A method for detecting phytosterol content in dried blood spots by liquid chromatography-mass spectrometry

Abstract

The application provides a liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood spots, and belongs to the technical field of detection.The application simplifies a sampling process, uses a disposable blood taking needle and a blood taking card for sampling, replaces traditional serum or plasma samples with obtained dried blood spots, punches a sampling area of the dried blood spot sample through a puncher with a diameter of 3.2 mm, takes three dried blood spot samples, extracts seven kinds of sterol substances contained in the dried blood spot samples through n-hexane, adopts liquid chromatography-mass spectrometry, carries out detection and quantification through chromatographic separation and a mass spectrometry detector, realizes rapid and accurate determination of cholesterols, beta-sitosterols, daidzein, campesterol, dihydrocholesterol, dehydrocholesterol and 7-alkenyl cholestanol, and the method is simple in operation, accurate in quantification, and has a recovery rate of 92.4%-96.7%.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and in particular relates to a liquid chromatography-mass spectrometry method for detecting the content of phytosterols in dried blood smears. Background Technology

[0002] Phytosterols (PS) are naturally occurring plant-derived compounds widely distributed in plants. The most common phytosterols in nature are sitosterol, campesterol, and stigmasterol. In healthy individuals, phytosterols can lower serum cholesterol, improve benign prostatic hyperplasia, improve gestational diabetes, reduce the risk of cancer, and regulate immunity. However, in patients with phytosterolemia, a disorder of phytosterol metabolism, excessive accumulation of phytosterols in the blood and tissues can lead to increased risks of atherosclerosis, early-onset coronary heart disease, hemolytic anemia, and thrombocytopenia. Therefore, it is necessary to limit the dietary intake of phytosterols in patients with phytosterolemia as early as possible to avoid irreversible damage to the body.

[0003] Phytosterols and cholesterol have highly similar structures, both based on cyclopentanoperhydrophenanthrene and containing hydroxyl groups. 7-Encholanol is an isomer of cholesterol, with the same molecular weight and similar retention times. Conventional detection methods struggle to achieve baseline separation between the two, and they are easily affected by high concentrations of cholesterol in the blood, impacting quantitative accuracy. Current methods typically require collecting large volumes of plasma or serum samples, which poses significant challenges and potential harm to certain populations, such as infants. Existing methods for detecting phytosterols in serum / plasma based on liquid chromatography-tandem mass spectrometry (LC-MS / MS), while optimizing chromatographic separation and pretreatment processes, still rely on liquid samples from venous blood collection. They are unsuitable for the small, minimally invasive, and easily transportable sampling method of dried blood smears, and lack specific extraction and purification protocols to address issues such as protein binding and impurity interference in the dried blood smear matrix. Therefore, existing methods are insufficient to meet the needs of infants and other special populations. Compared to blood sampling, dried blood smear sampling offers several advantages, including minimally invasive sampling (requiring only fingertip sampling, eliminating the need for venipuncture), convenient storage / transport (allowing for short-term preservation at room temperature after drying, without the need for cold chain transportation), and sample stability (inhibiting protease and bacterial growth in the dried state, extending the preservation time of substances within the sample). These advantages make it more suitable for testing infants and young children, and also more beneficial for large-scale screening of high-risk groups for phytosterolemia and long-term monitoring of individuals with phytosterolemia. Currently, clinical applications of dried blood smear sampling include blood glucose testing, insulin testing, and C-reactive protein testing. However, methods based on LC-MS / MS (liquid chromatography-tandem mass spectrometry) that are adapted to dried blood smear matrices and can simultaneously and accurately separate and detect multiple phytosterols such as cholesterol and 7-encholanol remain undeveloped. Existing serum testing technologies cannot be directly transferred to the detection of dried blood smear samples. Summary of the Invention

