WO2018219258A1 - 血清游离甘露糖和葡萄糖的高效液相色谱检测 - Google Patents

血清游离甘露糖和葡萄糖的高效液相色谱检测 Download PDF

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WO2018219258A1
WO2018219258A1 PCT/CN2018/088774 CN2018088774W WO2018219258A1 WO 2018219258 A1 WO2018219258 A1 WO 2018219258A1 CN 2018088774 W CN2018088774 W CN 2018088774W WO 2018219258 A1 WO2018219258 A1 WO 2018219258A1
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performance liquid
glucose
liquid chromatography
mannose
serum
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张丽娟
刘永
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青岛大学附属医院
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    • G01N30/02Column chromatography
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    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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    • G01N30/02Column chromatography
    • G01N30/88Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/8813Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
    • G01N2030/8836Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving saccharides
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    • G01N30/26Conditioning of the fluid carrier; Flow patterns
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Definitions

  • the invention belongs to the field of medicine, and particularly relates to the detection of monosaccharides in serum, in particular to high-performance liquid chromatography (HPLC) detection of serum free mannose and glucose.
  • HPLC high-performance liquid chromatography
  • the content of glucose (Glc) in human serum free monosaccharide is the highest, the normal range is 3.9 ⁇ 6.16mmol / L, and the content of mannose (Man) is about 1% of glucose content.
  • oligosaccharides consisting of 14 monosaccharides (containing 9 mannose, 3 glucose and 2 N-acetylglucosamine) transfer the most newly synthesized protein N in the endoplasmic reticulum - The sugar chain is attached to the site.
  • the analysis time of this method is only 8 min, however, it is also necessary to remove high glucose content through a very complicated pre-treatment step in order to detect mannose.
  • Tadao Taguchi et al. (Clinical Chemistry 49, No. 1, 2003, 181-183) used a post-column derivatization method to determine free mannose in plasma by high performance liquid phase method.
  • the column was an anion exchange column and the detector was fluorescently detected. Device.
  • the analysis time of the method is 57 min, and it is necessary to remove the high content of glucose through a relatively complicated pre-treatment step to detect the mannose; and post-column derivatization is adopted, so that the equipment is required to be high.
  • the common feature of these methods is that the high content of glucose needs to be removed first in order to detect mannose, so the pre-processing steps are cumbersome and the operation process is complicated.
  • Sato et al. (Research in Veterinary Science 84 (2008) 26–29) used ethyl paraben (ABEE) as a derivatization reagent for serum monosaccharides to separate different monosaccharides by high-performance liquid chromatography.
  • the detector detects glucose and the fluorescence detector detects mannose, thus achieving the simultaneous quantitative analysis of glucose and mannose in dog and chicken serum.
  • the method uses a serum dose of 100 ⁇ L, and it is necessary to quantify glucose and mannose by using two standard curves of the two detectors. This method has not been used for quantitative qualitative analysis of glucose and mannose in human serum.
  • the invention patent CN 103969371 B discloses "the application of a method for degrading blood and detecting monosaccharides in cancer detection".
  • the invention patent firstly degrades the polysaccharide sample and the glycoprotein into a monosaccharide component by acid degradation; and then detects the 8 monosaccharides obtained by PMP derivatization and relative degradation of the high-efficiency liquid.
  • the composition and properties of the sample have changed dramatically compared to the serum before degradation. Therefore, the method described in the patent is only applicable to the detection of serum after degradation, and is not suitable for direct detection of serum; in addition, the high-performance liquid chromatography used in the method has a long detection time and low efficiency.
  • the monosaccharide PMP derivatization method is currently the commonly used monosaccharide detection method. Although this method is widely used in various fields, it has not been reported to be applied to the detection of serum free mannose and glucose.
  • the method comprises the following steps: (1) creating alkaline conditions for PMP, (2) adding PMP, derivatization at 70 ° C, (3) adjusting the pH of the derivatized solution, and extracting residual PMP.
  • the reagent for the creation of the alkaline condition in the step (1) is sodium hydroxide and ammonia. Ammonia water is used as a basic condition to create a reagent. After the derivatization is completed, neutralization is not required.
  • the ammonia water after the reaction is removed by drying or evaporation, and the loss rate of the monosaccharide in the PMP extraction process is small.
  • the ammonia water cannot be completely removed, resulting in an alkaline extraction condition of the sample.
  • the PMP residue is severe and the chromatographic peak shape is extremely poor.
  • sodium hydroxide is used as a basic reagent, it is necessary to adjust the derivatized sample to neutral by hydrochloric acid and extract it under neutral conditions.
  • the residual of PMP was significantly reduced, the peak shape of the chromatogram was excellent; however, the recovery rate of mannose was only 80%, and the interference of impurity peaks after galactosamine and glucuronic acid seriously affected the quantitative results.
