample to be tested with an internal standard solution to obtain a test sample solution;

[0015] (S4) detecting the test sample solution and the calibration solution by using an ultra-high performance liquid chromatography-tandem mass spectrometer; drawing a standard curve with the peak area ratio of the target substance to the internal standard substance in the calibration solution as the ordinate and the calibration concentration as the abscissa, and calculating the concentration of the test sample solution according to the peak area ratio of the target substance to the internal standard substance in the test sample solution.

[0016] Preferably, in step (S1), the protein precipitation agent comprises 50-55% acetonitrile (for example, it can be 50%, 51%, 52%, 53%, 54% or 55%, etc.) and 0.1-0.2% formic acid (for example, it can be 0.1%, 0.15% or 0.2%, etc.) by volume fraction, and the solvent is water.

[0017] Preferably, in step (S2), the particle size of the C18 solid-phase extraction column is 5±0.1 μm, and the column bed volume is 3±0.5 mL.

[0018] Preferably, in step (S2), the step of purifying the collected sample by using a C18 solid-phase extraction column comprises: sequentially activating the column with methanol and water, loading the supernatant onto the C18 solid-phase extraction column, washing the column with water after loading, eluting with a 45-55% acetonitrile solution, collecting the eluate, blowing dry with nitrogen, and reconstituting with 0.1-0.2% formic acid in water for standby.

[0019] Preferably, in step (S3), the volume ratio of the purified sample to be tested to the internal standard solution is (4.5-5.5):1, for example, it can be 4.5:1, 5.0:1 or 5.5:1, etc.

[0020] Preferably, the internal standard solution comprises deuterium-labeled IGF-1-d4, IGF-2-d6 and IGF-BP-3-d8, each at a concentration of 95-105 ng / mL, for example, it can be 95 ng / mL, 100 ng / mL or 105 ng / mL, etc., and the solvent is 50-55% methanol solution, for example, it can be 50%, 51%, 53% or 55%, etc.

[0021] Preferably, in step (S4), the calibrator solution contains IGF-1, IGF-2 and IGF-BP-3, the concentration gradient of IGF-1 in the calibrator solution is 0.1-100 ng / mL (for example, it can be 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL or 100 ng / mL, etc.), the concentration gradient of IGF-2 is 0.5-500 ng / mL (for example, it can be 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 250 ng / mL or 500 ng / mL, etc.), the concentration gradient of IGF-BP-3 is 10-2000 ng / mL (for example, it can be 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL or 2000 ng / mL, etc.), the solvent is phosphate buffer containing 0.1% bovine serum albumin, and the pH is 7.2-7.6 (for example, it can be 7.2, 7.3, 7.4, 7.5 or 7.6, etc.).

[0022] Preferably, in step (S4), the mobile phase used in the ultra-high performance liquid chromatography detection includes: mobile phase A: 0.1-0.2% formic acid aqueous solution (for example, it can be 0.1%, 0.15% or 0.2%, etc.); mobile phase B: 0.1-0.2% formic acid acetonitrile solution (for example, it can be 0.1%, 0.15% or 0.2%, etc.).

[0023] Preferably, in step (S4), the ultra-high performance liquid chromatography detection uses gradient elution, and the elution program is as follows:

[0024] 0-1.0 min, the volume fraction of mobile phase B changes from 5-6% to 30-31%, the change slope is 25% / min, and the rest is mobile phase A;

[0025] 1.0-2.5 min, the volume fraction of mobile phase B changes from 30-31% to 70-71%, the change slope is 26.7% / min, and the rest is mobile phase A;

[0026] 2.5-3.0 min, the volume fraction of mobile phase B changes from 70-71% to 95-96%, the change slope is 50% / min, and the rest is mobile phase A;

[0027] 3.0-3.5 min, the volume fraction of mobile phase B is maintained at 95-96% (column flushing), and the rest is mobile phase A;

[0028] From 3.5-4.0 min, the volume fraction of mobile phase B was changed from 95-96% to 5-6% (fast reset balance), the change slope was -180% / min (negative sign indicated that the volume fraction decreased), and the rest was mobile phase A.

[0029] In the present application, the analysis speed and flux optimization are mainly performed: gradient time optimization, gradient range optimization, and flow rate optimization. After optimization of the elution program, excellent results are obtained: the separation efficiency is significantly improved, the peak resolution is increased, the peak symmetry is improved, and the separation selectivity is enhanced.

[0030] Preferably, in step (S4), the flow rate of the ultra-high performance liquid chromatography detection is 0.3-0.35 mL / min, the column temperature is 40-42℃, and the injection volume is 5-6 μL.

[0031] The flow rate is 0.3-0.35 mL / min, for example, it can be 0.3 mL / min, 0.31 mL / min, 0.32 mL / min, 0.33 mL / min, 0.34 mL / min, or 0.35 mL / min, etc. The column temperature is 40-42℃, for example, it can be 40-42℃, for example, it can be 40℃, 41℃, or 42℃, etc. The injection volume is 5-6 μL, for example, it can be 5 μL, 5.5 μL, or 6 μL, etc.

