Method for detecting ceramide and phosphatidyl ethanolamine in neonatal dried blood spots

Abstract

The invention relates to the technical field of assay and inspection, in particular to a method for detecting ceramide and phosphatidyl ethanolamine in neonatal dried blood spots, and the preparation method comprises the following steps: S1, drawing a standard curve equation: preparing an internal standard extract; preparing a mixed standard solution; preparing standard substance solutions with a series of concentrations; adding the internal standard extraction liquid into the standard substance solution, carrying out pretreatment, and carrying out HPLC-MS / MS detection to respectively obtain a ceramide standard curve and a phosphatidyl ethanolamine standard curve; and S2, detecting the contents of ceramide and phosphatidyl ethanolamine in the dried blood spot to be detected, namely adding the internal standard extract into the dried blood spot to be detected, performing pretreatment, performing HPLC-MS / MS detection, and obtaining the contents of ceramide and phosphatidyl ethanolamine according to a ceramide standard curve and a phosphatidyl ethanolamine standard curve respectively. The detection method provided by the embodiment of the invention has the characteristics of high accuracy, good sensitivity, stability and simplicity and convenience in operation.

Description

Technical Field

[0001] This invention relates to the field of laboratory testing technology, and in particular to a method for detecting ceramides and phosphatidylethanolamine in dried blood spots of newborns. Background Technology

[0002] Obesity is defined as the abnormal accumulation of adipose tissue, which can lead to cardiometabolic diseases such as type 2 diabetes, hypertension, and metabolic fatty liver disease. Global obesity rates among children and adolescents continue to rise and are projected to affect more than 250 million people by 2030.

[0003] Recent studies have revealed the important roles of lipids such as sphingomyelin and phosphatidylethanolamine in obesity complications. Ceramides (Cer) are a class of complex sphingomyelin compounds that play a crucial role in cell signaling and regulate cellular physiological functions. Phosphatidylethanolamine (PE) is a major component of the cell membrane, playing key roles in maintaining cell membrane structure, participating in signal transduction, supporting nerve function, and regulating metabolism. Recent clinical studies have shown that elevated levels of certain ceramides (Cer) and phosphatidylethanolamine (PE) are associated with hepatic steatosis; specifically, Cer (d18:0 / 24:0) and PE (18:0 / 18:1) levels are positively correlated with obesity.

[0004] Currently, studies based on the detection of ceramide or phosphatidylethanolamine levels primarily focus on adults or adolescents. However, obesity has a heritability rate as high as 67%; therefore, the levels of ceramides and / or phosphatidylethanolamine in the blood of newborns have become an important parameter for understanding their lipid levels.

[0005] Therefore, the search for a method to detect ceramides and phosphatidylethanolamine in neonatal dried blood spots has become a research hotspot. Summary of the Invention

[0006] To address at least one of the aforementioned technical problems, the present invention provides a method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots.

[0007] In one aspect, the present invention provides a method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots, comprising: S1: plotting standard curve equations, including: preparing an internal standard extract; diluting the ceramide standard stock solution and the phosphatidylethanolamine standard stock solution respectively to obtain a mixed standard solution of ceramide and phosphatidylethanolamine; diluting the mixed standard solution with artificial plasma at different multiples to obtain a series of standard solutions of different concentrations; adding the internal standard extract to the standard solutions, pretreatment, and performing HPLC-MS / MS detection to obtain ceramide standard curves and phosphatidylethanolamine standard curves respectively; S2: detecting the content of ceramide and phosphatidylethanolamine in the dried blood spots to be tested, including: adding the internal standard extract to the dried blood spots to be tested, pretreatment, and performing HPLC-MS / MS detection to obtain the content of ceramide and phosphatidylethanolamine according to the ceramide standard curve and the phosphatidylethanolamine standard curve respectively.