  • the present invention provides a method for the HPLC analysis of serum free mannose and glucose, which is applied to the determination of free glucose and mannose in serum.
  • the method has simple pre-treatment, analysis time period and accurate detection, which opens a new door for studying the relationship between serum free monosaccharide and disease.
  • the technical scheme of the invention a method for detecting high-performance liquid chromatography of serum free mannose and glucose, comprising the following steps:
  • the molar ratio to NaOH is from 1.5:1 to 2.5:1.
  • the derivatized solution was directly adjusted to acidity and the residual PMP was extracted, thereby eliminating the influence of the impurity peak on the quantification of the monosaccharide and reducing the loss rate of the monosaccharide.
  • Sample B was centrifuged at high speed to obtain sample C; the high speed centrifugation was carried out by centrifugation at a speed of 13,000 r/min for 10 min.
  • the sample C obtained in the step (1) is detected by high performance liquid chromatography; the detection method is an external standard method, and the rhamnose which is not present in the human body is used as the internal reference monosaccharide.
  • the external standard method includes the following steps:
  • 0.3M NaOH solution add 60 ⁇ L 0.5M PMP solution, react in oven at 70 °C for 1h; remove the reaction to cool to room temperature, add 80 ⁇ L 0.3M HCl, CHCl 3 for three times; centrifuge at 13000r/min for 10min; then use high performance liquid chromatography Performing detection and analysis, taking the concentration of monosaccharide as the abscissa and the corresponding peak area as the ordinate to draw a standard curve;
  • Detection of the sample to be tested Take 10 ⁇ L of the serum sample to be tested, add 20 ⁇ L of ultrapure water, 10 ⁇ L of 0.1 mg/ml rhamnose solution, and perform the sample loading; calculate the free nectar in the serum sample to be tested by using the standard curve obtained in step (1) Sugar and glucose content.
  • the high performance liquid chromatography was an Agilent 1260 high performance liquid phase system, and an Agilent Poroshell EC-C18 column: (4.6 x 100 mm 2.7 ⁇ m).
  • the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column.
  • the blockage By adding 0.1% trifluoroacetic acid to the mobile phase, the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column. The blockage.
  • the high performance liquid chromatography conditions are as follows: detection wavelength: 254 nm, bandwidth 4 nm; reference wavelength: 350 nm, bandwidth 100 nm; column temperature: 37 ° C; flow rate: 1 mL/min; injection volume: 20 ⁇ L.
  • a method for detecting high-performance liquid chromatography of serum free mannose and glucose in a protein glycosylation abnormal disease characterized in that the test sample is serum of a patient with abnormal protein glycosylation disease.
  • a method for detecting high-performance liquid chromatography of serum free mannose and glucose in a protein glycosylation abnormal disease characterized in that the protein glycosylation abnormal disease includes diabetes and gastric cancer.
  • the precision, stability, and reproducibility RSD of the serum free mannose and glucose high-performance liquid chromatography detection methods described in the patent application of the present invention are less than 2%; indicating the reliability of the method, For the determination of serum monosaccharide composition.
  • the high performance liquid chromatography method for detecting serum free mannose and glucose according to the present invention has the following advantages:
  • the pretreatment process of the sample in the invention is simpler, the detection time is shortened, and the detection efficiency is improved;
  • Figure 1 is a high performance liquid chromatographic peak of mannose, rhamnose and glucose.
  • the high performance liquid chromatography was an Agilent 1260 high performance liquid phase system, and an Agilent Poroshell EC-C18 column: (4.6 x 100 mm 2.7 ⁇ m).
  • the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column.
  • the blockage By adding 0.1% trifluoroacetic acid to the mobile phase, the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column. The blockage.
  • Peak 1 peak 2
  • peak 3 peak 4 are the peaks of PMP, Man, Rha and Glc.
  • Example 2 Normal human serum free mannose and glucose detection
  • Sample B was centrifuged at 13,000 r/min for 10 min to obtain sample C.
  • the high performance liquid chromatography was an Agilent 1260 high performance liquid phase system, and an Agilent Poroshell EC-C18 column (4.6 x 100 mm 2.7 ⁇ m).
  • the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column.
  • the blockage By adding 0.1% trifluoroacetic acid to the mobile phase, the pH of the rinse mobile phase is adjusted to be acidic, avoiding the precipitation of proteins and peptides in serum samples under near-neutral and alkaline conditions, thereby avoiding the column. The blockage.
  • Serum free monosaccharide derivatization 2 The reactants in the oven 1 step 1 were taken out, cooled to room temperature, 60 ⁇ L of 0.3 M HCl was added, and extracted 3 times with CHCl 3 to obtain a sample B.