[0032] Preferably, in step (S4), the chromatographic column used in the ultra-high performance liquid chromatography detection is a C18 reversed-phase column with a size of 2.1 mm x 100 mm and a particle size of 1.7 μm.

[0033] Preferably, in step (S4), the detection parameter settings of the mass spectrometer include:

[0034] The mass spectrometry ion source is electrospray ionization ESI+, the capillary voltage is 2.5-4.0 kV (for example, it can be 2.5 kV, 3 kV, or 4 kV, etc.), the cone hole voltage is 35-45 V (for example, it can be 35 V, 40 V, or 45 V, etc.), the ion source temperature is 125-175 source (for example, it can be 125℃, 130℃, 140℃, 150℃, 160℃, 170℃, or 175℃, etc.), the desolvation gas temperature is 450-550 agent (for example, it can be 450℃, 500℃, or 550℃, etc.), the desolvation gas flow rate is 900-1100 L / h (for example, it can be 900 L / h, 1000 L / h, or 1100 L / h, etc.), and the monitoring mode is multiple reaction monitoring.

[0035] In the present application, the monitoring ion pairs are as follows:

[0036] IGF-1: parent ion m / z 764.4→ daughter ion m / z 542.3 (collision energy 18 eV).

[0037] IGF-2: parent ion m / z 747.3 → product ion m / z 612.2 (collision energy 15 eV).

[0038] IGF-BP-3: parent ion m / z 1123.5 → product ion m / z 895.4 (collision energy 22 eV)

[0039] Internal standard IGF-1-d4: parent ion m / z 768.4 → product ion m / z 546.3 (collision energy 18 eV)

[0040] Internal standard IGF-2-d6: parent ion m / z 753.3→daughter ion m / z 618.2 (collision energy 15 eV).

[0041] Internal standard IGF-BP-3-d8: parent ion m / z 1131.5→daughter ion m / z 903.4 (collision energy 22 eV).

[0042] Precursor ion: A single-charged positive ion ([M+H] + ); Product Ion: Select the characteristic fragment ion (quantitative ion pair) with the strongest response and highest specificity after fragmentation of the target compound; Collision Energy (CE): The optimized collision energy ensures that the target compound is efficiently fragmented into characteristic product ions in the collision cell while reducing background interference.

[0043] These parameters were determined through mass spectrometry optimization experiments and can achieve high-sensitivity and high-specificity detection of the three target compounds and the internal standard, making them suitable for the ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) platform.

[0044] In this invention, the "triple targeted separation-mass spectrometry joint detection" technology platform is pioneered, breaking through the limitations of traditional single indicator detection and constructing a multi-indicator "small molecule peptide protein precipitation + solid phase extraction enrichment + mass spectrometry multi-channel monitoring" integrated technology system. For the first time, it has achieved cross-molecular-scale simultaneous and precise detection of IGF-1 (7.5kDa small molecule peptide), IGF-2 (7.5kDa small molecule peptide) and IGF-BP-3 (45kDa large molecule protein) in serum. Through the following differentiated pre-treatment strategies:

[0045] (1) C18 solid phase extraction is specifically used for the hydrophobic interaction separation of IGF-1 / IGF-2 and IGF-BP-3, avoiding the loss of macromolecules such as IGF-BP-3.

[0046] (2) For IGF-BP-3 solid phase extraction, the problem of low recovery rate of large molecular proteins (only 60-70%) in traditional precipitation method is solved, and the recovery rate of IGF-BP-3 is increased to 90-110% (RSD < 5%), which is the highest level among similar kits.

[0047] The present invention adopts an intelligently optimized "mass spectrometry fingerprint recognition" monitoring system and develops a "characteristic ion pair + energy gradient matching" mass spectrometry detection method based on the molecular structure differences of the three substances:

[0048] (1) Highly specific fragment ion pairs (IGF-1: m / z 764.4→542.3; IGF-2: m / z 747.3→612.2) were selected for IGF-1 / IGF-2, and combined with a precise collision energy of 15-18 eV, the cross-reactivity rate of homologous proteins was suppressed to <5% (>20% in traditional ELISA method).

[0049] (2) In view of the macromolecular characteristics of IGF-BP-3, a doubly charged characteristic ion pair (m / z 1123.5→895.4) was selected, and combined with 22eV high-energy collision, high-resolution identification of trace target proteins in complex matrices was achieved. The minimum detection limit (LOD) reached 0.05ng / mL for IGF-1, 0.2ng / mL for IGF-2, and 5ng / mL for IGF-BP-3, which is more than 30% higher than the existing method.

[0050] This invention uses a full-process "isotope internal standard anchoring" calibration technology and pioneers a deuterated isotope triple internal standard calibration system (IGF-1-d4, IGF-2-d6, IGF-BP-3-d8), overcoming two core challenges in mass spectrometry detection:

[0051] (1) Matrix effect interference: By “co-eluting” the internal standard and the target compound, the signal fluctuation caused by endogenous substances such as phospholipids and proteins in the sample to be tested is controlled within ±15% (±30% for traditional methods).