[0008] In some embodiments of the present invention, the step of preparing the internal standard extract includes: diluting and mixing a 500 μmol / L ceramide internal standard stock solution and a 500 μmol / L phosphatidylethanolamine internal standard stock solution with an extractant to obtain the internal standard extract; wherein the components of the extract include any two of isopropanol, methanol and water, and the volume ratio is (1~3):(1~3); and / or, the extract is preferably a mixture of isopropanol and methanol in a volume ratio of 1:1; and / or, the ceramide internal standard is Cer(d18:1 / 24:0)-d7; and / or, the phosphatidylethanolamine internal standard is PE(17:0 / 18:1)-d5.

[0009] In some embodiments of the present invention, the concentration of ceramide in the mixed standard solution is 20 μmol / L; and / or the concentration of phosphatidylethanolamine is 400 μmol / L.

[0010] In some embodiments of the present invention, the method for preparing artificial plasma includes: adding human serum albumin to a PBS solution to obtain artificial plasma with a human serum albumin mass fraction of 7%.

[0011] In some embodiments of the present invention, the concentration of ceramide in the standard solution is 0.04~2 μmol / L; and / or the concentration of phosphatidylethanolamine is 0.8~40 μmol / L.

[0012] In some embodiments of the present invention, the HPLC detection conditions in the HPLC-MS / MS detection include: chromatographic column: C18 column; and / or, column temperature: 30~50℃ (e.g., 32℃, 36℃, 39℃, 43℃, 48℃, etc.), preferably 40℃; and / or, flow rate: 0.2~0.8mL / min (e.g., 0.36mL / min, 0.58mL / min, 0.67mL / min, 0.71mL / min, 0.78mL / min, etc.), preferably 0.4mL / min; and / or, injection volume: 1~10μL (e.g., 2μL, 3μL, 6μL, 7μL, 9μL, etc.), preferably 1μL; and / or, mobile phase A: an aqueous solution containing 0.05%~0.2% formic acid (e.g., 0.05%~0.2% formic acid). The mobile phase A is a solution containing 0% to 0.1% formic acid (e.g., 0.02%, 0.04%, 0.07%, 0.08%, 0.09%, etc.), preferably an aqueous solution containing 0.1% formic acid; mobile phase B is an isopropanol solution containing 0% to 0.1% formic acid (e.g., 0.02%, 0.04%, 0.07%, 0.08%, 0.09%, etc.), preferably an isopropanol solution; and / or, the gradient elution conditions are: 0-1.20 min, mobile phase A (%): 60→5, mobile phase B (%): 40→95; 1.20-2.00 min, mobile phase A (%): 5→5, mobile phase B (%): 95→95; 2.00-2.10 min, mobile phase A (%): 5→60, mobile phase B (%): 95→40; 2.10-2.50 min, mobile phase A (%): 60→60, mobile phase B (%): 40→40.

[0013] In some embodiments of the present invention, in HPLC-MS / MS detection, the MS detection conditions include: positive ion detection mode of the ESI ion source; and / or, ion transmission tube temperature: 200~400℃ (e.g., 210℃, 280℃, 310℃, 360℃, 390℃, etc.), preferably 350℃; and / or, nebulization temperature: 250~550℃ (e.g., 210℃, 380℃, 410℃, 460℃, 490℃, etc.), preferably 450℃; and / or, sheath gas flow rate: 30~60 alb (e.g., 39 alb, 43 alb, 46 alb, etc.). 53 arb, 59 arb, etc.), preferably 45 arb; and / or, the gas flow rate of the auxiliary gas: 10~20 arb (e.g., 11 arb, 13 arb, 16 arb, 17 arb, 19 arb, etc.), preferably 15 arb; and / or, the gas flow rate of the backflush gas: 0~10 arb (e.g., 1 arb, 3 arb, 6 arb, 7 arb, 9 arb, etc.), preferably 5 arb; and / or, the spray voltage: 1.0~4.5 kV (e.g., 1.3 kV, 2.1 kV, 2.8 kV, 3.7 kV, 4.3 kV, etc.), preferably 3.5 kV.