  • Serum free monosaccharide derivatization 2 The reactants obtained in the oven 1 step were taken out, cooled to room temperature, and 85 ⁇ L of 0.3 M HCl was added thereto, and extracted with CHCl 3 three times to obtain a sample B.
  • Sample B was centrifuged at 13,000 r/min for 10 min to obtain sample C.
  • Sample C was detected by high performance liquid chromatography: The content of free mannose and glucose in the serum sample to be tested was calculated from the peak area obtained by the analysis and the standard curve obtained in Example 1.
  • the high performance liquid chromatography was an Agilent 1260 high performance liquid phase system, an Agilent Poroshell EC-C18 column (4.6 x 100 mm 2.7 ⁇ m).
  • Serum free monosaccharide derivatization 2 The reactants in the oven 1 step 1 were taken out, cooled to room temperature, 90 ⁇ L of 0.3 M HCl was added, and extracted 3 times with CHCl 3 to obtain a sample B.
  • Serum free monosaccharide derivatization 2 The reactants which were processed in the oven of step 1 were taken out, cooled to room temperature, and 70 ⁇ L of 0.3 M HCl was added thereto, and extracted with CHCl 3 three times to obtain a sample B.
  • Example 8 Detection of serum free mannose and glucose in gastric cancer patients
  • Sample B was centrifuged at 13,000 r/min for 10 min to obtain sample C.
  • the high performance liquid chromatography was an Agilent 1260 high performance liquid phase system, and an Agilent Poroshell EC-C18 column (4.6 x 100 mm 2.7 ⁇ m).
  • Serum free monosaccharide derivatization 2 The reactants obtained in the oven 1 step were taken out, cooled to room temperature, 100 ⁇ L of 0.3 M HCl was added, and extracted with CHCl 3 three times to obtain a sample B.
  • Example 10 Detection of serum free mannose and glucose in gastric cancer patients
  • Serum free monosaccharide derivatization 2 The reactants obtained in the oven 1 step removal were taken out, cooled to room temperature, 75 ⁇ L of 0.3 M HCl was added, and extracted with CHCl 3 three times to obtain a sample B.
  • the free mannose and glucose contents in the serum of 39 normal persons, 39 cases of diabetes, and 39 cases of gastric cancer patients detected by the method of the present invention were obtained.
  • the results are shown in Table 4.
  • the normal human serum mannose concentration is much lower than the mannose concentration of diabetic patients and gastric cancer patients. It is fully explained that the concentration of serum free mannose is indeed related to abnormal glycosylation diseases such as diabetes and gastric cancer; therefore, the method of the present invention is for studying the relationship between serum free monosaccharide and disease, and searching for clinical detection of diseases. Markers have a very important meaning.