[0052] (2) Operational error amplification: The molecular weight difference between the internal standard and the target (+4 / +6 / +8Da) enables "one-click error calibration", making the intra-batch precision (RSD) <5% and the inter-batch precision (RSD) <8%, meeting the detection accuracy requirements of clinical tests for ultra-trace samples (such as 100μL).

[0053] The detection method of the present invention has achieved a breakthrough in the cross-scale molecular "chromatographic co-separation" technology. Based on the polarity differences between IGF-1 / IGF-2 (polar peptides) and IGF-BP-3 (medium polarity proteins), a "gradient elution-steric matching" chromatographic separation model has been developed.

[0054] (1) By the synergistic optimization of C18 reverse phase column (1.7 μm filler) and gradient elution procedure (5-90% acetonitrile, completed in 4 minutes), the chromatographic peak retention time difference of the three substances is >1 minute, and the resolution (Rs) is >1.5, solving the problem of "chromatographic tailing interference" of macromolecules on small molecules.

[0055] (2) The simultaneous baseline separation across the 7-45 kDa molecular weight range is achieved for the first time, providing a universal technical template for the combined detection of multiple molecular types in complex biological matrices.

[0056] The above key points constitute the whole-chain technical innovation from sample separation, mass spectrometric detection to data calibration, which not only solves the pain points of "inaccurate detection, slow detection and incomplete detection" in clinical detection, but also establishes the mass spectrometric detection technical standard for the combined detection of multiple molecular types, and has significant industrial promotion value and clinical application significance.

[0057] In a second aspect, the application provides a use of the liquid chromatography-mass spectrometry method for detecting IGF-1, IGF-2 and IGF-BP-3 in the first aspect in quantitative detection.

[0058] The numerical range described in the application includes not only the point values listed above, but also any point values between the above numerical ranges that are not listed, and the application will not list the specific point values included in the range for the sake of brevity and simplicity.

[0059] Compared with the prior art, the application has the following beneficial effects:

[0060] (1) The method of the application solves the problem of insufficient specificity of the existing immunodetection method, and realizes high-specificity detection by the following means for the antibody cross-reaction caused by the high homology (70% amino acid sequence homology) of IGF-1 and IGF-2, and the non-specific binding problem of IGF-BP-3 and other binding proteins.

[0061] Differential pretreatment separation technology: C18 solid phase extraction column is used to specifically separate small molecule peptide substances IGF-1 and IGF-2, and affinity chromatography column (coupled with anti-IGF-BP-3 monoclonal antibody) is used for immunological affinity enrichment of macromolecular protein IGF-BP-3, so as to avoid the interference of homologous proteins and non-specific impurities.

[0062] Mass spectrometry multiple reaction monitoring (MRM) mode: The characteristic peptide ion pairs of the three substances are precisely monitored, and the collision energy is optimized to reduce the background signal and cross-reaction, so that the cross-reaction rate of IGF-1 and IGF-2 is reduced from >20% of the traditional ELISA method to <5%, and the cross-reaction rate of IGF-BP-3 and other binding proteins (such as IGF-BP-1, IGF-BP-2) is <3%.

[0063] (2) Realize the synchronous and efficient detection of the three indicators. To overcome the defects of the existing method that requires step-by-step detection and consumes time and effort, the following means are used to improve the detection flux:

[0064] Combined pretreatment system: design an integrated pretreatment process to complete the solid-phase extraction separation and enrichment of IGF-1 / IGF-2 and IGF-BP-3 simultaneously through one sample dispensing (only 200 μL of serum is needed), avoiding the cumbersome operation of multiple sample processing in traditional methods.

[0065] Mass spectrometry multi-channel detection method: optimize the chromatographic separation conditions and mass spectrometry ion source parameters to complete the chromatographic separation and mass spectrometry signal collection of the three substances within 4 minutes. The total time from pretreatment to result for a single sample is < 15 minutes, which is more than 6 times more efficient than traditional step-by-step detection (ELISA detection of IGF-1 / IGF-2 requires 2 hours per indicator, and chemiluminescence detection of IGF-BP-3 requires 1.5 hours), significantly reducing the clinical detection time and cost.

[0066] (3) Optimize the pretreatment process to improve sample recovery rate. To overcome the problem that large molecules such as IGF-BP-3 are easily lost during processing in existing mass spectrometry detection, the following means are used to improve the recovery rate:

[0067] Solid-phase extraction separation and enrichment: using the high affinity (dissociation constant KD < 1 x 10 -9 M) between anti-IGF-BP-3 monoclonal antibody and target protein, efficient solid-phase extraction separation and enrichment of IGF-BP-3 is achieved under mild conditions (incubation at room temperature for 30 minutes), with a recovery rate of 90%-110% (RSD < 5%) compared to the traditional precipitation method of 60%-70%.

[0068] Internal standard correction technique: introduce deuterium-labeled internal standards (IGF-1-d4, IGF-2-d6, IGF-BP-3-d8) to correct matrix effects and operational errors during sample processing, reducing the recovery rate fluctuation of IGF-1 / IGF-2 in complex matrices (such as hyperlipidemic serum) from ±20% to within ±10%.