[0014] In some embodiments of the present invention, the pretreatment includes incubating the sample at 25~45°C (e.g., 26°C, 28°C, 31°C, 36°C, 43°C, etc.) for 5~120 min (e.g., 46 min, 58 min, 89 min, 96 min, 118 min, etc.), preferably at 37°C for 60 min.

[0015] In some embodiments of the present invention, the detection method further includes: detecting the content of ceramide and phosphatidylethanolamine in the dried blood spot quality control product; wherein the preparation method of the dried blood spot quality control product includes: centrifuging a whole blood sample to obtain a precipitate; adding artificial plasma to the precipitate, mixing, centrifuging, and repeating at least three times to obtain blood cells; mixing red blood cells and artificial plasma to obtain a whole blood solution; and preparing the whole blood solution into a dried blood spot quality control product.

[0016] In some embodiments of the present invention, the ceramide is Cer(d18:0 / 24:0); and / or the phosphatidylethanolamine is PE(18:0 / 18:1).

[0017] The technical solution provided by this invention has the following advantages: (1) The detection method for ceramide and phosphatidylethanolamine in neonatal dried blood spots of the present invention has the characteristics of high sensitivity, strong specificity, simple sample handling, and fast instrument analysis speed. The specific explanation is as follows: ①Dried blood spot (DBS) samples were selected as analytical samples for detecting the levels of ceramide Cer (d18:0 / 24:0) and phosphatidylethanolamine PE (18:0 / 18:1) in newborns. This method has advantages such as low sample collection risk, small blood volume, convenient sample preservation and transportation, and long-term stability of detection indicators. It is the main sample collection method widely accepted in clinical practice for newborn screening projects.

[0018] ②Simple sample pretreatment: Since dried blood spots contain little blood, sample pretreatment is simple.

[0019] ③ High sensitivity, strong specificity, and fast analysis speed: Using high performance liquid chromatography-tandem mass spectrometry as the analytical method, it can quickly analyze the results and has the characteristics of high sensitivity and strong specificity; at the same time, it can also share dried blood spot specimens and mass spectrometry platform with existing neonatal inherited metabolic diseases.

[0020] (2) In the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots of the present invention, the preparation method of dried blood spot quality control is simple. Specifically, the background of the analyte is reduced by washing whole blood cell samples, and the washed blood cells are mixed with artificial plasma to obtain a whole blood solution. The whole blood is added to the analyte standard solution, and spots are applied to prepare standard dried blood spot samples.

[0021] (3) In the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots of the present invention, the dried blood spot extract can effectively extract the analytes from the dried blood spots. Specifically, based on the physicochemical properties of the analytes ceramide (e.g., Cer(d18:0 / 24:0)) and phosphatidylethanolamine (e.g., PE(18:0 / 18:1)), a mixture of isopropanol and methanol is selected for the extraction of the analytes from the dried blood spots. This can effectively extract the analytes from the dried blood spots into the solution, while effectively reducing the matrix components in the dried blood spots from entering the extract and reducing the matrix effect in mass spectrometry detection.

[0022] (4) In the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots of the present invention, the selected mobile phase can rapidly analyze the substances. Specifically, based on the physicochemical properties of the analytes ceramide (e.g., Cer(d18:0 / 24:0)) and phosphatidylethanolamine (e.g., PE(18:0 / 18:1)), isopropanol is selected as the organic phase, which can both match the dried blood spot extraction solution and reduce the solvent effect in chromatographic analysis; and allow the analytes to rapidly elute peaks, shortening the analysis time.

[0023] (5) The method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots of the present invention can understand the lipid level of newborns and provide data support for clinical recommendations on diet and exercise intervention for newborns with high lipid levels. Attached Figure Description

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, those skilled in the art can obtain other drawings based on these drawings without creative effort.