  • the method of the present invention compared with the existing HPLC detection method after monosaccharide derivatization, the method of the present invention shortens the detection time to about 20 minutes, improves the detection efficiency; and the data parallelism is good, which fully proves the present.
  • the precision, accuracy and repeatability of the method of the invention compared with the existing HPLC detection method after monosaccharide derivatization, the method of the present invention shortens the detection time to about 20 minutes, improves the detection efficiency; and the data parallelism is good, which fully proves the present. The precision, accuracy and repeatability of the method of the invention.

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Abstract

基于现有技术中血清游离甘露糖和葡萄糖检测的现状,提供了一种血清游离甘露糖和葡萄糖的高效液相色谱分析方法,将其应用于测定血清中的游离葡萄糖和甘露糖,使样品的前处理过程更加简单,缩短检测时间,提高检测效率。在检测血清样品时,甘露糖,鼠李糖和葡萄糖可以完全分离,而且甘露糖与葡萄糖在进行定量时相互之间不会产生影响,保证检测结果的准确性。

Description

血清游离甘露糖和葡萄糖的高效液相色谱检测 技术领域
本发明属于医药领域,具体涉及血清中单糖的检测,尤其涉及血清游离甘露糖和葡萄糖高效液相色谱(HPLC)检测。
背景技术
人体血清游离单糖中葡萄糖(Glc)的含量最高,正常范围为3.9~6.16mmol/L,甘露糖(Man)的含量约为葡萄糖含量的1%。在真核细胞中,由14个单糖组成的寡糖(含9个甘露糖,3个葡萄糖和2个N-乙酰葡糖胺)在内质网中转移倒大多数新合成的蛋白质的N-糖链连接位点上。在高尔基中9个甘露糖中的6个可以不同程度的被切除,寡糖作为底物形成高甘露糖型、杂化型或复杂性的N-糖链,而甘露糖则随连接有N-糖链的糖蛋白在高尔基网中移动,最终被释放在细胞之外。蛋白质的N-糖化机制解释了甘露糖广泛存在与动物血清中。研究表明,糖尿病患者血清游离单糖中,除葡萄糖明显升高外,甘露糖的含量也较正常人为高。研究报道,异常蛋白质糖基化不仅与糖尿病相关,也与其他代谢综合征,包括癌症和其他复杂疾病有关。因此,测量人血清中的葡萄糖与甘露糖浓度将为遗传性疾病的治疗以及糖尿病和其他蛋白质糖基化异常病人的诊断提供新型分子水平信息。
根据早期的研究报道(2008年以前),检测血液中甘露糖的方法包括高效液相法、毛细管电泳法和酶法。Janmes R.ETCHISION等人(Clinical Chemistry 43,No.3,1997,533–538)采用酶法通过配有脉冲电流检测器的高效阴离子交换色谱分析血清中的游离甘露糖。该方法首先需要通过非常复杂的前处理步骤将高含量的葡萄糖除去,才能对甘露糖进行检测,血清用量为200μL。Hubert A等人(Clinical Chemistry 47,No.7,2001,1319-1321)采用柱前衍生化方法通过毛细管电泳分析血清中游离甘露糖。该方法分析时间仅为8min,然而同样需要通过非常复杂的前处理步骤将高含量的葡萄糖除去,才能对甘露糖进行检测。Tadao Taguchi等人(Clinical Chemistry 49,No.1,2003,181-183)采用柱后衍生化方法通过高效液相法测定了血浆中游离甘露糖,色谱柱为阴离子交换柱,检测器为荧光检测器。该方法的分析时间为57min,需要通过较为复杂的前处理步骤将高含量的葡萄糖除去,才能对甘露糖进行检测;且采用柱后衍生,因此从对设备要求高。
综上所述,这些方法的共同特点是需要首先将高含量的葡萄糖除去,才能对甘露糖进行检测,因此前处理步骤繁琐,操作过程复杂。在2008年,Sato等人(Research in Veterinary Science 84(2008)26–29)采用对氨基苯甲酸乙酯(ABEE)作为血清单糖的衍生化试剂,高效液相法分 离不同单糖,通过紫外检测器检测葡萄糖、荧光检测器检测甘露糖,从而首次实现了对狗与鸡血清中葡萄糖与甘露糖的同时定量分析。该方法血清用量为100μL,需要用两种检测器两种标准曲线分别对葡萄糖与甘露糖进行定量。此方法尚未用于人血清中的葡萄糖与甘露糖定量定性分析。