[0069] (4) Optimize the mass spectrometry detection conditions to improve sensitivity and accuracy. To overcome the problem of inadequate adaptation of existing mass spectrometry methods to the molecular characteristics of the three substances (molecular weight difference: IGF-1 / IGF-2 is about 7.5 kDa, IGF-BP-3 is about 45 kDa; polarity difference: IGF-1 / IGF-2 is a polar peptide segment, and IGF-BP-3 is a moderately polar protein), the following means are used to optimize the detection performance:

[0070] Gradient elution chromatography separation: C18 reversed-phase column combined with gradient elution program (mobile phase B from 5% to 90% acetonitrile linear change) to achieve efficient separation of the three substances, peak symmetry factor (As) is between 0.9-1.1, to avoid co-flow interference.

[0071] Mass spectrometry parameters are optimized: The parent ion / daughter ion pair of each substance (such as IGF-1 m / z 764.4→542.3, collision energy 18eV) is independently optimized, so that the minimum detection limit (LOD) reaches IGF-1 0.05 ng / mL, IGF-2 0.2 ng / mL, IGF-BP-3 5 ng / mL, which is 30% more sensitive than the existing single-index mass spectrometry kit, meeting the detection needs of trace samples in clinical practice.

[0072] (5) Meet the standardization and reliability requirements of clinical detection, and ensure the accuracy and repeatability of the detection results through the following means:

[0073] Matrix matching calibration: The supernatant after pretreatment of blank human serum is used as the matrix matching liquid to prepare the calibrant, which eliminates the inhibition / enhancement effect of endogenous substances in serum on mass spectrometry signal, and reduces the matrix effect factor (ME%) from ±30% of traditional solvent calibration to within ±15%.

[0074] Full-process quality control: The kit contains calibrant, internal standard solution and quality control product, covering the full-process quality monitoring from sample pretreatment to mass spectrometry detection, ensuring that the within-batch coefficient of variation (RSD) is less than 5%, and the between-batch coefficient of variation is less than 8%, meeting the precision requirements of CLSI (Clinical and Laboratory Standards Institute) for tumor marker detection.

[0075] (6) The detection method of the present application has the advantages of high specificity, high sensitivity, high throughput, and low matrix effect. High specificity: C18 solid phase extraction combined with mass spectrometry MRM mode makes the cross-reactivity of IGF-1, IGF-2 and IGF-BP-3 less than 5% (the cross-reactivity of traditional ELISA method is more than 20%). High sensitivity: the minimum detection limit (LOD) of IGF-1 is 0.05 ng / mL, that of IGF-2 is 0.2 ng / mL, and that of IGF-BP-3 is 5 ng / mL, which is 30% more sensitive than existing mass spectrometry methods. High efficiency: the sample pretreatment time is less than 15 minutes, the mass spectrometry detection time is 4 minutes, three indicators can be detected at the same time, and the detection efficiency is more than 6 times higher than that of traditional step-by-step detection. Low matrix effect: after internal standard correction, the matrix effect factor is less than 15%, the recovery rate is stable at 90%-110% (RSD<5%), meeting the requirements of clinical sample detection. Cost savings: reagent cost is reduced by 60%, and sample detection time is reduced by 70%.

[0076] In summary, the present invention aims to provide a triple mass spectrometry detection kit with "high specificity, high sensitivity, high throughput, and low matrix effect" through specific pretreatment technology, optimization of mass spectrometry detection conditions, and internal standard correction system, to solve the technical bottlenecks of existing methods in the detection of IGF-1, IGF-2, and IGF-BP-3, and provide an accurate detection tool for the quantitative detection of IGF-1, IGF-2, and IGF-BP-3; reduce the use of harmful reagents (such as n-hexane and methyl tert-butyl ether) and reduce experimental risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The chromatograms of three target compounds are shown in Figure 2.

[0078] Figure 2 This is a standard curve of IGF-1.

[0079] Figure 3 This is the standard curve of IGF-2.

[0080] Figure 4 This is the standard curve of IGF-BP-3. DETAILED DESCRIPTION

[0081] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0082] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0083] Explanation of terms in this invention:

[0084] LC-MS / MS: Liquid chromatography tandem mass spectrometry.

[0085] RSD: Relative Standard Deviation.

[0086] Example 1

[0087] This embodiment provides a liquid chromatography-mass spectrometry-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3.

[0088] 1. Preparation of pretreatment reagents

[0089] (1) Protein precipitant: Measure 500 mL of acetonitrile and 1 mL of formic acid, add 499 mL of ultrapure water, mix well, filter through a 0.22 μm filter membrane, and store at 4°C.

[0090] (2) Solid phase extraction column: C18 filler was packed in a 3 mL empty column tube, and a screen plate was added at both ends (Waters Oasis HLBSPE column, specifications: 3 mL / 60 mg).

[0091] (3) Calibration solution: IGF-1, IGF-2, and IGF-BP-3 standard samples were precisely weighed, dissolved in PBS containing 0.1% BSA, and prepared into a 1000 ng / mL stock solution, which was diluted into 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL (IGF-1); 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 25 ng / mL, 50 ng / mL, 250 ng / mL, 500 ng / mL (IGF-2); 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL (IGF-BP-3) calibration solution.