[0026] Figure 1 This is a flowchart illustrating the preparation of dried blood spot quality control material in the method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention. Figure 2 The detection spectrum of Cer(d18:0 / 24:0) in neonatal dried blood spots is shown in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention. Figure 3 The detection spectrum of PE (18:0 / 18:1) in neonatal dried blood spots is shown in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention. Figure 4 The standard curve for Cer(d18:0 / 24:0) detection of dried blood spots in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention is shown. Figure 5 The standard curve for PE (18:0 / 18:1) detection of dried blood spots in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention; Figure 6 Macroscopic information of blood spots with different drop volumes in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention; Figure 7 The results of the extraction solutions obtained by different combinations of different types of extraction solvents in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots in this embodiment of the invention; Figure 8 The effect of different incubation times on extraction efficiency in the method for detecting ceramide and phosphatidylethanolamine in neonatal dried blood spots in this embodiment of the invention; Figure 9 This invention relates to the effect of different incubation temperatures on extraction efficiency in a method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots, as described in an embodiment of the present invention. Detailed Implementation

[0027] To better understand the above-mentioned objectives, features, and advantages of the present invention, the solutions of the present invention will be further described below. It should be noted that, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention, but the invention may also be practiced in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the invention, and not all embodiments.

[0029] Experimental reagents and equipment reagents ①Standards: Cer (d18:0 / 24:0) and PE (18:0 / 18:1) standards were purchased from Avanti. ② Deuterated internal standards: Cer(d18:1 / 24:0)-d7 and PE(17:0 / 18:1)-d5 were purchased from Avanti. ③ Methanol (chromatographic grade, Fisher); ④ Isopropanol (chromatographic grade, Fisher); ⑤ Formic acid (mass spectrometry grade, Fisher); ⑥ Dimethyl sulfoxide (chromatographic grade, Aladdin).

[0030] ⑦PBS (Solepro) ⑧ Human serum albumin (Aladdin) ⑨ Whatman 903 filter paper (Cytiva) Note: The internal standard for deuteration corresponding to Cer(d18:0 / 24:0) is Cer(d18:1 / 24:0)-d7; the internal standard for deuteration corresponding to PE(18:0 / 18:1) is PE(17:0 / 18:1)-d5, both purchased from Avanti. equipment: ① YS EXACT 9900MD High Performance Liquid Chromatography-Tandem Mass Spectrometry Detection System (Shandong Yingsheng Biotechnology Co., Ltd.) ② MCA3.6P-2CCN-M 1 / 1,000,000 electronic balance (Sartorius) ③ Sorvall Legeug Micro 21R High-Speed ​​Refrigerated Centrifuge (Thermo Fisher) ④IQ7005 Pure Water System (Millipore) ⑤ Pipettes (Eppendorf) ⑥ST07-2 Microplate Thermostatic Oscillator (MIULAB) ⑦ MS3DS025 Mixer (IKA) ⑧201 HemoCue® Hb+ System (Hemocue, Sweden) ⑨ ChromCore 120 C18 (30*2.1mm, 3.0 μm) chromatographic column (nanospectral analysis) ⑩ The mass spectrometer is used with TraceFinder 5.1 software.

[0031] Detection methods for ceramides and phosphatidylethanolamine in neonatal dried blood spots A method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots includes the following steps: 1.1 Solution Preparation 1.1.1 Preparation of Standard Stock Solutions Accurately weigh 3.26 mg of Cer(d18:0 / 24:0) standard, dissolve it in isopropanol-dimethyl sulfoxide (2:1, v / v) and bring the volume to 10 mL. Dissolve by sonication to prepare a standard stock solution with a concentration of 500 μmol / L. Aliquot the solution and store at -20℃.

[0032] Accurately weigh 7.46 mg of PE (18:0 / 18:1) standard, dissolve it in methanol and bring the volume to 10 mL, sonicate to dissolve, prepare a standard stock solution with a concentration of 1000 μmol / L, aliquot and store at -20℃.

[0033] 1.1.2 Preparation of Internal Standard Stock Solution Cer(d18:1 / 24:0)-d7 internal standard stock solution: Accurately weigh 1 mg of Cer(d18:1 / 16:0)-d7 solid, dissolve it in isopropanol-dimethyl sulfoxide (2:1, v / v) by sonication to prepare a stock solution with a concentration of 500 μmol / L, aliquot and store at -20℃.

