WO2022257950A1 - 红细胞叶酸的检测方法 - Google Patents

红细胞叶酸的检测方法 Download PDF

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WO2022257950A1
WO2022257950A1 PCT/CN2022/097535 CN2022097535W WO2022257950A1 WO 2022257950 A1 WO2022257950 A1 WO 2022257950A1 CN 2022097535 W CN2022097535 W CN 2022097535W WO 2022257950 A1 WO2022257950 A1 WO 2022257950A1
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folic acid
red blood
blood cell
concentration
erythrocyte
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施红军
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西湖维泰(杭州)诊断技术有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86

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  • the invention relates to the field of folic acid detection, in particular to a method for detecting red blood cell folic acid.
  • Folate levels are closely related to reproductive health.
  • the clinical detection of folic acid level is divided into serum (plasma) detection and red blood cell detection.
  • serum (plasma) detection technology is relatively mature, and there are three detection methods: microbial method, protein binding method and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
  • the microbial method utilizes the principle that the growth rate of Lactobacillus is sensitive to folic acid, and reflects the concentration of folic acid added in the culture solution by measuring the turbidity of the bacteria after a certain period of growth (Grossowicz, N., et al., Determination of folic acid metabolites in normal subjects and in patients with nutritional megaloblastic anemia. Proc Soc Exp Biol Med, 1962.109: p.770-3.).
  • the microbiological method is recognized by the clinical nutrition community as the gold standard for folic acid detection, and is often used for performance comparison by new methods (Yetley, E.A., et al., Biomarkers of folate status in NHANES: a roundtable summary. Am J Clin Nutr, 2011.94( 1): p.303S-312S).
  • the main disadvantage of the microbiological method is that it takes a long time to detect, generally takes 3 days, and requires high aseptic operation, which is not conducive to commercial promotion.
  • the main disadvantage of serological testing is that it reflects the recent intake of folic acid, which is easily affected by transient dietary factors and has large fluctuations.
  • the reference value of its concentration for avoiding birth defects has not been widely recognized by the academic community.
  • WHO pointed out in a nutritional guideline for the prevention of neural tube defects that the serum or plasma folic acid threshold of women of childbearing age is not sufficient to prevent neural tube defects; the red blood cell folic acid concentration must reach 906 nmol/L or more to maximize the prevention of neural tube defects ( Cordero, A.M., et al., Optimal serum and red blood cell folate concentrations in women of reproductive age for prevention of neural tube defects: World Health Organization guidelines. MMWR Morb Mortal Wkly Rep, 2015.64(415): p. ).
  • red blood cell folic acid Compared with serum folic acid, red blood cell folic acid reflects the long-term storage level of folic acid in the body, and is not easily affected by recent folic acid intake. It is considered by many scholars to be a better indicator of folic acid (Farrell, C.J., S.H. folate: what to do in clinical practice? Clin Chem Lab Med, 2013.51(3): p.555-69.).
  • the detection of erythrocyte folic acid based on the protein binding method has the problem of low accuracy.
  • Owen et al. (2003) compared the detection data of 5 different brands of folic acid detection kits and found that the difference in the value of erythrocyte folic acid among different detection kits was as high as 250%. , significantly higher than the serum folic acid difference of 40% (Owen,W.E.and W.L.Roberts,Comparison of five automated serum and whole blood folate assays.Am J Clin Pathol,2003.120(1):p.121-6.).
  • the inaccuracy of red blood cell folic acid detection severely limits its wide application in clinical practice.
  • 5-methyltetrahydrofolate acts as a methyl donor and directly participates in the remethylation of homocysteine, which is the main active state of folic acid in cells.
  • 5-MTHF concentration is directly affected by the activity of methylenetetrahydrofolate reductase (MTHFR).
  • MTHFR 677 site C->T polymorphism will reduce the activity of the enzyme by 70%, significantly reduce the concentration of 5-MTHF, thereby increasing the risk of neural tube defects (Yan, L., et al., Association of the Maternal MTHFR C677T Polymorphism with Susceptibility to Neural Tube Defects in Offsprings: Evidence from 25 Case-Control Studies.PLoS ONE,2012.7(10):p.e41689.).
  • Mass spectrometry detection can accurately identify a single intermediate small molecule in the folic acid metabolism process through its unique chromatographic separation using the mass-to-charge ratio of the primary and secondary target ions, so the detection is more accurate.
  • Whole blood is made up of blood cells (mainly red blood cells) and a fluid portion, plasma.
  • Folic acid exists in both intracellular and plasma, and plasma folic acid accounts for about 6% of whole blood folic acid, and this ratio has individual differences (Zhang, M., et al., Population RBC folate concentrations can be accurately estimated from measured whole blood folate, measured hemoglobin, and predicted serum folate—cross-sectional data from the NHANES 1988–2010. The American Journal of Clinical Nutrition, 2020.111(3):p.601-612.).
  • the first method is to extrapolate erythrocyte folate concentration from whole blood folate concentration. Specifically, first a small portion of whole blood was taken out to measure the hematocrit; then a small portion of whole blood was taken out to measure the concentration of folic acid in the whole blood. Then divide whole blood folic acid concentration by hematocrit to estimate red blood cell folic acid concentration (Smith, D.E., et al., Quantitative determination of erythrocyte folate vitamin distribution by liquid chromatography-tandem mass spectrometry. Clin Chem Lab Med, 2006.44(4): p.450-9; De Bruyn, E., B.
  • the current routine is to add 4-9 times the volume of 1% ascorbic acid to a portion of EDTA whole blood, incubate at 37°C or room temperature for about 30-120 minutes, and then add it to the solid-phase extraction column. After washing, the folic acid was eluted with a solution containing methanol, acetonitrile, acetic acid and ascorbic acid.
  • a solution containing methanol, acetonitrile, acetic acid and ascorbic acid Such purified and concentrated folic acid can be directly used (Fazili, Z. and C.M.Pfeiffer, Measurement of Folates in Serum and Conventionally Prepared Whole Blood Lysates: Application of an Automated 96-Well Plate Isotope-Dilution Tandem Mass Spectrometry Method.
  • the purpose of the present invention is to provide a new detection method of red blood cell folic acid.