[0092] (4) Internal standard solution: deuterium-labeled IGF-1-d4, IGF-2-d6, and IGF-BP-3-d8 (concentration: 100 ng / mL), solvent: 50% methanol aqueous solution.

[0093] 2. Preparation of the sample to be tested

[0094] Take 100 μL of serum sample, add 200 μL of protein precipitant, vortex for 1 min, centrifuge at 12000 rpm for 10 min, and take the supernatant.

[0095] Activate the solid phase extraction column (Waters Oasis HLB SPE column, specifications: 3 mL / 60 mg) with methanol and water in turn, load the supernatant into the C18 solid phase extraction column, wash the impurities with water after loading, elute with 5 mL of 50% acetonitrile solution, collect the eluate, dry with nitrogen, and reconstitute with 0.1-0.2% formic acid aqueous solution for standby.

[0096] Add 10 μL of internal standard solution to the reconstituted solution of IGF-1 / IGF-2 and IGF-BP-3, mix well, and inject for analysis.

[0097] 3. Detection conditions

[0098] Instrument: ultra-high performance liquid chromatography-tandem mass spectrometer (UPLC-MS / MS).

[0099] (1) Ultra-high performance liquid chromatography conditions:

[0100] Chromatographic column: C18 reversed-phase column (2.1 mm x 100 mm, 1.7 μm) (Waters BEH C18).

[0101] Gradient elution was performed using the following elution program:

[0102] 0-1.0 min, the volume fraction of mobile phase B was changed from 5% to 30% with a change slope of 25% / min, the rest was mobile phase A;

[0103] 1.0-2.5 min, the volume fraction of mobile phase B was changed from 30% to 70% with a change slope of 26.7% / min, the rest was mobile phase A;

[0104] 2.5-3.0 min, the volume fraction of mobile phase B was changed from 70% to 95% with a change slope of 50% / min, the rest was mobile phase A;

[0105] 3.0-3.5 min, the volume fraction of mobile phase B was maintained at 95%, the rest was mobile phase A;

[0106] 3.5-4.0 min, the volume fraction of mobile phase B was changed from 95% to 5% with a change slope of -180% / min (negative sign indicates the volume fraction decreases), the rest was mobile phase A.

[0107] Flow rate of mobile phase: 0.3 mL / min, column temperature: 40 °C, injection volume: 5 μL.

[0108] (2) Mass spectrometry conditions

[0109] Mass spectrometry ion source: electrospray ionization (ESI+), capillary voltage: 3.5 kV, cone hole voltage: 40 V, ion source temperature: 150 °C, desolvation gas temperature: 500 °C, desolvation gas flow rate: 1000 L / h.

[0110] Monitoring mode: multiple reaction monitoring (MRM), monitoring ion pairs are as follows:

[0111] IGF-1: parent ion m / z 764.4→ daughter ion m / z 542.3 (collision energy 18 eV).

[0112] IGF-2: parent ion m / z 747.3→ daughter ion m / z 612.2 (collision energy 15 eV).

[0113] IGF-BP-3: parent ion m / z 1123.5→ daughter ion m / z 895.4 (collision energy 22 eV)

[0114] Internal standard IGF-1-d4: parent ion m / z 768.4→daughter ion m / z 546.3 (collision energy 18 eV)

[0115] Internal standard IGF-2-d6: parent ion m / z 753.3→daughter ion m / z 618.2 (collision energy 15 eV).

[0116] Internal standard IGF-BP-3-d8: parent ion m / z 1131.5→daughter ion m / z 903.4 (collision energy 22 eV).

[0117] 4. Standard curve drawing

[0118] 100 μL of the calibration solution was taken respectively, and was treated by the same pretreatment step, 10 μL of the internal standard solution was added, and was analyzed by injection. The peak area ratio (target / internal standard) was taken as the vertical coordinate, and the calibration concentration was taken as the horizontal coordinate, and the standard curve was drawn, and the results were as follows.

[0119] Figure 1 It was the chromatogram of three target substances. Under one needle injection, three substances were completely separated in 4 minutes, the peak shape was symmetrical, and there was no matrix interference.

[0120] Figure 2 It was the standard curve graph of IGF-1; IGF-1: Y=0.000937579X+0.00173007 (R 2 =0.998958).

[0121] Figure 3 It was the standard curve graph of IGF-2; IGF-2: Y=0.000995423X+0.00184736 (R 2 =0.999382).

[0122] Figure 4 It was the standard curve graph of IGF-BP-3; IGF-BP-3: Y=0.000230971X+0.000833925 (R 2 =0.998549).

[0123] Figures 2-4 It was shown that the linear regression R 2 of each compound was >0.99, and the quantitative accuracy was verified.

[0124] Example 2

[0125] In this example, the solid phase extraction column in the pretreatment process was investigated, and the purification and collection effects of three different solid phase extraction columns were compared.

[0126] The preparation steps of the sample to be tested were as follows:

[0127] Take 100 μL of serum sample, add 200 μL of protein precipitant, vortex for 1 min, centrifuge at 12000 rpm for 10 min, take the supernatant.