[0034] PE(17:0 / 18:1)-d5 internal standard stock solution: Accurately weigh 1 mg of PE(d18:1 / 18:0)-d5 solid, dissolve it in methanol to prepare a stock solution with a concentration of 500 μmol / L, aliquot and store at -20℃.

[0035] 1.1.3 Preparation of internal standard extraction solution Take 30 μL and 10 μL of Cer(d18:1 / 24:0)-d7 internal standard stock solution and PE(17:0 / 18:1)-d5 internal standard stock solution, respectively, with a concentration of 500 μmol / L, and add 100 mL of isopropanol-methanol (1:1, v / v). Mix well to obtain the internal standard extraction solution. The concentration of Cer(d18:1 / 24:0)-d7 in the internal standard extraction solution is 150 nmol / L, and the concentration of PE(17:0 / 18:1)-d5 is 50 nmol / L.

[0036] 1.2 Preparation of standard solutions 1.2.1 Preparation of artificial plasma: Weigh 3.5 g of human serum albumin and add 50 mL of PBS solution to prepare a 7% human serum albumin solution as artificial plasma, which is used as a blank matrix for preparing standard solutions.

[0037] 1.2.2 Preparation of a series of standard working solutions of different concentrations Take the Cer(d18:0 / 24:0) and PE(18:0 / 18:1) stock solutions prepared in 1.1.1, dilute them with 75% methanol to prepare a mixed standard solution C6 with a Cer(d18:0 / 24:0) concentration of 20 μmol / L and a PE(18:0 / 18:1) concentration of 400 μmol / L. C6 is then serially diluted with 75% methanol to obtain mixed standard working solutions. The concentrations of the series of working solutions are shown in Table 1 below. The concentration unit in Table 1 is μmol / L.

[0038] Table 1 Concentration of Mixed Standard Working Solutions 1.2.3 Preparation of standard solutions Take 0.9 mL of artificial plasma and add it to a 1.5 mL EP tube. Prepare 6 parallel aliquots. Add 0.1 mL of each of the series of working solutions C1 to C6 prepared in 1.2.2 to each aliquot. Vortex mix for 3 min to obtain standard solutions. The concentrations of each standard solution are shown in Table 2. The concentration unit in Table 2 is μmol / L.

[0039] Table 2 Concentration of Standard Solutions 1.3 Preparation of dried blood spot quality control material The preparation process of dried blood spot quality control product is as follows: Figure 1 As shown. The preparation process of a dried blood spot quality control product includes the following steps: 1.3.1 Preparation of low-background whole blood samples Take a low-background EDTA-anticoagulated whole blood sample and centrifuge at 2000g for 10 minutes at 18-24℃. Remove the supernatant plasma solution. Add an equal volume of artificial plasma according to the volume of the lower blood cell layer. Gently invert the sample 5-10 times to ensure complete mixing of red blood cells and artificial plasma. Centrifuge again at 2000g for 10 minutes to remove blood cells and plasma components. Repeat the washing step twice. Finally, mix the artificial plasma with the plasma-removed whole blood cells, controlling the hematocrit to 50-55%. Use this whole blood solution to prepare quality control samples by adding standard solutions of different concentrations.

[0040] 1.3.2 Preparation of a series of whole blood quality control products at various concentrations Prepare three levels of quality control samples, with low, medium, and high concentration levels, named LQC, MQC, and HQC respectively. The preparation process is as follows: HQC whole blood sample: Take the mixed standard solution C6 prepared in step 1.2.2 above, add it to the low background whole blood solution prepared in step 1.3.1, dilute C6 by 13 times, that is, the volume ratio of C6 solution to whole blood solution is 1:12, mix well, and then prepare HQC whole blood sample.

[0041] MQC whole blood sample: Take the HQC whole blood sample and dilute it 3 times with low background whole blood solution, that is, the volume ratio of HQC solution to whole blood solution is 1:2. After mixing evenly, prepare the MQC whole blood sample.