  • the method of the invention mainly includes red blood cell preparation, transportation, folic acid extraction and new methods of concentration calculation before mass spectrometry detection, so that folic acid in red blood cells can be measured by LC-MS/MS method.
  • the present invention provides a kind of detection method of erythrocyte folic acid, and this method comprises the following steps:
  • Erythrocyte folic acid concentration folic acid concentration in erythrocyte suspension/hematocrit of erythrocyte suspension; or
  • Erythrocyte folic acid concentration folic acid concentration in erythrocyte lysate/(hemoglobin concentration in erythrocyte lysate*0.0029).
  • the isotonic non-toxic liquid refers to a pH-neutral isotonic non-toxic liquid that can be used in the rinsing process of red blood cells.
  • the isotonic non-toxic liquid includes, but is not limited to, physiological saline, phosphate buffer and the like.
  • S1 includes the following steps: place the fasting venous blood sample in an anticoagulant tube (for example, EDTA-K2 anticoagulant tube), store it temporarily in the dark at 0 to 4°C, and store it at 0 to 4°C within 72 hours.
  • an anticoagulant tube for example, EDTA-K2 anticoagulant tube
  • S1' includes the following steps: place the fasting venous blood sample in an anticoagulant tube (for example, EDTA-K2 anticoagulant tube), store it temporarily in the dark at 0 to 4°C, and store it at 0 to 4 hours within 72 hours.
  • an anticoagulant tube for example, EDTA-K2 anticoagulant tube
  • Transport to the local sample disposal point in the dark at °C After receiving the sample at the disposal point, complete the following operations: conventional centrifugation to separate plasma (for example, 2000g, 10min), discard the supernatant, and add isotonic non-toxic liquid (preferably saline); Mix well, centrifuge (for example, 2000g, 10min), discard the supernatant, collect red blood cells, and freeze at -40 to -80°C (preferably at -80°C or in dry ice); transport the sample to the testing laboratory in dry ice; detect After the laboratory receives the sample, the following operations are completed: add 1% ascorbic acid solution twice the volume of red blood cells to the frozen red blood cells, thaw and suspend the red blood cells to make red blood cell lysate; detect the hemoglobin concentration of the red blood cell lysate.
  • conventional centrifugation to separate plasma for example, 2000g, 10min
  • isotonic non-toxic liquid preferably saline
  • Mix well centrifuge (for example, 2000
  • the folic acid includes various intermediate forms in the folic acid metabolic pathway, including but not limited to folic acid (folic acid), 5-methyltetrahydrofolic acid (5-methyltetrahydrofolic acid), tetrahydrofolic acid (tetrahydrofolic acid) acid), 5-formyltetrahydrofolic acid (5-formyltetrahydrofolic acid), 5,10-methylenetetrahydrofolic acid (5,10-methenyltetrahydrofolic acid), etc.
  • the folic acid extract in the step (2) of the present invention refers to an aqueous solution that can provide an antioxidant environment and contains an enzyme capable of hydrolyzing folic acid polyglutamate into folic acid monoglutamate.
  • the folic acid extract comprises 1% ascorbic acid (or DTT, TCEP, ⁇ -mercaptoethanol), 1 ⁇ g/ml GGH recombinase (or animal plasma or tissue extract containing GGH enzyme, or animal tissue Purified GGH enzyme), isotope-labeled 5-methyltetrahydrofolate internal standard.
  • step (2) of the present invention the process of performing LC-MS/MS detection of folic acid on the supernatant is known to those skilled in the art. For example, it is performed by reversed-phase column-based (ultra)high-performance liquid chromatography coupled with triple quadrupole mass detection. Specific chromatographic separation and mass spectrometry detection parameters have been reported in many documents, and the parameter settings of different examples are slightly different, but basically similar.
  • the LC-MS/MS detection conditions are as follows:
  • Chromatographic conditions use ACQUITY UPLC BEH C18 Column, 1.7 ⁇ m, 2.1mm X 50mm for separation, mobile phase water (0.1% formic acid) as phase A, methanol (0.1% formic acid) as phase B, column temperature 40°C; The sample volume is 5 ⁇ L, the flow rate is 0.5ml/min, and gradient elution is performed;
  • Mass spectrometry conditions Electrospray ionization source was used to scan mass spectrometry in positive ion acquisition mode and multiple reaction monitoring mode.
  • the chemical reagent capable of precipitating protein refers to an organic or inorganic solvent capable of causing protein precipitation.
  • the chemical reagents capable of precipitating proteins include, but are not limited to, trichloroacetic acid (TCA), methanol, acetonitrile, zinc sulfide solution, chloroform-methanol mixture, and the like.
  • step (2) includes: adding folic acid extract (1% ascorbic acid, 1 ⁇ g/ml GGH recombinant enzyme, isotope-labeled 5-methyltetrahydrofolate internal standard), incubated at 37°C for 1h; then added trichloroacetic acid (TCA) solution (for example, 10% TCA solution), let stand (for example, 30 minutes), centrifuged ( For example, 13000rpm, 4°C, 10min), take the supernatant for folic acid LC-MS/MS detection, and calculate the folic acid concentration in the erythrocyte suspension or erythrocyte lysate according to the standard concentration.
  • TCA trichloroacetic acid
  • FIG. 1 is a schematic flow chart of a detection method according to an embodiment of the present invention.
  • Fig. 2 is a schematic flowchart of a detection method according to another embodiment of the present invention.
  • Figure 3 is a comparison chart of the detection results of the detection method of the present invention and the Beckman Coulter Access Folate assay red blood cell folic acid detection method in Example 1.
  • Fig. 4 is the figure that the distribution of red blood cell folic acid detected by the detection method of the present invention is compared with the detection results of the commercial kit Access Folate Kit [A98032] (Beckman Coulter) and the microbiological method in Example 1.
  • the left picture is the detection of the present invention
  • the middle picture is the detection of the Beckman kit
  • the right picture is the detection of the microbial method.
  • Example 5 is a graph showing the effect of comparing the 4°C standard shipping procedure in Example 1 and the dry ice freezing shipping procedure in Example 2 on the results of red blood cell folic acid.
  • red blood cell suspension Use a 2ml EDTA-K2 anticoagulant tube to draw a tube of fasting venous blood, gently invert it 5 times and shake well, store it in a refrigerator at 4°C in the dark, and transport it to the processing point within 72 hours at 4°C to complete the preparation of red blood cells.