发明专利CN 103969371 B公开了“一种血液降解得到并检测单糖的方法在癌症检测中的应用”。该发明专利首先将血清样品通过酸降解,将其中的多糖和糖蛋白降解为单糖组分;然后经过PMP衍生和高效液相对降解后得到的8种单糖进行检测。相比降解前的血清,样品组分与性质已经发生巨大变化。因此,该专利所述的方法仅仅适用于降解后血清的检测,不适用于血清的直接检测;此外,该方法采用的高效液相色谱的检测时间长,效率低。受到检测方法的限制,血清中游离甘露糖的深入研究受到了一定的阻碍。因此,建立高效准确的血清中游离甘露糖的检测方法,对于研究血清游离单糖与疾病之间的关系、寻找疾病临床检测的标志物有着非常重要的意义。
单糖的PMP衍生法是目前较为常用的单糖检测方法,虽然该方法在各个领域应用比较广泛,但目前尚无报道将该方法应用于血清游离甘露糖和葡萄糖的检测。该方法包括以下几个步骤:(1)为PMP创建碱性条件,(2)加入PMP,在70℃进行衍生,(3)调节衍生后的溶液pH,将残余PMP萃取出来。步骤(1)中碱性条件的创建试剂有氢氧化钠和氨水。氨水作为碱性条件创建试剂,衍生完成后不需要进行中和,通常是通过烘干或蒸干的方式将反应后的氨水除去,单糖在PMP萃取过程中损失率较小。然而,氨水无法完全去除,导致样品的萃取条件为碱性,进行液相检测时,PMP残余严重,色谱峰型极差。以氢氧化钠作为碱性创建试剂时,需要将衍生后的样品通过盐酸调节成中性,在中性条件下进行萃取。虽然PMP的残余明显减少,色谱峰峰型优异;然而甘露糖的回收率仅为80%,半乳糖胺及葡萄糖醛酸后有杂质峰的干扰,严重影响了定量结果。
在PMP衍生后单糖进行HPLC分析时,国内外报道中均采用的是全多孔型的C18色谱柱。现有PMP衍生后单糖的HPLC分析方法不但耗时较长,而且无法将多种单糖组分的样品在短时间内完成分析。
发明内容
基于现有技术中血清游离甘露糖和葡萄糖检测的现状,本发明提供了一种血清游离甘露糖和葡萄糖HPLC分析方法,将其应用于测定血清中的游离葡萄糖和甘露糖。本方法前处理简单,分析时间段,检测准确,为研究血清游离单糖与疾病之间的关系打开了一扇崭新的大门。
本发明的技术方案:血清游离甘露糖和葡萄糖的高效液相色谱检测方法,包括以下步骤:
(1)血清游离单糖衍生:
①取适量血清样品,向其中依次加入超纯水、鼠李糖(Rha)溶液、氢氧化钠溶液、1-苯基-3甲基-5-吡唑啉酮(PMP)溶液,混合后得到pH=7-14的体系,混合后离心处理,在70℃的温度条件的烘箱中处理1h,得到样品A;所述血清的体积为10μL、超纯水的体积为20μL、鼠李糖溶液的浓度为0.1mg/ml,鼠李糖溶液的体积为10μL;所述NaOH和PMP的摩尔比为2:5;
②将样品A冷却至室温,加入适量HCl溶液,混合后得到pH=1-7的体系,离心处理,然后采用三氯甲烷萃取3次,每次取上清液,得到样品B;所述HCl和NaOH的摩尔比为1.5:1-2.5:1。将衍生后的溶液直接调节至酸性后萃取残余PMP,从而消除了杂质峰对单糖定量的影响,降低了单糖的损失率。
③将样品B高速离心处理,得到样品C;所述高速离心处理为在13000r/min的速度下离心10min。
(2)将步骤(1)得到的样品C采用高效液相色谱进行检测;检测方法为外标法,同时采用人体中不存在的鼠李糖作为内参单糖。所述外标法包括以下几个步骤:
①制作标准曲线:取40μL浓度为0.5、0.25、0.10、0.05、0.025、0.01、0.005、0.0025、0.001、0.0005mg/mL的甘露糖(Man)、葡萄糖(Glc)标准品的混合溶液,加入40μL 0.3M NaOH溶液,再加入60μL 0.5M PMP溶液,70℃烘箱反应1h;取出反应物冷却至室温,加入80μL 0.3M HCl,CHCl 3萃取三次;13000r/min离心10min;然后分别采用高效液相色谱进行检测分析,以单糖的浓度为横坐标,以相应的峰面积为纵坐标,绘制标准曲线;
②检测待测样品:取10μL待测血清样品,加入20μL超纯水,10μL 0.1mg/ml鼠李糖溶液,衍生上样;利用步骤(1)得到的标准曲线计算待测血清样品中游离甘露糖和葡萄糖的含量。
其中,所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱:(4.6×100mm 2.7μm)。高效液相色谱条件如下:(1)检测波长:254nm;参比波长:360nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;PH=5.5的醋酸盐在该条件下没有杂质析出,避免了色谱柱的堵塞,保证了在外标法的线性范围内的完美峰型。梯度洗脱:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L乙酸铵缓冲溶液(PH=5.5)的浓度梯度:85%→78%→76%→85%;该梯度模式可以将8种单糖完美的分离开,并尽量缩短分析时间;(3)柱温:25-50℃;(4)流速1.0mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。通过向流动相中加入 0.1%的三氟乙酸,将冲洗流动相的pH调节成酸性,避免了血清样品中蛋白和多肽类物质在近中性和碱性的条件下析出,从而避免了色谱柱的堵塞。
优选的是,所述高效液相色谱条件如下:检测波长:254nm,带宽4nm;参比波长:350nm,带宽100nm;柱温:37℃;流速:1mL/min;进样体积:20μL。
一种血清游离甘露糖和葡萄糖的高效液相色谱检测方法在蛋白质糖基化异常疾病中的应用,其特征在于:所述检测样品为蛋白质糖基化异常疾病患者的血清。
一种血清游离甘露糖和葡萄糖的高效液相色谱检测方法在蛋白质糖基化异常疾病中的应用,其特征在于:所述蛋白质糖基化异常疾病包括糖尿病、胃癌。
方法学性能的考察:
为了充分验证上述检测方法的实用性,发明人从精密度、准确性和重复性三个角度进行了考察。
(1)精密度试验
将浓度为0.5mg/mL的2种单糖混合溶液衍生,连续进样5次,记录甘露糖、葡萄糖的峰面积,计算其精密度RSD值。从表1-1得知,2种单糖的RSD值依次为1.88%,,0.40%,表明实验的精密度良好。
表1-1 精密度实验