[0042] LQC whole blood sample: Take MQC whole blood sample and dilute it 4.16 times with low background whole blood solution, that is, the volume ratio of MQC solution to whole blood solution is 6:25. After mixing evenly, LQC whole blood sample is prepared.

[0043] 1.3.3 Preparation of a series of concentrated dried blood spot quality control products Take the HQC, MQC, and LQC whole blood quality control samples prepared in step 1.3.2 above. Take 60 μL each time and slowly drop the blood onto Whatman 903 filter paper, aligning it with the center of the sampling circle on the collection card, avoiding air bubbles. Place the prepared dried blood spot quality control samples on a dry, clean, flat, non-absorbent surface for 3-4 hours to air dry naturally. Check the dried blood spot quality control samples: the blood should completely penetrate the back of the filter paper, the blood spot diameter should be approximately 12 mm, and there should be no overlap. Place the qualified dried blood spot quality control samples into a plastic-sealed bag containing desiccant and seal it for later use. The concentration of the dried blood spot quality control samples is shown in Table 3.

[0044] Table 3 Concentration of dried blood spot quality control material 1.4 Sample Pretreatment 1.4.1 Dried blood spot samples from newborns to be tested Dried blood spots were collected from the heels of newborns. Two dried blood spots were punched into each well using a 3mm punch and inserted into a 96-well polystyrene plate. 100 μL of internal standard extraction buffer was added to each well. The plate was then capped and incubated at 37°C and 750 rpm for 1 hour. Afterward, the 96-well polystyrene plate was removed from the shaker and allowed to stand at room temperature for 5 minutes. The capping membrane was removed, and 70 μL of the supernatant was transferred to a sample vial for HPLC-MS / MS analysis. The chromatogram of Cer(d18:0 / 24:0) in the dried blood spots is shown below. Figure 2 As shown; the detection spectrum of PE (18:0 / 18:1) in the obtained dried blood spots is as follows. Figure 3 As shown.

[0045] 1.4.2 Quality Control of Dried Blood Spots Two dried blood spot quality controls prepared in section 1.3.3 were punched into each well of a polystyrene 96-well plate using a 3 mm diameter punch. 100 μL of internal standard extraction buffer was added to each well. The plate was then capped and incubated at 37°C and 750 rpm for 1 h. Subsequently, the polystyrene 96-well plate was removed from the shaker and allowed to stand at room temperature for 5 min. The capping membrane was removed, and 70 μL of the supernatant was transferred to a sample vial for HPLC-MS / MS analysis.

[0046] 1.4.3 Pretreatment of Standard Solutions 5.5 μL of each of the six-level standard solutions prepared in 1.2.3 above was added to a polystyrene 96-well plate. 100 μL of internal standard extraction buffer was added to each well. The plate was then capped and incubated at 37°C and 750 rpm for 1 h. Subsequently, the polystyrene 96-well plate was removed from the shaker and allowed to stand at room temperature for 5 min. The capping membrane was removed, and 70 μL of the supernatant was transferred to a sample vial for HPLC-MS / MS analysis. The resulting Cer(d18:0 / 24:0) standard curve for dried blood spot detection is shown below. Figure 4 As shown; the standard curve for blood spot detection PE (18:0 / 18:1) is as follows. Figure 5 As shown.

[0047] 2. Detection conditions for high performance liquid chromatography-tandem mass spectrometry 2.1 High Performance Liquid Chromatography Conditions Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: Isopropanol; Chromatographic column: ChromCore 120 C18 (30*2.1mm, 3.0 μm); Column temperature: 40℃; Flow rate: 0.4 mL / min; Injection volume: 1 μL; The gradient elution procedure is shown in Table 4.

[0048] Table 4 Mobile Phase Gradient Table 2.2 Mass Spectrometry Conditions In electrospray ionization mode, positive ion mode scanning was performed using selected reaction monitoring (SRM) to detect Cer (d18:0 / 24:0) and PE (18:0 / 18:1) and their internal standard signals. The ion source parameters are shown in Table 5 below, and the ion pair parameters are shown in Table 6 below.