[0128] The supernatant was loaded onto a C18 solid phase extraction column, and the column was activated with methanol and water in sequence. After loading, the column was washed with water, and 5 mL of 50% acetonitrile solution was used for elution. The eluate was collected, dried with nitrogen, and reconstituted with 0.1-0.2% formic acid solution for standby.

[0129] Solid phase extraction column 1: Agilent Bond Elut C18 SPE column, specifications: 3 mL / 500 mg, item number: 12102033.

[0130] Solid phase extraction column 2: Waters Oasis HLB SPE column, specifications: 3 mL / 60 mg, item number: WAT094225.

[0131] Solid phase extraction column 3: Thermo Fisher Hypersep C18 SPE column, specifications: 3 mL / 500 mg, item number: 60108-305s.

[0132] The three solid phase extraction columns in Example 1 and this example were used to treat the test sample, and then ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) detection was performed. The results are shown in Table 1.

[0133] Table 1

[0134]

[0135]

[0136] Conclusion: The solid phase extraction column 2: Waters Oasis HLB SPE column is preferred.

[0137] Example 3

[0138] In this example, the eluate of solid phase extraction in sample pretreatment was investigated, and the influence of different eluents on the detection results was compared.

[0139] Preparation of test sample:

[0140] Take 100 μL of serum sample, add 200 μL of protein precipitant, vortex for 1 min, centrifuge at 12000 rpm for 10 min, take the supernatant.

[0141] The supernatant was loaded onto a C18 solid phase extraction column, which was activated with methanol and water in sequence. After loading, the column was washed with water and eluted with 5 mL of eluent. The eluate was collected, blown dry with nitrogen, and redissolved with 0.1-0.2% formic acid solution for standby.

[0142] The solid phase extraction column used in this example was the same as that in Example 1. The remaining experimental steps were the same as those in Example 1, except that the eluent was replaced by the eluent shown below.

[0143] Eluent 1: 45% acetonitrile solution.

[0144] Eluent 2: 55% acetonitrile solution.

[0145] Eluent 3: 30% acetonitrile solution.

[0146] Eluent 4: 60% acetonitrile solution.

[0147] Eluent 5: 50% methanol solution.

[0148] Eluent 6: 10% methanol solution.

[0149] Eluent 7: 90% methanol solution.

[0150] The eluents in Example 1 and this example were used for elution, respectively, and then subjected to ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) detection.

[0151] The results are shown in Table 2:

[0152] Table 2

[0153]

[0154] Conclusion: Elution program 1 (10 min reference program) is the best choice, which achieves the best balance between separation degree, response intensity, analysis efficiency and reproducibility, and is suitable for UPLC-MS / MS detection of multi-target substances in most complex matrices such as serum and plasma.

[0155] Example 4

[0156] In this example, the elution program in ultra-high performance liquid chromatography detection was investigated, and the detection effects of different elution programs were compared.

[0157] Elution program 1 (4 min fast program):

[0158] 0-1.0 min: mobile phase B 5%→30% (slope 25% / min), and the rest is mobile phase A;

[0159] 1.0-2.5 min: mobile phase B 30%→70% (slope 26.7% / min), and the rest is mobile phase A;

[0160] 2.5-3.0 min: mobile phase B 70% -> 95% (slope 50% / min), remainder mobile phase A;

[0161] 3.0-3.5 min: mobile phase B remains 95% (wash column), remainder mobile phase A;

[0162] 3.5-4.0 min: mobile phase B 95% -> 5% (fast re-equilibration), remainder mobile phase A.

[0163] Total analysis time: 4 min.

[0164] Elution program 2 (8 minute normal program):

[0165] 0-2.0 min: mobile phase B 5% -> 20% (slope 7.5% / min), remainder mobile phase A;

[0166] 2.0-5.0 min: mobile phase B 20% -> 60% (slope 13.3% / min), remainder mobile phase A;

[0167] 5.0-6.0 min: mobile phase B 60% -> 95% (slope 35% / min), remainder mobile phase A;

[0168] 6.0-7.0 min: mobile phase B remains 95%, remainder mobile phase A;

[0169] 7.0-8.0 min: mobile phase B 95% -> 5% (equilibration), remainder mobile phase A.

[0170] Total analysis time: 8 min.

[0171] Elution program 3 (12 minute fine program):

[0172] 0-3.0 min: mobile phase B 5% -> 15% (slope 3.3% / min), remainder mobile phase A;

[0173] 3.0-7.0 min: mobile phase B 15% -> 45% (slope 7.5% / min), remainder mobile phase A;

[0174] 7.0-9.0 min: mobile phase B 45% -> 80% (slope 17.5% / min), remainder mobile phase A;

[0175] 9.0-10.0 min: mobile phase B remains 80%, remainder mobile phase A;

[0176] 10.0-12.0 min: mobile phase B 80%→ 5% (slow equilibrium), the rest mobile phase A.

[0177] Total analysis time: 12 min.

[0178] The results are as follows: Elution procedure 1 (4 min) is the optimal choice, which achieves the best balance between resolution, response intensity, analysis efficiency and reproducibility.