  • centrifuge at 2000g for 10min at room temperature discard the plasma
  • add 4ml of pre-cooled normal saline shake well, centrifuge at 2000g for 10min, and discard the supernatant.
  • pre-cooled normal saline about twice the volume of red blood cells, shake gently to suspend the red blood cells, and make red blood cell normal saline suspension (hereinafter referred to as red blood cell suspension).
  • Mass spectrometry conditions Electrospray ionization source was used to scan mass spectrometry in positive ion acquisition mode and multiple reaction monitoring mode. Analyte transition information:
  • the concentration of folic acid in the red blood cell suspension was calculated according to the standard concentration.
  • RBC folic acid concentration folic acid concentration in erythrocyte suspension/hematocrit of erythrocyte suspension
  • the same EDTA blood was divided into 5 tubes of equal volume, 1ml in each tube, and sent to a Maternal and Child Health Hospital in Beijing on the day of blood collection for Beckman Coulter Access Folate assay red blood cell folic acid test. After the test is completed, send it to the processing point at 4°C within 48 hours. After processing according to the above red blood cell preparation procedure, go to the SCIEX machine 6500+LC-MS/MS mass spectrometer detection, using the folic acid calculation formula in (3) above to obtain the red blood cell folic acid concentration.
  • Figure 3 is a comparison chart of the detection results of the detection method of the present invention and the Beckman Coulter Access Folate assay red blood cell folic acid detection method in Example 1.
  • This study detected the basic red blood cell folic acid of 75 women of childbearing age (without folic acid supplementation), and sequenced their MTHFR 677 loci to obtain three genes of MTHFR 677 C/C, C/T, and T/T Erythrocyte folic acid distribution in type population.
  • Ni et al(2017.80(3):p.147-153.) analyzed the red blood cell folic acid of 330 people using the protein-binding commercial kit Access Folate Kit[A98032](Beckman Coulter), and simultaneously detected their MTHFR 677 The polymorphisms of loci were used to obtain the distribution status of erythrocyte folic acid in the three genotypes.
  • Crider et al (2011) used microbiological methods to detect the basal red blood cell folic acid levels of 932 women of childbearing age, and at the same time detected their MTHFR 677 polymorphisms.
  • Fig. 4 is the graph that uses the red blood cell folic acid distribution that detection method of the present invention detects in embodiment 1 and uses commercial kit Access Folate Kit [A98032] (Beckman Coulter) and microbiological method detection result to compare.
  • the left figure is the detection method of the present invention
  • the middle figure is the Beckman kit detection
  • the right figure is the microbial method detection.
  • the results of the detection by the method of the present invention show that the erythrocyte 5-MTHF level of the population of the folate metabolizing enzyme MTHFR 677T/T genotype is significantly lower than that of the C/C type and the C/T type population, and there is a significant difference.
  • Microbiological method can also detect the difference of red blood cell folic acid 5-MTHF level in MTHFR 677T/T genotype population compared with C/C type and C/T type population, indicating that MTHFR 677T/T can reduce red blood cell folic acid 5-MTHF level .
  • the reduced level of erythrocyte 5-MTHF coincides with the reduced enzymatic activity of the MTHFR C677T mutation.
  • an alternative procedure of dry ice freezing transportation can be used.
  • the remaining red blood cells were immediately frozen at -80°C or in dry ice, and transported to the processing point on dry ice.
  • red blood cell lysate After receiving the frozen blood sample at the processing point, add 1% ascorbic acid about twice the volume of red blood cells before the sample is thawed, shake well to thaw and suspend the red blood cells, and make red blood cell lysate. Take out 100 ⁇ l of erythrocyte lysate and detect hemoglobin by absorbance photometry. Take out 100 ⁇ l of red blood cell lysate for folic acid extraction, or freeze at -80°C until folic acid extraction.
  • Red blood cell folic acid concentration folic acid concentration in red blood cell lysate / (red blood cell lysate hemoglobin concentration * 0.0029)
  • the inventor collected the blood of 9 people, and each sample was divided into two equally, one of which was operated according to Example 1 and transported at 4°C, and the other was transported with red blood cells rinsed according to Example 2 and then frozen in dry ice. After receiving the samples, extract folic acid according to the respective procedures, and perform LC-MS/MS detection and calculation.
  • Example 5 is a graph showing the effect of comparing the 4°C standard shipping procedure in Example 1 and the dry ice freezing shipping procedure in Example 2 on the results of red blood cell folic acid.