[0049] Table 5 Ion source parameters for mass spectrometry Table 6 Ion-pair parameters for mass spectrometry Note: "*" represents quantitative ions.

[0050] 3.1. Investigation of the preparation process of dried blood spots 3.1.1 Examination of Whole Blood Droplet Volume The effects of adding 50 μL, 60 μL, and 70 μL of whole blood to Whatman 903 filter paper were investigated. The blood dropped onto the filter paper was required to be absorbed from the front and permeate to the back, with a blood spot diameter greater than 10 mm. The experiment showed that the 60 μL volume was close to the diameter of clinically collected neonatal dried blood spot specimens, and the permeation effect was consistent on both sides. Therefore, 60 μL was selected as the addition volume for preparing standard dried blood spots. Specific information on the blood spots is shown in Table 7 below. Figure 6 .in, Figure 6 This is macroscopic information of blood spots with different drop volumes in the method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots according to an embodiment of the present invention.

[0051] Table 7. Parameter information of standard dried blood spots 3.1.2 Investigation of the extract The selected extraction solvent was used for the extraction of calibrators, quality control samples, and dried blood spot samples from newborns. The extraction efficiency was determined based on the peak area of ​​the detection indicators. During method development, different combinations of extraction solvents were tested, including mixtures of isopropanol, methanol, and water in different proportions. Isopropanol-methanol (1:1, v / v) was ultimately selected as the sample extraction solvent. The extraction efficiency is shown in Table 8. Figure 7 As shown.

[0052] Table 8. Detection results of different combinations of different extraction solvents. 3.1.3 Examination of incubation time Isopropanol-methanol (1:1, v / v) was used as the extraction solvent in this experiment to investigate the effect of different incubation times on the extraction efficiency of the compounds. Incubation times of 5, 15, 30, 45, 60, and 120 min were examined, and the optimal extraction time was evaluated using the peak area of ​​the compounds. A final incubation time of 60 min was selected. The experimental results are shown in Table 9. Figure 8 .

[0053] Table 9. Effect of different incubation times on extraction efficiency 3.1.4 Investigation of incubation temperature Isopropanol-methanol (1:1, v / v) was used as the extraction solution in the experiment, and the incubation time was 60 min. The effect of different incubation temperatures on the extraction efficiency of the compounds was investigated. The incubation temperatures were room temperature (25℃), 37℃, and 45℃, and the optimal extraction temperature was evaluated by the peak area of ​​the compounds. Since excessively high temperatures would accelerate the evaporation of the extract, an incubation temperature of 37℃ was ultimately selected. The experimental results are shown in Table 10. Figure 9 .

[0054] Table 10 Effect of different incubation temperatures on extraction efficiency In summary, the embodiments of the present invention establish a liquid chromatography-tandem mass spectrometry method for the detection of ceramide Cer (d18:0 / 24:0) and phosphatidylethanolamine PE (18:0 / 18:1) in neonatal dried blood spots, which has the characteristics of high accuracy, good sensitivity, stability and simple operation.

[0055] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0056] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting ceramides and phosphatidylethanolamine in neonatal dried blood spots, characterized in that, include: S1: Plot the equation of the standard curve, including: Prepare the internal standard extraction solution; The ceramide standard stock solution and the phosphatidylethanolamine standard stock solution were diluted separately to obtain a mixed standard solution of ceramide and phosphatidylethanolamine. The mixed standard solution was diluted with artificial plasma at different ratios to obtain a series of standard solutions of different concentrations. The internal standard extract was added to the standard solution. After pretreatment, HPLC-MS / MS was performed to obtain the standard curves for ceramide and phosphatidylethanolamine, respectively. S2: Detection of the content of ceramide and phosphatidylethanolamine in the dried blood spot to be tested, including: The internal standard extract was added to the dried blood spot to be tested. After pretreatment, HPLC-MS / MS was performed to detect the contents of ceramide and phosphatidylethanolamine, respectively, according to the standard curves of ceramide and phosphatidylethanolamine.