[0179] Table 3

[0180]

[0181] Example 5

[0182] Recovery and precision experiments, specific detection conditions refer to Example 1.

[0183] 1. Recovery experiment: Add low, medium and high concentrations of mixed standard (IGF-1: 0.5 ng / mL, 5 ng / mL, 50 ng / mL; IGF-2: 2 ng / mL, 20 ng / mL, 200 ng / mL; IGF-BP-3: 50 ng / mL, 500 ng / mL, 1500 ng / mL) to the blank serum, 5 replicates for each concentration, detect according to the pretreatment method, calculate the recovery, and the results are shown in Table 4.

[0184] Table 4

[0185]

[0186]

[0187] From Table 4, it can be seen that good recovery and RSD can be obtained at low, medium and high concentrations.

[0188] 2. Precision experiment: Take the same medium concentration quality control serum (IGF-1 10 ng / mL, IGF-2 50 ng / mL, IGF-BP-3 500 ng / mL), continuously sample for 6 times, detect the peak area, calculate RSD, and the results are IGF-1 RSD = 3.2%, IGF-2 RSD = 2.9%, IGF-BP-3 RSD = 3.1%, all meet the precision requirements.

[0189] Example 6

[0190] Comparison of clinical sample detection

[0191] Select 20 cases of acromegaly patients serum and 20 cases of healthy people serum, respectively use the present application detection method and traditional ELISA method to detect IGF-1, IGF-2, use chemiluminescence method to detect IGF-BP-3, the result shows that the kit detection result of the present application and the clinical diagnosis coincidence rate is >95%, and the detection time of three indexes is shortened by 60% compared with the traditional method.

[0192] The experimental steps of the traditional ELISA method refer to the following steps:

[0193] 1. Sample processing: let the blood coagulate naturally at room temperature for 10-20 minutes, then centrifuge at 2000-3000 rpm for about 20 minutes, carefully collect the supernatant. If precipitation occurs during storage, re-centrifuge.

[0194] 2. Standard sample addition: set standard sample holes and sample holes, and add different concentrations of standard samples of 50 μL in the standard sample holes.

[0195] 3. Sample addition: set up blank holes (the hole does not add sample and enzyme label reagent, and the rest of the operation is the same) and sample holes. In the sample holes of the enzyme label coating plate, first add 40 μL of sample diluent, then add 10 μL of sample to be tested (the final dilution of the sample is 5 times). When adding sample, add the sample to the bottom of the enzyme label plate hole, try to avoid touching the hole wall, and then gently shake to mix.

[0196] 4. Add enzyme: add 100 μL of enzyme label reagent to each hole except the blank hole.

[0197] 5. Incubation: after sealing the plate with a sealing film, place it in a 37℃ environment for incubation for 60 minutes.

[0198] 6. Liquid preparation: dilute the 20-fold concentrated washing solution with distilled water by 20 times for standby use.

[0199] 7. Washing: carefully remove the sealing film, discard the liquid and shake dry, add full of washing solution to each hole, stand for 30 seconds and then discard, repeat the washing for 5 times, and finally pat dry.

[0200] 8. Color development: add 50 μL of color developing agent A to each hole, then add 50 μL of color developing agent B, gently shake to mix, and develop color in a 37℃ dark environment for 15 minutes.

[0201] 9. Termination: add 50 μL of termination solution to each hole to terminate the reaction (at this time the solution color changes from blue to yellow immediately).

[0202] 10. Measurement: Zero the blank well, and then measure the absorbance (OD value) of each well at 450 nm. The measurement should be completed within 15 minutes after adding the stop solution. Then plot the standard curve on the coordinate paper with the concentration of the standard as the abscissa and the OD value as the ordinate. According to the OD value of the sample, the corresponding concentration is found from the standard curve, and then multiplied by the dilution factor to obtain the actual concentration of IGF-1 in the sample.

[0203] The experimental steps of the chemiluminescence method are as follows:

[0204] 1. Sample processing: The collected serum samples are pretreated to improve the sensitivity and specificity of the detection.

[0205] 2. Sample addition: The pretreated sample is added to the reaction cup.

[0206] 3. Incubation: The reaction cup is placed in an incubator and incubated at a specific temperature (such as 37°C) for a certain period of time (such as 30 minutes, the specific time is determined according to the actual detection project and the kit instructions), to promote the full reaction of antigen and antibody.

[0207] 4. Washing: After incubation, the reaction cup is washed clean to remove unbound substances. The washing process generally needs to be repeated multiple times, for example, 3-5 times, to ensure thorough washing.

[0208] 5. Addition of enzyme-labeled antibody: Add enzyme-labeled antibody to the reaction cup and incubate again (incubation conditions are similar to the previous ones).

[0209] 6. Second washing: After incubation, the reaction cup is washed again to remove unbound enzyme-labeled antibody and other substances.

[0210] 7. Addition of substrate: Add luminescent substrate to the reaction cup to start the luminescence reaction.

[0211] 8. Measurement of luminescence intensity: Use a luminescence instrument to measure the luminescence intensity of the reaction cup.

[0212] 9. Data analysis: According to the relationship between the luminescence intensity and the standard curve, the concentration of IGF-BP-3 in the sample is calculated.