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Abstract

一种红细胞叶酸的检测方法，包括以下步骤：(1)红细胞准备程序，其中制成红细胞悬液，检测红细胞悬液的红细胞压积，或者制成红细胞裂解液，检测红细胞裂解液的血红蛋白浓度；(2)叶酸提取和检测，其中对叶酸提取所得上清液进行叶酸LC-MS/MS检测，并根据标准品浓度计算出红细胞悬液或红细胞裂解液中叶酸的浓度；(3)叶酸浓度计算，其中根据以下公式计算叶酸浓度：红细胞叶酸浓度＝红细胞悬液中叶酸浓度/红细胞悬液的红细胞压积；或红细胞叶酸浓度＝红细胞裂解液中叶酸浓度/(红细胞裂解液血红蛋白浓度*0.0029)。

Description

红细胞叶酸的检测方法 技术领域
本发明涉及叶酸检测领域，具体而言，涉及红细胞叶酸的检测方法。
背景技术
叶酸水平与生殖健康密切相关。叶酸水平临床检测分血清(血浆)检测和红细胞检测两种。
目前血清(血浆)检测技术比较成熟，其检测方法有三种：微生物法、蛋白结合法和液相色谱-串联质谱法(LC-MS/MS)。
微生物法利用乳杆菌生长速度对叶酸敏感的原理，通过测量经过一定时长生长后的细菌的浑浊度来反映培养液中添加的叶酸的浓度(Grossowicz,N.,et al.,Determination of folic acid metabolites in normal subjects and in patients with nutritional megaloblastic anemia.Proc Soc Exp Biol Med,1962.109:p.770-3.)。
微生物法被临床营养学界公认为叶酸检测的金标准，常被新方法用来进行性能对比(Yetley,E.A.,et al.,Biomarkers of folate status in NHANES:a roundtable summary.Am J Clin Nutr,2011.94(1):p.303S-312S)。微生物法的主要缺点是检测耗时长，一般需要3天时间，对无菌操作要求高，不利于商业推广。
自从上世纪70年代，基于蛋白结合原理的商业检测试剂盒得到广泛应用一直到今天。国际上主要的试剂盒厂家包括Roche
Figure PCTCN2022097535-appb-000001
Folate III和Beckman Coulter Access Folate assay等，其检测主要是基于内源性叶酸和外源性标记的叶酸对叶酸结合蛋白的竞争性结合原理,利用化学发光产生信号的蛋白结合法(protein binding assay)。
近年来基于液相色谱-串联质谱(LC-MS/MS)的检测方法也逐渐被广泛用于血清叶酸检测，并显示出非常好的特异性和敏感度(Verstraete,J.,et al.,Clinical determination of folates:recent analytical strategies and challenges.Anal Bioanal Chem,2019.411(19):p.4383-4399.)。
血清学检测的主要缺点在于其反映的是近期叶酸摄入量，容易受一过性膳食因素的影响，波动性大，其浓度对避免出生缺陷的参考价值并没有得到学术界的广泛认可。WHO在一份预防神经管缺陷的营养学指南中指出育龄妇女的血清或血浆叶酸阈值对预防神经管缺陷是不充分的；红细胞叶酸浓度必须达到906nmol/L以上才能最大化的预防神经管缺陷(Cordero,A.M.,et al.,Optimal serum and red blood cell folate concentrations in  women of reproductive age for prevention of neural tube defects:World Health Organization guidelines.MMWR Morb Mortal Wkly Rep,2015.64(15):p.421-3.)。
相对血清叶酸，红细胞叶酸反映了体内叶酸长期存储水平，不易受近期叶酸摄入的影响，被很多学者认为是更好的叶酸指标(Farrell,C.J.,S.H.Kirsch,and M.Herrmann,Red cell or serum folate:what to do in clinical practice？Clin Chem Lab Med,2013.51(3):p.555-69.)。
但基于蛋白结合法的红细胞叶酸检测存在精准度低的问题，如Owen等(2003)比较了5种不同品牌的叶酸检测试剂盒的检测数据，发现不同检测试剂盒间红细胞叶酸值差异高达250％，显著高于血清叶酸差异值40％(Owen,W.E.and W.L.Roberts,Comparison of five automated serum and whole blood folate assays.Am J Clin Pathol,2003.120(1):p.121-6.)。红细胞叶酸检测的不精准性严重限制了其在临床上的广泛应用。
蛋白结合法检测更重要的缺陷的在于其检测是总叶酸，不能区分叶酸在体内各种不同的中间代谢产物，而不同的中间代谢物具有不同的功能。特别地来讲，中间代谢物之一5-甲基四氢叶酸(5-MTHF)作为甲基的供体，直接参与同型半胱氨酸再甲基化，是叶酸在细胞内主要的活性态。5-MTHF浓度直接受亚甲基四氢叶酸还原酶(MTHFR)活性的影响。MTHFR 677位点C->T多态性会降低酶的活性70％，显著降低5-MTHF的浓度，从而提升神经管缺陷的风险(Yan,L.,et al.,Association of the Maternal MTHFR C677T Polymorphism with Susceptibility to Neural Tube Defects in Offsprings:Evidence from 25 Case-Control Studies.PLoS ONE,2012.7(10):p.e41689.)。而使用蛋白结合法(Bio-Rad QuantaPhase II radioassay)检测到的红细胞总叶酸浓度在MTHFR677不同基因型的人群中呈现出TT>CT>CC型的反常分布，这与被WHO认为是叶酸检测金标准的微生物法的检测结果是截然相反的(Crider et al:MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large,population-based,double-blind trial of folic acid supplementation.The American Journal of Clinical Nutrition 2011,93(6):1365-1372)。
质谱检测可以通过其特有的色谱分离连用一级和二级目标离子质荷比精确地鉴定叶酸代谢过程中的单个中间小分子，因此检测更加精准。