[0004] Since current detection methods cannot adequately address the testing needs of special populations such as infants and young children, this invention proposes a liquid chromatography-mass spectrometry (LC-MS) method for detecting phytosterol content in dried blood smears. This invention can simultaneously detect cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-lactosterol, aiming to achieve accurate qualitative and quantitative detection of these seven sterols in dried blood smears, thus meeting the testing needs of special populations such as infants and young children.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a liquid chromatography-mass spectrometry (LC-MS) method for detecting phytosterol content in dried blood smears, comprising the steps of sample pretreatment, LC-MS detection, and calculation, wherein: The sample pretreatment includes taking dried blood smear samples, obtaining dried blood spots by punching holes, adding internal standard and extraction solvent for extraction, separating and taking the supernatant, concentrating and redissolving to obtain the test solution; The liquid chromatography-tandem mass spectrometry detection includes detecting the test solution using liquid chromatography-tandem mass spectrometry to obtain detection results. In the liquid chromatography detection conditions, the chromatographic column is a PFP chromatographic column, the mobile phase includes an aqueous phase and an organic phase, and isocratic elution is used. The calculations were performed based on the detection results of liquid chromatography-tandem mass spectrometry to determine the content of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in the dried blood smear.

[0006] The method of this invention relies on the pretreatment and purification of dried blood smear samples to achieve the extraction and enrichment of target substances, combined with isocratic liquid chromatography separation using a PFP column to achieve effective peak separation of seven sterols, and then completes the qualitative and quantitative analysis of the target substances through atmospheric pressure chemical ionization-tandem mass spectrometry in multiple reaction monitoring mode. Finally, the precise content of seven sterols, including cholesterol and β-sitosterol, in dried blood smears is calculated using the internal standard method. Overall, the method utilizes the high resolution, high sensitivity, and high specificity of liquid chromatography-mass spectrometry to achieve the accurate detection of trace sterols in the dried blood smear matrix.

[0007] Furthermore, in the sample pretreatment step, the dried blood smear sample is collected by fingertip blood collection, the collected blood is dropped onto filter paper, and then air-dried to obtain a dried blood smear sample.

[0008] Furthermore, in the sample pretreatment step, the diameter of the holes is 3.2 mm, and the number of holes is 3.

[0009] Furthermore, in the sample pretreatment step, the extraction solvent is n-hexane, and KOH-ethanol solution is added before extraction to carry out a hydrolysis reaction.

[0010] Furthermore, the sample pretreatment step further includes a grinding process by adding steel balls during extraction.

[0011] Furthermore, in the detection conditions of the liquid chromatography, the organic phase is methanol, and the isocratic elution program is 0-32 min.

[0012] Furthermore, in the liquid chromatography-tandem mass spectrometry detection step, the mass spectrometry conditions include: ionization mode of atmospheric pressure chemical ionization source and scanning mode of multiple reaction monitoring mode.

[0013] Furthermore, in the liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection step, the tandem mass spectrometry detection parameters for cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol are shown in Table 1: Table 1 Note: Marked with " "" indicates a quantitative ion.

[0014] Furthermore, in the sample pretreatment step, the internal standard is stigmasterol-D6, and its mass spectrometry parameters include: a precursor ion of 401 m / z and daughter ions of 83 m / z. m / z, 95 m / z and 303 m / z.

[0015] Furthermore, in the calculation step, the internal standard method is used for quantification, and a standard curve is plotted with the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard as the ordinate and the concentration as the abscissa.

[0016] More specifically, the present invention provides a liquid chromatography-mass spectrometry method for detecting the content of phytosterols in dried blood smears, comprising the following steps: 1. Sample preparation (1) Blood collection personnel should wash their hands and wear gloves; (2) Massage or apply heat to the subject's left ring finger, and disinfect the skin with 75% alcohol. After the skin is dry, collect blood. (3) Use a disposable blood collection needle to prick the inside of the tip of the left ring finger (if there is a wound or other situation that makes it impossible to collect a sample, other fingers can be selected), wipe away the first drop of blood with a dry cotton ball, and collect the second drop of blood; (4) Place the blood collection area of ​​the filter paper in contact with the blood drop, but do not touch the skin. Let the blood naturally seep into the back of the filter paper. Do not smear. Only drop the blood once in each blood collection area and collect at least three blood spots. (5) Place the blood smear in clean air, avoid direct sunlight, and air dry naturally for 3-4 hours until it turns dark brown to obtain a dried blood smear sample. Store it in a sealed bag. The quality requirements followed in the sample preparation process are as follows: One needle per person; A qualified dried blood spot on filter paper should have the following characteristics: each blood spot should be larger than 8 mm in diameter; there should be no overlap between blood spots; blood droplets should naturally penetrate the filter paper, and the blood spots should be consistent on both sides; the blood spots should be uncontaminated.