Figure PCTCN2018088774-appb-000001
(2)稳定性试验
将浓度为0.1mg/mL的2种单糖混合溶液衍生,分别在0h、2h、4h、8h、16h、24h进样,并记录甘露糖、葡萄糖的峰面积,计算其精密度RSD值。从表1-2得知,2种单糖的RSD值依次为0.30%,0.44%,从而表明实验的的稳定性良好。
表1-2 稳定性实验
Figure PCTCN2018088774-appb-000002
(3)重复性实验
分别取3份20个正常人的混合血清样品,通过该方法衍生后进样分析,记录甘露糖、葡萄糖,计算其精密度RSD值,从表1-3得知2种单糖的RSD值依次为1.59%,1.67%,从而 表明实验的重复性良好。
表1-3 重复性实验
Figure PCTCN2018088774-appb-000003
综上所述,本发明专利申请所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法的精密度、稳定性以及重复性实验的RSD均小于2%;表明该方法的可靠性,可以用于血清单糖组成的测定。
甘露糖影响因素实验:
(1)甘露糖回收率试验:
在20个混合血清样品中加入10μl 10、50、100、500μmol/L的甘露糖标准溶液,衍生上样,考察在衍生过程中以及血清环境是否对甘露糖的检测产生影响。实验结果表明,衍生过程以及血清环境几乎不影响甘露糖的回收与检测,SD在4.3%以下,甘露糖回收率接近100%(表2-1)。
表2-1 血清甘露糖回收率实验
加入Man的浓度(μmol/L), Man的回收率(%) 标准差(%)
10 101.5 2.7
50 97.4 4.3
100 101.2 3.0
500 101.2 0.5
(2)葡萄糖影响试验:
有研究表明,葡萄糖在弱碱性条件下会产生甘露糖的互变异构体。为验证在血清游离的葡萄糖与甘露糖PMP衍生的弱碱性条件下是否也存在该反应,向20例糖尿病患者混合血清样品中加入0.5、0.25、0.1mg/mL葡萄糖溶液衍生后上样。由表2-2可知,甘露糖的检测并没有随葡萄糖含量的高低而产生改变,从而表明,高浓度的葡萄糖在衍生条件下对甘露糖的测定不会产生影响。
表2-2 不同浓度葡萄糖对甘露糖检测的影响
加入Glc的浓度(mg/mL) Man测定的浓度(μmol/L)
0 44.7
0.1 45.2
0.25 46.9
0.5 42.9
本发明的有益效果:与现有技术相比,本发明所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法具有下述优势:
(1)与其他检测方法相比,本发明中样品的前处理过程更加简单,缩短了检测时间,提高了检测效率;
(2)与现有单糖衍生后的HPLC检测方法相比,本发明的分析时间更短,仅需要20min即可完成分析;
(3)采用本发明检测血清样品时,甘露糖,鼠李糖和葡萄糖可以完全分离,而且甘露糖与葡萄糖在进行定量时相互之间不会产生影响;从而保证了检测结果的准确性。
说明书附图
附图1是甘露糖、鼠李糖和葡萄糖的高效液相色谱峰。
具体实施方式
下面结合实施例对本发明做进一步的说明。
实施例1:绘制外标法标准曲线
(1)单糖衍生:取40μL浓度为0.5、0.25、0.10、0.05、0.025、0.01、0.005、0.0025、0.001、0.0005mg/mL的甘露糖、葡萄糖标准品的混合溶液,加入40μL 0.3M NaOH溶液,再加入60μL 0.5M PMP溶液,70℃烘箱反应1h;取出反应物冷却至室温,加入80μL 0.3M HCl,CHCl 3萃取三次;13000r/min离心10min;
(2)采用高效液相色谱进行检测分析,以单糖的浓度为横坐标,以相应的峰面积为纵坐标,绘制标准曲线(见表3)。
其中,所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱:(4.6×100mm 2.7μm)。高效液相色谱条件如下:(1)检测波长:254nm,带宽4nm;参比波长:350nm,带宽100nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;PH=5.5的醋酸盐在该条件下没有杂质析出,避免了色谱柱的堵塞,保证了在外标法的线性范围内的完美峰型。梯度洗脱:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L乙酸铵缓冲溶液(PH=5.5)的浓度梯度:85%→78%→76%→85%;该梯度模式可以将8种单糖完美的分离开,并尽量缩短分析时间;(3)柱温:37℃;(4)流速:1mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。通过向流动相中加入0.1%的三氟乙酸,将冲洗流动相的pH调节成酸性,避免了血清样品中蛋白和多肽类物质在近中性和碱性的条件下析出,从而避免了色谱柱的堵塞。
表3 甘露糖与葡萄糖外标法检测的标准曲线
Figure PCTCN2018088774-appb-000004
由图1可知,采用上述条件的高效液相色谱分析方法,甘露糖,鼠李糖和葡萄糖可以完全的分离开,色谱峰型优异。谱峰1,谱峰2,谱峰3,谱峰4依次为PMP,Man,Rha以及Glc的色谱峰。
实施例2:正常人血清游离甘露糖和葡萄糖检测
(1)血清游离单糖衍生:
①取10μL 13例正常人血清样品,向其中依次加入20μL超纯水,10μL 0.1mg/ml鼠李糖溶液,40μL 0.3M NaOH,60μL 0.5M 1-苯基-3甲基-5-吡唑啉酮,混合离心处理,70℃烘箱反应1h,得到样品A;
②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入80μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。将衍生后的溶液直接调节至酸性后萃取残余PMP,从而消除了杂质峰对单糖定量的影响,降低了单糖的损失率。