近年来有多篇文献报导使用LC-MS/MS技术检测全血叶酸，其检测结果比化学发光法更接近叶酸检测金标准微生物法的检测结果，显示了更高的精准度(Verstraete,J.,et al.,Clinical determination of folates:recent analytical strategies and challenges.Anal Bioanal Chem,2019.411(19):p.4383-4399.)。但是目前还没有报道将质谱检测技术运用到红细胞叶酸的检测方法中。
全血由血细胞(主要是红细胞)和液体部分血浆构成。细胞内和血浆中都有叶酸的存在，其中血浆叶酸约占全血叶酸的6％左右，且此比例具有个体差异(Zhang,M.,et al.,Population RBC folate concentrations can be accurately estimated from measured whole blood folate,measured hemoglobin,and predicted serum folate—cross-sectional data from the NHANES 1988–2010.The American Journal of Clinical Nutrition,2020.111(3):p.601-612.)。
文献中有两种方法估算红细胞叶酸浓度。第一种方法是根据全血叶酸浓度推算红细胞叶酸浓度。具体地，首先取出一小份全血，测量红细胞压积；再取出一小份全血，测得全血叶酸浓度。再将全血叶酸浓度除以红细胞压积以估算红细胞叶酸浓度(Smith,D.E.,et al.,Quantitative determination of erythrocyte folate vitamer distribution by liquid chromatography-tandem mass spectrometry.Clin Chem Lab Med,2006.44(4):p.450-9；De Bruyn,E.,B.Gulbis,and F.Cotton,Serum and red blood cell folate testing for folate deficiency:new features？European Journal of Haematology,2014.92(4):p.354-359.)。这种方法由于忽略了血浆叶酸的贡献及占比的差异性，会造成红细胞叶酸浓度高估和不准确。第二种方法是先测得红细胞压积和全血叶酸浓度，再将同一管血离心取得血浆，测得血浆叶酸浓度，再根据公式“红细胞叶酸＝[全血叶酸-血浆叶酸(1-红细胞压积)]/红细胞压积”估算出红细胞叶酸(Hoffbrand,A.V.,B.F.A.Newcombe,and D.L.Mollin,Method of assay of red cell folate activity and the value of the assay as a test for folate deficiency.Journal of Clinical Pathology,1966.19(1):p.17-28；Huang,Y.,et al.,Quantification of key red blood cell folates from subjects with defined MTHFR 677C>T genotypes using stable isotope dilution liquid chromatography/mass spectrometry.Rapid Commun Mass Spectrom,2008.22(16):p.2403-12.)。这种方法的缺陷是需要分别做血浆和全血的叶酸检测，增加了检测误差和成本。
在全血叶酸提取方面，目前常规做法是往一份EDTA全血中加入4-9倍体积的1％抗坏血酸，37℃或常温孵育30-120分钟左右后，将之加入固相萃取柱子中，经过洗涤后用含有甲醇、乙腈、乙酸、抗坏血酸的溶液将叶酸洗脱下来。这样经过纯净和浓缩后的叶酸可直接(Fazili,Z.and C.M.Pfeiffer,Measurement of Folates in Serum and Conventionally Prepared Whole Blood Lysates:Application of an Automated 96-Well Plate Isotope-Dilution Tandem Mass Spectrometry Method.Clinical Chemistry,2004.50(12):p.2378-2381.)或氮气吹干再复溶后进入液相色谱-串联质谱进行检测(Huang,Y.,et al.,Quantification of key red blood cell folates from subjects with defined MTHFR 677C>T genotypes using stable isotope dilution liquid chromatography/mass spectrometry.Rapid  Commun Mass Spectrom,2008.22(16):p.2403-12.)。这些提取方法程序繁琐，即增加了成本，降低了效率，也有可能因步骤多而增加了检测误差。
发明内容
针对现有技术的不足，本发明人进行了广泛深入的研究，并完成了本发明。
本发明的目的是提供一种新的红细胞叶酸的检测方法。本发明方法主要包括质谱检测前的红细胞准备，运输，叶酸提取和浓度计算的新方法，使红细胞中的叶酸可以被LC-MS/MS方法测量。
一方面，本发明提供了一种红细胞叶酸的检测方法，该方法包括以下步骤：
(1)红细胞准备程序
S1，将空腹静脉血样置于抗凝管中，置于0至4℃保存和运输，最长72小时内进行如下操作：使用0至4℃等渗无毒性液体润洗红细胞至少1次并制成红细胞悬液；检测红细胞悬液的红细胞压积(HCT)；或者，
S1’，将空腹静脉血样置于抗凝管中，置于0至4℃保存或运输，最长72小时内进行如下操作：使用0至4℃等渗无毒性液体润洗红细胞至少1次，离心，收集红细胞，在-40至-80℃(优选在-80℃或在干冰中)冻存(按此条件冻存后可长时间保存和运输)；向冻存的红细胞中加入两倍红细胞体积的1％抗坏血酸溶液，使红细胞解冻混悬，制成红细胞裂解液；检测红细胞裂解液的血红蛋白浓度；
(2)叶酸提取和检测
分别向S1中得到的红细胞悬液或S1’中得到的红细胞裂解液和叶酸标准品溶液中加入叶酸提取液，37℃孵育30min-2h；再使用能够沉淀蛋白质的化学试剂去蛋白，得到上清液，直接对上清液进行叶酸LC-MS/MS检测，并根据标准品浓度计算出红细胞悬液或红细胞裂解液中叶酸的浓度；
(3)叶酸浓度计算
根据以下公式计算叶酸浓度
红细胞叶酸浓度＝红细胞悬液中叶酸浓度/红细胞悬液的红细胞压积；或
红细胞叶酸浓度＝红细胞裂解液中叶酸浓度/(红细胞裂解液血红蛋白浓度*0.0029)。