[0017] 2. Sample pretreatment (1) Punch three holes in the sampling area of ​​the dried blood smear sample using a 3.2 mm diameter punch and place it in a 2.0 mL EP tube; (2) Add two steel balls, add 200 μL of KOH-ethanol solution, and add 20 μL of internal standard stigmasterol-D6; (3) Grind for 3 min, vortex mix, sonicate in water bath, and react in a 70℃ water bath; (4) After taking it out, add ultrapure water and n-hexane, vortex to mix, centrifuge and take the supernatant; (5) Add n-hexane to the original EP tube again, vortex to mix, centrifuge and take the supernatant, and combine the two supernatants; (6) Freeze-dry, add methanol to reconstitute, and vortex mix. (7) Place the reconstituted solution in the inner liner tube to obtain the test solution, which is used for the detection of six non-cholesterol sterols on the instrument; (8) After diluting the reconstituted solution by 1000 times stepwise, place it in the inner liner tube for use in cholesterol detection.

[0018] 3. Preparation of standard products (1) Mixed standard stock solution: Weigh 10.0 mg (accurate to 0.0001 g) of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol and 7-enylcholesterol standards respectively, dissolve them in methanol and make up to 50 mL, store at 4 °C protected from light, shelf life 12 months, stock solution concentration is 200 μg / mL; (2) Mixed standard intermediate solution: Take 0.5 mL of each standard stock solution, dilute to 50 mL with methanol, mix well, and prepare immediately before use. The concentration is 2 μg / mL. (3) Mixed standard series working solutions: Pipette 0.005 mL, 0.01 mL, 0.025 mL, 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, and dilute to volume with methanol to obtain a series of standard working solutions with concentrations of 1.0 ng / mL, 2 ng / mL, 5 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 50.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, 500.0 ng / mL, and 1000.0 ng / mL, respectively. Prepare the solutions immediately before use.

[0019] 4. Liquid Chromatography Conditions Column: Phenomenex Kinetex PFP (4.6×250mm, 5μm); Mobile phase A: Primary water; Mobile phase B: Methanol; Elution program: isocratic elution, 0-32 min, 90% mobile phase B; Flow rate: 0.5 mL / min.

[0020] 5. Mass spectrometry conditions Ionization mode: Atmospheric pressure chemical ionization source (APCI); Scanning method: Multiple response monitoring (MRM); Evaporation temperature: 450℃; Ion transport capillary temperature: 350℃; The quantitative and qualitative ions, collision energies, and lens voltage parameters of each compound are shown in Table 2.

[0021] Table 2 Note: Marked with " "" indicates a quantitative ion.

[0022] 6. Quantitative Calculation After data acquisition, a standard curve was plotted with the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard as the ordinate and the concentration as the abscissa. Quantification was performed using the internal standard method. By substituting the peak areas of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in the sample into the standard curve, the precise content of each analyte in the sample could be calculated.