2. The detection method according to claim 1, characterized in that, The step of preparing the internal standard extraction solution includes: The 500 μmol / L ceramide internal standard stock solution and the 500 μmol / L phosphatidylethanolamine internal standard stock solution were diluted and mixed with the extraction buffer to obtain the internal standard extract. The components of the extract include any two of isopropanol, methanol and water, and the volume ratio is (1~3):(1~3); And / or, the extract is preferably a mixture of isopropanol and methanol in a volume ratio of 1:1; And / or, the ceramide internal standard is Cer(d18∶1 / 24∶0)-d7; And / or, the phosphatidylethanolamine internal standard is PE(17:0 / 18:1)-d5.

3. The detection method according to claim 1 or 2, characterized in that, In the mixed standard solution The concentration of ceramide was 20 μmol / L; And / or, the concentration of phosphatidylethanolamine is 400 μmol / L.

4. The detection method according to any one of claims 1 to 3, characterized in that, The method for preparing the artificial plasma includes: Human serum albumin was added to PBS solution to obtain artificial plasma with a human serum albumin mass fraction of 7%.

5. The detection method according to any one of claims 1 to 4, characterized in that, In the standard solution The concentration of ceramide is 0.04~2 μmol / L; And / or, the concentration of phosphatidylethanolamine is 0.8~40 μmol / L.

6. The detection method according to any one of claims 1 to 5, characterized in that, In the HPLC-MS / MS detection, the HPLC detection conditions include: Chromatographic column: C18 column; And / or, column temperature: 30~50℃, preferably 40℃; And / or, flow rate: 0.2~0.8 mL / min, preferably 0.4 mL / min; And / or, the injection volume is 1~10μL, preferably 1μL; And / or, mobile phase A: an aqueous solution containing 0.05%~0.2% formic acid, preferably an aqueous solution containing 0.1% formic acid; mobile phase B: an isopropanol solution containing 0%~0.1% formic acid, preferably an isopropanol solution; And / or, the gradient elution conditions are: 0-1.20 min, mobile phase A (%): 60→5, mobile phase B (%): 40→95; 1.20-2.00 min, mobile phase A (%): 5→5, mobile phase B (%): 95→95; 2.00-2.10 min, mobile phase A (%): 5→60, mobile phase B (%): 95→40; 2.10-2.50 min, mobile phase A (%): 60→60, mobile phase B (%): 40→40.

7. The detection method according to any one of claims 1 to 6, characterized in that, In the HPLC-MS / MS detection, the MS detection conditions include: Positive ion detection mode of ESI ion source; And / or, ion transport tube temperature: 200~400℃, preferably 350℃; And / or, atomization temperature: 250~550℃, preferably 450℃; And / or, the gas flow rate of the sheath gas: 30~60 ab, preferably 45 ab; And / or, the gas flow rate of the auxiliary gas: 10~20 ab, preferably 15 ab; And / or, the backflush gas flow rate: 0~10 arb, preferably 5 arb; And / or, spray voltage: 1.0~4.5kV, preferably 3.5kV.

8. The detection method according to any one of claims 1 to 7, characterized in that, The preprocessing includes: Incubate the sample at 25-45℃ for 5-120 min, preferably at 37℃ for 60 min.

9. The detection method according to any one of claims 1 to 8, characterized in that, The detection method further includes: detecting the content of ceramide and phosphatidylethanolamine in the dried blood spot quality control sample; The preparation method of the dried blood spot quality control product includes: The whole blood sample was centrifuged to obtain a precipitate; Artificial plasma was added to the precipitate, mixed well, centrifuged, and repeated at least three times to obtain blood cells; The red blood cells and artificial plasma are mixed to obtain a whole blood solution; The whole blood solution was prepared into a dried blood spot quality control product.

10. The detection method according to any one of claims 1 to 9, characterized in that, The ceramide is Cer(d18:0 / 24:0); And / or, the phosphatidylethanolamine is PE (18:0 / 18:1).

Images