③将样品B在13000r/min的速度下离心10min,得到样品C。
(2)采用高效液相色谱检测样品C:根据分析得到的峰面积以及实施例1得到的标准曲线计算待测血清样品中游离甘露糖和葡萄糖的含量。
其中,所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱(4.6×100mm 2.7μm)。高效液相色谱条件如下:(1)检测波长:254nm,带宽4nm;参比波长:350nm,带宽100nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;PH=5.5的醋酸盐在该条件下没有杂质析出,避免了色谱柱的堵塞,保证了在外标法的线性范围内的完美峰型。梯度洗脱:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L乙酸铵缓冲溶液(PH=5.5)的浓度梯度:85%→78%→76%→85%;该梯度模式可以将8种单糖完美的分离开,并尽量缩短分析时间;(3)柱温37℃;(4)流速:1.0mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。通过向流动相中加入0.1%的三氟乙酸,将冲洗流动相的pH调节成酸性,避免了血清样品中蛋白和多肽类物质在近中性和碱性的条件下析出,从而避免了色谱柱的堵塞。
实施例3:正常人血清游离甘露糖和葡萄糖检测
与实施例2不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入60μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温25℃。
实施例4:正常人血清游离甘露糖和葡萄糖检测
与实施例2不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入85μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温30℃。
实施例5:糖尿病患者血清游离甘露糖和葡萄糖检测
(1)血清游离单糖衍生:
①取10μL 13例糖尿病患者血清样品,向其中依次加入20μL超纯水,10μL 0.1mg/ml鼠李糖溶液,40μL 0.3M NaOH,60μL 0.5M 1-苯基-3甲基-5-吡唑啉酮,混合离心处理,70℃烘箱反应1h,得到样品A;
②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入80μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
③将样品B在13000r/min的速度下离心10min,得到样品C。
(2)采用高效液相色谱检测样品C:根据分析得到的峰面积以及实施例1得到的标准曲线计算待测血清样品中游离甘露糖和葡萄糖的含量。所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱(4.6×100mm 2.7μm)。高效液相色谱条件如下:(1)检测波长:254nm;参比波长:360nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;梯度洗脱:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L乙酸铵缓冲溶液(PH=5.5)的浓度梯度:85%→78%→76%→85%;(3)柱温:37℃;(4)流速:1.0mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。
实施例6:糖尿病患者血清游离甘露糖和葡萄糖检测
与实施例5不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入90μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温35℃。
实施例7:糖尿病患者血清游离甘露糖和葡萄糖检测
与实施例5不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入70μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温40℃。
实施例8:胃癌患者血清游离甘露糖和葡萄糖检测
(1)血清游离单糖衍生:
①取10μL 13例胃癌患者血清,血清样品,向其中依次加入20μL超纯水,10μL 0.1mg/ml鼠李糖溶液,40μL 0.3M NaOH,60μL 0.5M 1-苯基-3甲基-5-吡唑啉酮,混合离心处理,70℃烘箱反应1h,得到样品A;
②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入80μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
③将样品B在13000r/min的速度下离心10min,得到样品C。
(2)采用高效液相色谱检测样品C:根据分析得到的峰面积以及实施例1得到的标准曲线计算待测血清样品中游离甘露糖和葡萄糖的含量。
其中,所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱(4.6×100mm 2.7μm)。高效液相色谱条件如下:(1)检测波长:254nm;参比波长:360nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;梯度洗脱:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L乙酸铵缓冲溶液(PH=5.5)的浓度梯度:85%→78%→76%→85%;(3)柱温:37℃;(4)流速:1.0mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。