[0023] The patent with publication number CN114942292A uses liquid serum / plasma as the detection target, employing a C18 chromatographic column, acetonitrile-ethanol mobile phase, and a strong column washing procedure. Its aim is to address the interference of high cholesterol concentrations in liquid blood on the quantitative analysis of 7-enylcholine and to achieve baseline stability for multi-needle injections. However, its pretreatment requires complex steps such as ultrasonic saponification and nitrogen blowing reconstitution, relying on venous blood collection and cold chain transportation, making it unsuitable for micro-sampling and large-scale screening. This invention uses dried blood smears as the sole detection matrix, employing a PFP chromatographic column and a methanol-water isocratic elution system. Through steel ball grinding, KOH-ethanol hydrolysis, and n-hexane extraction, it achieves efficient release of sterols from dried blood smears. It achieves baseline separation of 7-enylcholine and cholesterol without the need for strong column washing. It also supports minimally invasive finger-prick blood collection, room temperature storage and transportation, requires less sample volume, has simpler pretreatment, and is more versatile, making it specifically suitable for screening scenarios involving infants and high-risk groups. Compared with existing technologies, this invention has the following advantages and technical effects: This invention simplifies the sampling process by using disposable blood collection needles and cards to collect dried blood smears instead of traditional serum or plasma samples. A 3.2mm diameter punch is used to perforate the sampling area of ​​the dried blood smear, yielding three dried blood spots. Seven sterols contained in the dried blood spots are extracted with hexane. Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used for chromatographic separation and mass spectrometry detection and quantification. This method achieves rapid and accurate determination of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol. The method is simple to operate, provides accurate quantification, and achieves a recovery rate of 92.4%-96.7%. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is the standard curve for cholesterol. Figure 2 The standard curve for β-sitosterol; Figure 3 This is the standard curve for stigmasterol; Figure 4 The standard curve for rapeseed oil sterols; Figure 5 This is the standard curve for dihydrocholesterol; Figure 6 This is the standard curve for dehydrocholesterol; Figure 7 The standard curve for 7-enylcholine; Figure 8 This is a chromatogram of cholesterol standards; Figure 9The chromatogram of β-sitosterol standard; Figure 10 Chromatogram of stigmasterol standard; Figure 11 Chromatogram of standard rapeseed oil sterols; Figure 12 The chromatogram of dihydrocholesterol standard; Figure 13 The chromatogram of dehydrocholesterol standard; Figure 14 Chromatogram of 7-enylcholine standard; Figure 15 Chromatogram of stigmasterol-D6 standard; Figure 16 The chromatogram shows cholesterol in an actual dried blood sample. Figure 17 The chromatogram of β-sitosterol in an actual dried blood smear sample is shown. Figure 18 The chromatogram of stigmasterol in an actual dried blood smear sample; Figure 19 The chromatogram of rapeseed oil sterols in an actual dried blood smear sample is shown. Figure 20 The chromatogram of dihydrocholesterol in an actual dried blood smear sample; Figure 21 The chromatogram of dehydrocholesterol in an actual dried blood smear sample; Figure 22 This is a chromatogram of 7-enylcholine in an actual dried blood smear sample. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] This invention provides a liquid chromatography-mass spectrometry (LC-MS) method for detecting phytosterol content in dried blood smears, comprising steps of sample pretreatment, LC-MS detection, and calculation, wherein: Sample pretreatment includes taking dried blood smear samples, obtaining dried blood spots by punching holes, adding internal standard and extraction solvent for extraction, separating, taking the supernatant, concentrating and redissolving to obtain the test solution; Liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection involves detecting the analyte using LC-MS / MS to obtain detection results. In the LC detection conditions, the chromatographic column is a PFP column, and the mobile phase includes an aqueous phase and an organic phase, with isocratic elution. The content of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in dried blood smears was calculated based on the detection results of liquid chromatography-tandem mass spectrometry.

[0031] In the method of this embodiment of the invention, (1) the blood smear sample in the sample pretreatment is a dry blood spot matrix carried by filter paper, which contains interfering substances such as proteins and impurities. The core of the pretreatment is to break the matrix interference, efficiently extract seven kinds of sterol substances and achieve sample purification, so as to eliminate the matrix effect for subsequent liquid chromatography-mass spectrometry.

[0032] (2) The technical principle of liquid chromatography-tandem mass spectrometry detection is separation and qualitative and precise detection. This step is divided into two core links: liquid chromatography separation and tandem mass spectrometry detection. By utilizing the separation capability of chromatography and the high sensitivity and high specificity of mass spectrometry, effective peak separation and precise qualitative / quantitative analysis of seven sterol substances can be achieved.

[0033] The principle of liquid chromatography separation: A PFP column (pentafluorophenyl column) is used as the stationary phase, and an aqueous phase + organic phase is used as the mobile phase in isocratic elution mode. The PFP column achieves effective retention and separation of seven structurally similar sterols (cholesterol, β-sitosterol, stigmasterol, etc.) through multiple interactions such as hydrophobic interaction, π-π interaction, and hydrogen bonding, avoiding detection interference caused by co-elution. The stable mobile phase ratio of isocratic elution ensures symmetrical chromatographic peaks and stable retention times, providing a reliable retention time basis for subsequent qualitative mass spectrometry detection.

[0034] The principle of tandem mass spectrometry detection: Atmospheric pressure chemical ionization (APCI) source is used to ionize sterols in the test solution. This ionization mode is suitable for moderately polar to nonpolar sterols and can efficiently generate stable quasi-molecular ions. Then, multiple reaction monitoring (MRM) mode is used for detection. That is, the characteristic precursor ions (m / z) of each sterol are first screened by primary mass spectrometry, and then the precursor ions are fragmented into characteristic daughter ions by secondary mass spectrometry. Specific quantitative and qualitative ions are selected for monitoring.