本发明步骤(1)中，所述等渗无毒性液体是指能够用于红细胞润洗过程的pH中性的等渗无毒性液体。优选地，所述等渗无毒性液体包括但不限于，生理盐水、磷酸缓冲液等。
在一个实施方式中，S1包括以下步骤：将空腹静脉血样置于抗凝管(例如，EDTA-K2抗凝管)中，置于0至4℃避光暂存，72小时内0至4℃下避光运输至检测实验室；检 测实验室收到样本后，完成以下操作：常规离心分离血浆(例如，2000g，10min)，弃上清，加入等渗无毒性液体(优选生理盐水)；混匀，离心(例如，2000g，10min)，弃上清，加入两倍红细胞体积的等渗无毒性液体(优选生理盐水)，制作成红细胞等渗无毒性液体悬液。
在一个实施方式中，S1’包括以下步骤：将空腹静脉血样置于抗凝管(例如，EDTA-K2抗凝管)中，置于0至4℃避光暂存，72小时内0至4℃下避光运输至本地样本处置点：处置点收到样本后，完成以下操作：常规离心分离血浆(例如，2000g，10min)，弃上清，加入等渗无毒性液体(优选生理盐水)；混匀，离心(例如，2000g，10min)，弃上清，收集红细胞，在-40至-80℃(优选在-80℃或在干冰中)冻存；样本干冰内运输至检测实验室；检测实验室收到样本后，完成以下操作：向冻存的红细胞中加入两倍红细胞体积的1％抗坏血酸溶液，使红细胞解冻混悬，制成红细胞裂解液；检测红细胞裂解液的血红蛋白浓度。
本发明步骤(2)中，所述叶酸，包括叶酸代谢路径中各种中间形态，包括但不限于叶酸(folic acid)，5甲基四氢叶酸(5-methyltetrahydrofolic acid)，四氢叶酸(tetrahydrofolic acid)，5-甲酰基四氢叶酸(5-formyltetrahydrofolic acid)，5,10-亚甲基四氢叶酸(5,10-methenyltetrahydrofolic acid)等。
本发明步骤(2)中所述叶酸提取液是指能提供抗氧化环境，并且含有能将叶酸多聚谷氨酸盐水解为叶酸单谷氨酸盐的酶的水溶液。在一个实施方式中，所述叶酸提取液包含1％抗坏血酸(或DTT，TCEP，β-巯基乙醇)，1μg/ml GGH重组酶(或含有GGH酶的动物血浆或组织提取液，或从动物组织中纯化的GGH酶)，同位素标记的5-甲基四氢叶酸内标。
本发明步骤(2)中，对上清液进行叶酸LC-MS/MS检测的过程为本领域技术人员已知的。例如，它由基于反相色谱柱的(超)高效液相色谱，联用三重四极杆质谱检测完成。具体色谱分离和质谱检测参数在多篇文献中有报导，不同的实例参数设置略有不同，但基本类似。
在一个实施方式中，LC-MS/MS检测条件如下：
仪器：SCIEX
Figure PCTCN2022097535-appb-000002
6500+LC-MS/MS
色谱条件：色谱柱使用ACQUITY UPLC BEH C18 Column,1.7μm,2.1mm X 50mm进行分离，流动相用水(0.1％甲酸)作为A相，甲醇(0.1％甲酸)作为B相，柱温40℃；进样量为5μL，流速0.5ml/min，进行梯度洗脱；
质谱条件：采用电喷雾离子源，以正离子采集模式通过多反应监测模式进行质谱扫描。
本发明步骤(2)中，所述能够沉淀蛋白质的化学试剂是指能导致蛋白沉淀的有机或无机溶剂。优选地，所述能够沉淀蛋白质的化学试剂包括但不限于，三氯乙酸(TCA)，甲醇，乙腈，硫化锌溶液，氯仿-甲醇混合液等。
在一个实施方式中，步骤(2)包括：分别向S1中得到的红细胞悬液或S1’中得到的红细胞裂解液和叶酸标准品溶液中加入叶酸提取液(1％抗坏血酸，1μg/ml GGH重组酶，同位素标记的5-甲基四氢叶酸内标)，37℃孵育1h；再加入三氯乙酸(TCA)溶液(例如，10％TCA溶液)，静置(例如，30分钟)，离心(例如，13000rpm，4℃，10min)，取上清液进行叶酸LC-MS/MS检测，并根据标准品浓度计算出红细胞悬液或红细胞裂解液中叶酸的浓度。
经验证，本发明的检测有更高的准确度和临床价值。
在上文中已经详细地描述了本发明，但是上述实施方式本质上仅是例示性，且并不欲限制本发明。此外，本文并不受前述现有技术或发明内容或以下实施例中所描述的任何理论的限制。
除非另有明确说明，在整个申请文件中的数值范围包括其中的任何子范围和以其中给定值的最小子单位递增的任何数值。除非另有明确说明，在整个申请文件中的数值表示对包括与给定值的微小偏差以及具有大约所提及的值以及具有所提及的精确值的实施方案的范围的近似度量或限制。除了在详细描述最后提供的工作实施例之外，本申请文件(包括所附权利要求)中的参数(例如，数量或条件)的所有数值在所有情况下都应被理解为被术语“大约”修饰，不管“大约”是否实际出现在该数值之前。“大约”表示所述的数值允许稍微不精确(在该值上有一些接近精确；大约或合理地接近该值；近似)。如果“大约”提供的不精确性在本领域中没有以这个普通含义来理解，则本文所用的“大约”至少表示可以通过测量和使用这些参数的普通方法产生的变化。例如，“大约”可以包括小于或等于10％，小于或等于5％，小于或等于4％，小于或等于3％，小于或等于2％，小于或等于1％或者小于或等于0.5％的变化。
附图说明
图1为本发明的一个实施方式的检测方法的流程示意图。
图2为本发明的另一个实施方式的检测方法的流程示意图。
图3为实施例1中使用本发明检测方法与Beckman Coulter Access Folate assay红细胞叶酸检测方法的检测结果的对比图。
图4为实施例1中使用本发明检测方法检测的红细胞叶酸分布与使用商业试剂盒Access Folate Kit[A98032](Beckman Coulter)检测结果和微生物法进行对比的图。其 中，左图为本发明检测；中图为贝克曼试剂盒检测，右图为微生物法检测。
图5为显示比较实施例1中的4℃标准运输程序和实施例2中的干冰冷冻运输程序对红细胞叶酸结果的影响的图。
具体实施方式
在下文中，将通过实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。
下述实施例所使用的实验方法如无特殊说明，均为常规方法。
下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
Figure PCTCN2022097535-appb-000003
实施例
实施例1：
本发明检测方法
(1)标准红细胞准备程序
用2ml EDTA-K2抗凝管抽取1管空腹静脉血，轻缓颠倒5次摇匀，置于4℃冰箱避光暂存，72小时内4℃运输至处理点完成红细胞准备。处理点收到血液样本后，在常温2000g下离心10min，吸弃血浆，加入4ml预冷的生理盐水，充分摇匀，在2000g下离心10min，吸弃上清液。再加入大约两倍红细胞体积的预冷生理盐水，轻轻摇匀使红细胞混悬，制作成红细胞生理盐水悬液(以下简称红细胞悬液)。取出500μl红细胞悬液进行血常规检测，或离心法测得HCT(红细胞压积)。再取出100μl红细胞悬液进行叶酸提取，或-80℃冻存直到叶酸提取。
(2)叶酸提取和检测