实施例9:胃癌患者血清游离甘露糖和葡萄糖检测
与实施例8不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入100μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温45℃。
实施例10:胃癌患者血清游离甘露糖和葡萄糖检测
与实施例8不同的是,
(1)血清游离单糖衍生:②取出步骤①烘箱处理完毕的反应物,冷却至室温,加入75μL 0.3M HCl,采用CHCl 3萃取3次,得到样品B。
(2)采用高效液相色谱检测样品C:高效液相色谱条件如下:(3)柱温50℃。
表4 实施例2-10检测得到的血清游离葡萄糖及甘露糖的含量
Figure PCTCN2018088774-appb-000005
根据实施例2-10,得到了通过本发明所述的方法检测的39例正常人、39例糖尿病及39例胃癌患者血清中游离甘露糖和葡萄糖含量,结果详见表4。根据实施例2-4结果可知,正常人血清甘露糖浓度远低于糖尿病患者和胃癌患者的甘露糖浓度。充分说明,血清游离甘露糖的浓度的确与糖尿病、胃癌等蛋白质糖基化异常疾病有关系;因此,本发明所述的方法对于研究血清游离单糖与疾病之间的关系、寻找疾病临床检测的标志物有着非常重要的意义。
此外,由表4可知,与现有单糖衍生后的HPLC检测方法相比,本发明所述的方法将检测时间缩短到20min左右,提高了检测效率;而且数据平行性好,充分证明了本发明所述方法的精密度、准确性和重复性。

Claims (9)

  1. 血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:包括以下步骤:
    (1)血清游离单糖衍生:
    ①取适量血清样品,向其中依次加入超纯水、鼠李糖溶液、氢氧化钠溶液、1-苯基-3甲基-5-吡唑啉酮溶液,混合后得到pH=7-14的体系,混合后离心处理,特定温度下反应一定时间,得到样品A;
    ②将样品A冷却至室温,加入适量HCl溶液,混合后得到pH=1-7的体系,离心处理,然后采用三氯甲烷萃取3次,每次取上清液,得到样品B;
    ③将样品B高速离心处理,得到样品C;
    (2)将步骤(1)得到的样品C采用高效液相色谱进行检测;检测方法为外标法,同时采用人体中不存在的鼠李糖作为内参单糖。
  2. 根据权利要求1所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:步骤(1)中所述NaOH和1-苯基-3甲基-5-吡唑啉酮的摩尔比为2:5;所述HCl和NaOH的摩尔比为1.5:1-2.5:1。
  3. 根据权利要求1所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:步骤(1)中所述血清的体积为10μL、超纯水的体积为20μL、鼠李糖溶液的浓度为0.1mg/ml,鼠李糖溶液的体积为10μL;①中所述的特定温度为70℃,反应时间为1h;③中所述高速离心处理为在13000r/min的速度下离心10min。
  4. 根据权利要求1-3中任意一项所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:所述高效液相色谱为Agilent 1260高效液相系统,Agilent Poroshell EC-C18色谱柱(4.6×100mm 2.7μm);高效液相色谱条件如下:(1)检测波长:254nm;参比波长:360nm;(2)流动相:PH=5.5的醋酸盐为盐相,乙腈作为有机相;梯度洗脱;(3)柱温:25-50℃;(4)流速:1.0mL/min;(5)进样体积:20μL;(7)色谱柱冲洗溶剂:含有0.1%的三氟乙酸的水和乙腈冲洗色谱柱。
  5. 根据权利要求4所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:步骤(2)中所述梯度洗脱的梯度模式参数为:时间梯度:0→10→15→20min;乙腈的浓度梯度:15%→22%→24%→15%;0.10mol/L PH=5.5乙酸铵缓冲溶液的浓度梯度:85%→78%→76%→85%。
  6. 根据权利要求4所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:所述高效液相色谱条件如下:检测波长:254nm,带宽4nm;参比波长:350nm,带宽100nm;柱温:37℃;流速:1mL/min;进样体积:20μL。
  7. 根据权利要求4所述的血清游离甘露糖和葡萄糖的高效液相色谱检测方法,其特征在于:
    所述外标法包括以下几个步骤:
    (1)制作标准曲线:取40μL浓度为0.5、0.25、0.10、0.05、0.025、0.01、0.005、0.0025、0.001、0.0005mg/mL的甘露糖、葡萄糖标准品的混合溶液,加入40μL 0.3M NaOH溶液,再加入60μL 0.5M PMP溶液,70℃烘箱反应1h;取出反应物冷却至室温,加入80μL 0.3M HCl,CHCl 3萃取三次;13000r/min离心10min;
    然后分别采用高效液相色谱进行检测分析,以单糖的浓度为横坐标,以相应的峰面积为纵坐标,绘制标准曲线;
    (2)检测待测样品:取10μL待测血清样品,加入20μL超纯水,10μL 0.1mg/ml鼠李糖溶液,衍生上样;利用步骤(1)得到的标准曲线计算待测血清样品中游离甘露糖和葡萄糖的含量。
  8. 一种血清游离甘露糖和葡萄糖的高效液相色谱检测方法在蛋白质糖基化异常疾病中的应用,其特征在于:所述检测样品为蛋白质糖基化异常疾病患者的血清。
  9. 一种血清游离甘露糖和葡萄糖的高效液相色谱检测方法在蛋白质糖基化异常疾病中的应用,其特征在于:所述蛋白质糖基化异常疾病包括糖尿病、胃癌。
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