[0035] Since the parent and daughter ions of different sterols have unique mass-to-charge ratio characteristics, the MRM mode can effectively eliminate the interference of other impurities in the matrix, achieve specific qualitative analysis of seven target substances, and at the same time, achieve quantitative detection of target substances by monitoring the signal intensity of characteristic ions.

[0036] (3) The internal standard method is used for quantification. The core principle is to use the linear relationship between the ratio of the peak area of ​​the standard and the peak area of ​​the internal standard and the concentration to eliminate systematic errors such as extraction loss in the pretreatment process and signal fluctuation in mass spectrometry detection, so as to achieve accurate quantification of target substances in dried blood smears. First, a series of mixed standard working solutions of seven sterol substances with different concentrations are prepared. The same internal standard as in the sample is added. After the same pretreatment and liquid chromatography-mass spectrometry detection, a standard curve is plotted with the peak area of ​​the standard / internal standard as the ordinate and the concentration of the standard as the abscissa to obtain the linear regression equation. The peak areas of each target substance and the peak areas of the internal standard obtained from the detection of the actual dried blood smear sample are substituted into the linear regression equation. The accurate content of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol and 7-enylcholesterol in dried blood smears can be calculated.

[0037] All raw materials used in the embodiments of this invention were commercially available. Cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, 7-enylcholesterol, and stigmasterol-D6 were purchased from Alta Company, with CAS numbers 57-88-5, 83-46-5, 83-48-7, 474-62-4, 80-97-7, 313-04-2, 80-99-9, and 83-48-7, respectively.

[0038] The technical solution of the present invention will be further illustrated by the following embodiments.

[0039] Example 1 A liquid chromatography-mass spectrometry (LC-MS) method for detecting phytosterol content in dried blood smears includes the following steps: 1. Sample preparation (1) Blood collection personnel should wash their hands and wear gloves; (2) Massage or apply heat to the subject's left ring finger, and disinfect the skin with 75% alcohol. After the skin is dry, collect blood. (3) Use a disposable blood collection needle to prick the inside of the tip of the left ring finger (if there is a wound or other situation that makes it impossible to collect a sample, other fingers can be selected), wipe away the first drop of blood with a dry cotton ball, and collect the second drop of blood; (4) Place the blood collection area of ​​the filter paper in contact with the blood drop, but do not touch the skin. Let the blood naturally seep into the back of the filter paper. Do not smear. Only drop the blood once in each blood collection area and collect at least three blood spots. (5) Place the blood smear in clean air, avoid direct sunlight, and air dry naturally for 3-4 hours until it turns dark brown to obtain a dried blood smear sample. Store it in a sealed bag. The quality requirements followed in the sample preparation process are as follows: One needle per person; A qualified dried blood spot on filter paper should have the following characteristics: each blood spot should be larger than 8 mm in diameter; there should be no overlap between blood spots; blood droplets should naturally penetrate the filter paper, and the blood spots should be consistent on both sides; the blood spots should be uncontaminated.

[0040] 2. Sample pretreatment (1) Punch three holes in the sampling area of ​​the dried blood smear sample using a 3.2 mm diameter punch and place it in a 2.0 mL EP tube; (2) Add two steel balls, add 200 μL of KOH-ethanol solution, and add 20 μL of internal standard stigmasterol-D6; (3) Grind for 3 min, vortex mix, sonicate in water bath, and react in a 70℃ water bath; (4) After taking it out, add ultrapure water and n-hexane, vortex to mix, centrifuge and take the supernatant; (5) Add n-hexane to the original EP tube again, vortex to mix, centrifuge and take the supernatant, and combine the two supernatants; (6) Freeze-dry, add methanol to reconstitute, and vortex mix. (7) Place the reconstituted solution in the inner liner tube to obtain the test solution, which is used for the detection of six non-cholesterol sterols on the instrument; (8) After diluting the reconstituted solution by 1000 times stepwise, place it in the inner liner tube for use in cholesterol detection.