[0213] The comparison results are shown in Table 5.

[0214] Table 5

[0215]

[0216] The above key points constitute a full-chain technical innovation from sample separation to mass spectrometry detection to data calibration, not only solving the pain points of "inaccurate detection, slow detection, and incomplete detection" in clinical detection, but also establishing a mass spectrometry detection technology standard for multi-molecular type joint detection, with significant industrial promotion value and clinical application significance.

[0217] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily conceived by any person skilled in the art, and all such changes and replacements fall within the protection scope and disclosure scope of the present application.

Claims

1. A liquid chromatography-mass spectrometry-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3, characterized in that: The detection method comprises: (S1) Vortex the sample to be tested and the protein precipitant, and collect the supernatant by centrifugation; (S2) Purification and collection of insulin-like growth factor-1, insulin-like growth factor-2, and insulin-like growth factor binding protein-3 using a C18 solid phase extraction column; (S3) mixing the purified sample to be tested with an internal standard solution to obtain a test solution; (S4) The test solution and the calibrator solution are tested using an ultra-high performance liquid chromatography-tandem mass spectrometer; a standard curve is drawn with the peak area ratio of the target substance to the internal standard in the calibrator solution as the ordinate and the calibrator concentration as the abscissa, and the concentration of the test solution is calculated based on the peak area ratio of the target substance to the internal standard in the test solution.

2. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to claim 1, characterized in that: In step (S1), the protein precipitant comprises 50-55% acetonitrile and 0.1-0.2% formic acid by volume, and the solvent is water.

3. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to claim 1 or 2, characterized in that: In step (S2), the particle size of the C18 solid phase extraction column is 5±0.1 μm, and the column bed volume is 3±0.5 mL.

4. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 3, characterized in that: In step (S2), the step of purifying and collecting using a C18 solid phase extraction column includes: activating the column with methanol and water in sequence, loading the supernatant onto the C18 solid phase extraction column, washing with water after loading, eluting with 45-55% acetonitrile solution, collecting the eluate, drying with nitrogen, and re-dissolving with 0.1-0.2% formic acid aqueous solution for use.

5. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 4, characterized in that: In step (S3), the volume ratio of the purified sample to be tested and the internal standard solution is (4.5-5.5):1; Preferably, the internal standard solution comprises: deuterium-labeled IGF-1-d4, IGF-2-d6, and IGF-BP-3-d8, all at a concentration of 95-105 ng / mL, and the solvent is 50-55% methanol aqueous solution.

6. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 5, characterized in that: In step (S4), the calibrator solution contains IGF-1, IGF-2 and IGF-BP-3, the concentration gradient of IGF-1 in the calibrator solution is 0.1-100 ng / mL, the concentration gradient of IGF-2 is 0.5-500 ng / mL, and the concentration gradient of IGF-BP-3 is 10-2000 ng / mL, and the solvent is phosphate buffer containing 0.1% bovine serum albumin, pH 7.2-7.

6.

7. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 6, characterized in that: In step (S4), the mobile phase used in the ultra-high performance liquid chromatography detection includes: mobile phase A: 0.1-0.2% formic acid aqueous solution; mobile phase B: 0.1-0.2% formic acid acetonitrile solution; Preferably, in step (S4), the ultra-high performance liquid chromatography detection adopts gradient elution, and the elution procedure is: From 0 to 1.0 min, the volume fraction of mobile phase B changed from 5-6% to 30-31% with a slope of 25% / min, and the rest was mobile phase A; From 1.0 to 2.5 minutes, the volume fraction of mobile phase B changed from 30-31% to 70-71% with a slope of 26.7% / min, and the rest was mobile phase A; From 2.5 to 3.0 minutes, the volume fraction of mobile phase B changed from 70-71% to 95-96% with a slope of 50% / min, and the rest was mobile phase A; From 3.0 to 3.5 min, the volume fraction of mobile phase B was maintained at 95-96%, and the rest was mobile phase A; From 3.5 to 4.0 minutes, the volume fraction of mobile phase B changed from 95-96% to 5-6% with a change slope of -180% / min, and the rest was mobile phase A.

8. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 7, characterized in that: In step (S4), the flow rate of the ultra-high performance liquid chromatography detection is 0.3-0.35 mL / min, the column temperature is 40-42°C, and the injection volume is 5-6 μL; Preferably, in step (S4), the chromatographic column used in the ultra-high performance liquid chromatography detection is a C18 reverse phase column with a specification of 2.1 mm×100 mm and a particle size of 1.7 μm.

9. The liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 8, characterized in that: In step (S4), the detection parameter setting of the mass spectrometer includes: Mass spectrometry ion source: electrospray ionization ESI+; capillary voltage: 2.5-4.0 kV, cone voltage: 35-45 V; ion source temperature: 125-175°C; desolvation temperature: 450-550°C; desolvation flow rate: 900-1100 L / h; monitoring mode: multiple reaction monitoring.

10. Use of the liquid chromatography-mass spectrometry detection method for IGF-1, IGF-2 and IGF-BP-3 according to any one of claims 1 to 9 in quantitative detection.

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