分别向100μl红细胞悬液和叶酸标准品溶液中加入400μl叶酸提取液(1％抗坏血酸，1μg/ml GGH重组酶，同位素标记的5-甲基四氢叶酸内标)，37℃孵育1h。再加入500μl 10％三氯乙酸(TCA)，静置30分钟，13000rpm，4℃离心10min，取100μl上清液进行叶酸LC-MS/MS检测。
LC-MS/MS检测条件如下：
仪器：SCIEX
Figure PCTCN2022097535-appb-000004
6500+LC-MS/MS
色谱条件：色谱柱使用ACQUITY UPLC BEH C18 Column,1.7μm,2.1mm X 50mm进行分离，流动相用水(0.1％甲酸)作为A相，甲醇(0.1％甲酸)作为B相，柱温40℃。进样量为5μL，流速0.5ml/min，进行梯度洗脱。
质谱条件：采用电喷雾离子源，以正离子采集模式通过多反应监测模式进行质谱扫描。分析物离子对信息：
Figure PCTCN2022097535-appb-000005
根据标准品浓度计算出红细胞悬液中叶酸的浓度。
(3)叶酸浓度计算
红细胞叶酸浓度＝红细胞悬液中叶酸浓度/红细胞悬液的红细胞压积
结果：
(1)使用上述红细胞准备程序后经SCIEX
Figure PCTCN2022097535-appb-000006
6500+LC-MS/MS质谱仪检测结果与北京某妇幼保健院的Beckman Coulter Access Folate assay红细胞叶酸检测结果对比：
同一份EDTA血液分装成等量的5管，每管1ml，于采血日当天送北京某妇幼保健院进行Beckman Coulter Access Folate assay红细胞叶酸检测。检测完毕后，48小时内4℃寄往处理点，按上述红细胞准备程序处理后，上机SCIEX
Figure PCTCN2022097535-appb-000007
6500+LC-MS/MS质谱仪检测，用本上述(3)中的叶酸计算公式得出红细胞叶酸浓度。
图3为实施例1中使用本发明检测方法与Beckman Coulter Access Folate assay红细胞叶酸检测方法的检测结果的对比图。
从图3可以看出，两种检测方法的CV值相当(均<10％)，体现出本发明具有优良的精准度。但本发明检测数据与Beckman Coulter Access Folate assay检测的结果显著不同(Beckman试剂盒测得均值1586nmol/L,本发明测得均值489nmol/L，差异3.2倍，p<0.0001)。此外，用本发明方法检测了75名育龄妇女的基础红细胞叶酸(未进行叶酸增补)，测得红细胞叶酸中位值500nmol/L(42-1403nmol/L,2.5th-97.5th percentile)。Chen et al(2019)用叶酸检测金标准的微生物法检测了565例育龄妇女的基础红细胞叶酸，中位值533nmol/L(253-1244nmol/L 2.5th-97.5th percentile)(Chen,M.Y.,et al.,Defining the plasma folate concentration associated with the red blood cell folate concentration threshold for optimal neural tube defects prevention:a population-based,randomized trial of folic acid supplementation.Am J Clin Nutr,2019.109(5):p.1452-1461.)。因此用本发明测得红细胞叶酸分布比Beckman Coulter Access Folate assay检测的结果更接近文献中报导的用微生物法的检测结果。
(2)使用本发明检测方法检测的红细胞叶酸分布分别与文献中使用商业试剂盒Access Folate Kit[A98032](Beckman Coulter)的检测结果(Ni,J.,et al.,Association between the MTHFR C677T polymorphism,blood folate and vitamin B12 deficiency,and elevated serum total homocysteine in healthy individuals in Yunnan Province,China.Journal of the Chinese Medical Association,2017.80(3):p.147-153.)和使用微生物法得到的检测结果进行对比(Crider et al:MTHFR 677C→T genotype is associated with folate and homocysteine concentrations in a large,population-based,double-blind trial of folic acid supplementation.The American Journal of Clinical Nutrition 2011,93(6):1365-1372)。
本项研究检测了75名育龄妇女的基础红细胞叶酸(未进行叶酸增补)，并对她们的MTHFR 677位点进行测序，从而得出MTHFR 677 C/C，C/T，T/T三种基因型人群的红细胞叶酸分布。Ni et al(2017.80(3):p.147-153.)使用蛋白结合法的商业试剂盒Access Folate Kit[A98032](Beckman Coulter)分析了330人的红细胞叶酸，并同时检测了他们的MTHFR 677位点的多态性，从而得出红细胞叶酸在三种基因型中的分布状态。Crider et al(2011)使用微生物法检测了932育龄妇女的基础红细胞叶酸水平，并同时检测了她们的MTHFR 677位点的多态性。
图4为实施例1中使用本发明检测方法检测的红细胞叶酸分布与使用商业试剂盒Access Folate Kit[A98032](Beckman Coulter)和微生物法检测结果进行对比的图。其中，左图为本发明方法检测；中图为贝克曼试剂盒检测，右图为微生物法检测。
从图4可以看出，本发明方法检测的结果显示叶酸代谢酶MTHFR 677T/T基因型的人群红细胞5-MTHF水平比C/C型和C/T型人群显著降低，存在显著差别。微生物 法也能检测出MTHFR 677T/T基因型人群的红细胞叶酸5-MTHF水平相对于C/C型和C/T型人群的差别，表明MTHFR 677T/T对红细胞叶酸5-MTHF水平具有降低作用。红细胞5-MTHF水平降低与MTHFR C677T突变导致其酶活性降低吻合。而现有的商业试剂盒不能检出上述差别。因为MTHFR 677T/T导致的5-MTHF水平降低已知和神经管缺陷风险升高相关(Yan,L.,et al.,Association of the Maternal MTHFR C677T Polymorphism with Susceptibility to Neural Tube Defects in Offsprings:Evidence from 25 Case-Control Studies.PLoS ONE,2012.7(10):p.e41689.)，因此本发明的检测有更高的准确度和临床价值。