[0041] 3. Preparation of standard products (1) Mixed standard stock solution: Weigh 10.0 mg (accurate to 0.0001 g) of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol and 7-enylcholesterol standards respectively, dissolve them in methanol and make up to 50 mL, store at 4 °C protected from light, shelf life 12 months, stock solution concentration is 200 μg / mL; (2) Mixed standard intermediate solution: Take 0.5 mL of each standard stock solution, dilute to 50 mL with methanol, mix well, and prepare immediately before use. The concentration is 2 μg / mL. (3) Mixed standard series working solutions: Pipette 0.005 mL, 0.01 mL, 0.025 mL, 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, 1.0 mL, 2.5 mL, and 5.0 mL of the mixed standard intermediate solution into 10 mL volumetric flasks, and dilute to volume with methanol to obtain a series of standard working solutions with concentrations of 1.0 ng / mL, 2 ng / mL, 5 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 50.0 ng / mL, 100.0 ng / mL, 200.0 ng / mL, 500.0 ng / mL, and 1000.0 ng / mL, respectively. Prepare the solutions immediately before use. The sample supernatant and standard were simultaneously loaded into vials and fed into the instrument.

[0042] 4. Liquid Chromatography Conditions Column: Phenomenex Kinetex PFP (4.6×250mm, 5μm); Mobile phase A: Primary water; Mobile phase B: Methanol; Elution program: isocratic elution, 0-32 min, 90% mobile phase B; Flow rate: 0.5 mL / min.

[0043] 5. Mass spectrometry conditions Ionization mode: Atmospheric pressure chemical ionization source (APCI); Scanning method: Multiple response monitoring (MRM); Evaporation temperature: 450℃; Ion transport capillary temperature: 350℃; The quantitative and qualitative ions, collision energies, and lens voltage parameters of each compound are shown in Table 3. Table 3 Mass Spectrometry Parameters Table 3 contains entries marked with " "" indicates a quantitative ion.

[0044] 6. Quantitative Calculation After data collection, a standard curve is plotted with the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard as the ordinate and the concentration as the abscissa. The internal standard method is used for quantification. By substituting the peak areas of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in the sample into the standard curve, the precise content of each analyte in the sample can be calculated.

[0045] Methodological validation: (1) Linear relationship Figures 1 to 7 Table 4 shows the standard curves for cholesterol (CHOL), β-sitosterol (BSS), stigmasterol (SMT), campesterol (CAMP), dihydrocholesterol (DHC), dehydrocholesterol (24DHC), and 7-enylcholesterol (LTS). Table 4 also shows the linear range (1-1000 ng / mL) and correlation coefficient (R²) for the seven analytes. 2 ).

[0046] Table 4. Linear range and correlation coefficient of the seven analytes It can be seen that, within the concentration range of 1.0-1000 ng / mL, the peak area ratio of each analyte showed a good linear relationship with the concentration. Correlation coefficient R 2 All values ​​≥0.99 indicate that the method has good linearity within this concentration range, meets the linearity requirements for quantitative analysis of biological samples, and is suitable for the accurate quantification of phytosterol content in dried blood smears.

[0047] (2) Limit of detection and limit of quantitation Table 5 shows the results of the determination of the limit of detection (LOD) and limit of quantitation (LOQ) for seven analytes.

[0048] Table 5. Limits of Detection and Limits of Quantification for Seven Analogous Substances As shown in Table 5, the detection limits for each analyte are between 0.300 and 1.50 ng / mL, and the quantitation limits are between 1.00 and 5.00 ng / mL. This indicates that the method of the present invention has high sensitivity and can meet the detection requirements for trace amounts of phytosterols in dried blood smears, and is especially suitable for trace samples such as those from infants and young children.

[0049] (3) Recovery rate test Table 6 shows the spiked recoveries of the seven analytes.

[0050] Table 6. Spike recoveries of the seven analytes As shown in Table 6, the recoveries of each analyte were between 92.4% and 96.7%, with a relative standard deviation (RSD) ≤ 11.5%; the recoveries were in the range of 85% to 110%, which meets the requirements for quantitative analysis of biological samples, indicating that the method of the present invention has good accuracy.

[0051] (4) Precision test Table 7 shows the intra-batch and inter-batch errors for the seven test samples.

[0052] Table 7. Intra-batch and inter-batch errors of seven analytes As shown in Table 7, the intra-batch error is between 2.4% and 8.7%, and the inter-batch error is between 3.2% and 12.4%. This indicates that the method of the present invention has good precision, meets the repeatability requirements for biological sample detection, and is stable and reliable.

[0053] (5) Qualitative identification Figures 8-15 The figures show chromatograms of standards for cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, 7-enylcholesterol, and stigmasterol-D6 (SMT-d6). It can be seen that each compound exhibits symmetrical peak shapes and stable retention times under the liquid chromatography conditions described in this invention. The internal standard stigmasterol-D6 and the analytes show no chromatographic interference and can be effectively distinguished. This indicates that the chromatographic conditions established in this invention are suitable for the qualitative identification of seven plant sterols and the internal standard.

[0054] Test results: Figures 16-22 The images show the chromatograms of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in actual dried blood sample. It can be seen that all seven target compounds were detected in the actual dried blood sample, with good peak shapes and no interference from impurity peaks; the retention times are similar to those of the standard chromatogram. Figures 8-15 The results were consistent with the findings, further verifying the specificity and accuracy of the method; indicating that the method of the present invention can be effectively applied to the detection of phytosterol content in real dried blood smear samples.

[0055] The precise content of each analyte in the sample was further calculated, and the results are shown in Table 8.

[0056] Table 8 Precise content of each analyte in the sample In summary, this invention successfully establishes a liquid chromatography-tandem mass spectrometry method for detecting cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in dried blood smears. This method is simple to operate, highly sensitive (detection limit as low as 0.300 ng / mL), has a wide linear range (1-1000 ng / mL), good accuracy (recovery rate 92.4%-96.7%), and high precision (intra-batch error ≤8.7%, inter-batch error ≤12.4%), and can meet the accurate quantitative detection needs of seven phytosterols in dried blood smear samples from special populations such as infants and young children.

[0057] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A liquid chromatography-mass spectrometry method for detecting the content of phytosterols in dried blood smears, characterized in that, The steps include sample pretreatment, liquid chromatography-tandem mass spectrometry detection, and calculation, among which: The sample pretreatment includes taking dried blood smear samples, obtaining dried blood spots by punching holes, adding internal standard and extraction solvent for extraction, separating and taking the supernatant, concentrating and redissolving to obtain the test solution; The liquid chromatography-tandem mass spectrometry detection includes detecting the test solution using liquid chromatography-tandem mass spectrometry to obtain detection results. In the liquid chromatography detection conditions, the chromatographic column is a PFP chromatographic column, the mobile phase includes an aqueous phase and an organic phase, and isocratic elution is used. The calculations were performed based on the detection results of liquid chromatography-tandem mass spectrometry to determine the content of cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enylcholesterol in the dried blood smear.

2. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the sample pretreatment step, the dried blood sample is collected by fingertip blood collection, the collected blood is dropped onto filter paper and allowed to air dry naturally to obtain the dried blood sample.

3. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the sample pretreatment step, the diameter of the holes is 3.2 mm, and the number of holes is 3.

4. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the sample pretreatment step, the extraction solvent is n-hexane, and KOH-ethanol solution is added before extraction to carry out a hydrolysis reaction.

5. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, The sample pretreatment steps include a grinding process involving the addition of steel balls during extraction.

6. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the detection conditions of the liquid chromatography, the organic phase is methanol, and the isocratic elution program is 0-32 min.

7. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the liquid chromatography-tandem mass spectrometry detection step, the mass spectrometry conditions include: ionization mode of atmospheric pressure chemical ionization source and scanning mode of multiple reaction monitoring mode.

8. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the liquid chromatography-tandem mass spectrometry (LC-MS / MS) detection step, the tandem mass spectrometry detection parameters for cholesterol, β-sitosterol, stigmasterol, campesterol, dihydrocholesterol, dehydrocholesterol, and 7-enencholesterol are as follows: Among them The ions are quantitative.

9. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the sample pretreatment step, the internal standard is stigmasterol-D6, and its mass spectrometry parameters include: precursor ion 401 m / z, and daughter ions 83 m / z. m / z, 95 m / z and 303 m / z.

10. The liquid chromatography-mass spectrometry method for detecting phytosterol content in dried blood smears according to claim 1, characterized in that, In the calculation steps, the internal standard method is used for quantification, and a standard curve is plotted with the ratio of the peak area of ​​the standard to the peak area of ​​the internal standard as the ordinate and the concentration as the abscissa.

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