实施例2：
本发明检测方法
(1)红细胞替代准备程序
对于偏远地区冬季无4℃冷链运输条件的，可以采用干冰冷冻运输的替代程序。用2ml EDTA-K2抗凝管抽取1管空腹静脉血，轻缓颠倒5次摇匀，置于4℃冰箱避光暂存，2小时内在常温2000g下离心10min，吸弃血浆，加入4ml预冷的生理盐水，充分摇匀，在2000g下离心10min，吸弃上清液。剩余红细胞立即冻存于-80℃或干冰中，干冰运输至处理点。处理点收到冷冻的血液样本后在样本还未解冻前加入大约两倍红细胞体积的1％抗坏血酸，充分摇匀使红细胞解冻混悬，制作成红细胞裂解液。取出100μl红细胞裂解液用吸光光度法进行血红蛋白检测。再取出100μl红细胞裂解液进行叶酸提取，或-80℃冻存直到叶酸提取。
(2)叶酸提取和检测
分别向100μl红细胞裂解液和叶酸标准品溶液中加入400μl叶酸提取液(1％抗坏血酸，1μg/ml GGH重组酶，同位素标记的5-甲基四氢叶酸内标)，37℃孵育1h。再加入500μl 10％三氯乙酸(TCA)，静置30分钟，13000rpm，4℃离心10min，取100μl上清液进行叶酸LC-MS/MS检测(LC-MS/MS检测方法同实施例1)。根据标准品浓度计算出红细胞悬液中叶酸的浓度。
(3)叶酸浓度计算
红细胞叶酸浓度＝红细胞裂解液中叶酸浓度/(红细胞裂解液血红蛋白浓度*0.0029)
注：0.0029为红细胞血红蛋白与红细胞压积的换算系数，即HCT＝Hg*0.0029。
结果：
研究了实施例1中的4℃标准运输程序和实施例2中的干冰冷冻运输程序对红细胞 叶酸检测结果的影响
发明人采集了9个人的血液，每份样本平分为两份，其中一份按实施例1操作，4℃运输，另一份将红细胞按实施例2润洗后干冰冷冻运输。收到样本后按各自程序提取叶酸，进行LC-MS/MS检测和计算。
图5为显示比较实施例1中的4℃标准运输程序和实施例2中的干冰冷冻运输程序对红细胞叶酸结果的影响的图。
从图5可以看出，显示二者检测结果高度线性相关(r2＝0.95,p<0.0001)。与4℃标准运输程序相比，干冰冷冻运输程序测得的红细胞叶酸平均误差9.2％。

Claims (11)

  1. 一种红细胞叶酸的检测方法，该方法包括以下步骤：
    (1)红细胞准备程序
    S1，将空腹静脉血样置于抗凝管中，置于0至4℃保存和运输，最长72小时内进行如下操作：使用0至4℃等渗无毒性液体润洗红细胞至少1次并制成红细胞悬液；检测红细胞悬液的红细胞压积；或者，
    S1’，将空腹静脉血样置于抗凝管中，置于0至4℃保存或运输，最长72小时内进行如下操作：使用0至4℃等渗无毒性液体润洗红细胞至少1次，离心，收集红细胞，在-40至-80℃冻存；向冻存的红细胞中加入两倍红细胞体积的1％抗坏血酸溶液，使红细胞解冻混悬，制成红细胞裂解液；检测红细胞裂解液的血红蛋白浓度；
    (2)叶酸提取和检测
    分别向S1中得到的红细胞悬液或S1’中得到的红细胞裂解液和叶酸标准品溶液中加入叶酸提取液，37℃孵育30min-2h；再使用能够沉淀蛋白质的化学试剂去蛋白，得到上清液，直接对上清液进行叶酸LC-MS/MS检测，并根据标准品浓度计算出红细胞悬液或红细胞裂解液中叶酸的浓度；
    (3)叶酸浓度计算
    根据以下公式计算叶酸浓度
    红细胞叶酸浓度＝红细胞悬液中叶酸浓度/红细胞悬液的红细胞压积；或
    红细胞叶酸浓度＝红细胞裂解液中叶酸浓度/(红细胞裂解液血红蛋白浓度*0.0029)。
  2. 根据权利要求1所述的红细胞叶酸的检测方法，其中，本发明步骤(1)中，所述等渗无毒性液体是指能够用于红细胞润洗过程的pH中性的等渗无毒性液体。
  3. 根据权利要求2所述的红细胞叶酸的检测方法，其中，所述等渗无毒性液体为生理盐水或磷酸缓冲液。
  4. 根据权利要求1所述的红细胞叶酸的检测方法，其中，S1包括以下步骤：将空腹静脉血样置于抗凝管中，置于0至4℃避光暂存，72小时内0至4℃下避光运输至检测实验室；检测实验室收到样本后，完成以下操作：离心分离血浆，弃上清，加入等渗无毒性液体；混匀，离心，弃上清，加入两倍红细胞体积的等渗无毒性液体，制作成红细胞等渗无毒性液体悬液。
  5. 根据权利要求1所述的红细胞叶酸的检测方法，其中，S1’包括以下步骤：将空腹静脉血样置于抗凝管中，置于0至4℃避光暂存，72小时内0至4℃下避光运输至本地样本处置点：处置点收到样本后，完成以下操作：离心分离血浆，弃上清，加入等渗无毒性液体；混匀，离心，弃上清，收集红细胞，在-40至-80℃冻存；样本干冰内运输至检测实验室；检测实验室收到样本后，完成以下操作：向冻存的红细胞中加入两倍红细胞体积的1％抗坏血酸溶液，使红细胞解冻混悬，制成红细胞裂解液；检测红细胞裂解液的血红蛋白浓度。
  6. 根据权利要求1所述的红细胞叶酸的检测方法，其中，步骤(2)中，所述叶酸提取液是指能提供抗氧化环境，并且含有能将叶酸多聚谷氨酸盐水解为叶酸单谷氨酸盐的酶的水溶液。
  7. 根据权利要求1所述的红细胞叶酸的检测方法，其中，所述叶酸提取液包含：1％抗坏血酸或DTT或TCEP或β-巯基乙醇；1μg/ml GGH重组酶或含有GGH酶的动物血浆或组织提取液或从动物组织中纯化的GGH酶；同位素标记的5-甲基四氢叶酸内标。
  8. 根据权利要求1所述的红细胞叶酸的检测方法，其中，步骤(2)中，对上清液进行叶酸LC-MS/MS检测的检测条件如下：
    仪器：SCIEX
    Figure PCTCN2022097535-appb-100001
    6500+LC-MS/MS
    色谱条件：色谱柱使用ACQUITY UPLC BEH C18 Column,1.7μm,2.1mm X 50mm进行分离，流动相用水(0.1％甲酸)作为A相，甲醇(0.1％甲酸)作为B相，柱温40℃；进样量为5μL，流速0.5ml/min，进行梯度洗脱；
    质谱条件：采用电喷雾离子源，以正离子采集模式通过多反应监测模式进行质谱扫描。
  9. 根据权利要求1所述的红细胞叶酸的检测方法，其中，步骤(2)中，所述能够沉淀蛋白质的化学试剂是指能导致蛋白沉淀的有机或无机溶剂。
  10. 根据权利要求1所述的红细胞叶酸的检测方法，其中，所述能够沉淀蛋白质的化学试剂包括三氯乙酸，甲醇，乙腈，硫化锌溶液，氯仿-甲醇混合液。
  11. 根据权利要求1所述的红细胞叶酸的检测方法，其中，步骤(2)包括：分别 向S1中得到的红细胞悬液或S1’中得到的红细胞裂解液和叶酸标准品溶液中加入叶酸提取液，37℃孵育1h；再加入三氯乙酸溶液，静置，离心，取上清液进行叶酸LC-MS/MS检测，并根据标准品浓度计算出红细胞悬液或红细胞裂解液中叶酸的浓度。
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