【摘要】 目的 建立高效液相色谱串联质谱（high performance liquid chromatography-tandem mass spectrometry,HPLC-MS/MS）定量检测血清同型半胱氨酸（homocysteine,Hcy）谱的检测方法及在慢性肾脏病（chronic kidney disease,CKD）中的临床应用。方法 选取2024年1月—2025年1月初次就诊于河北医科大学第二医院的临床诊断为CKD的患者308例（CKD组）和健康体检人群103例（CON组）为研究对象，检测2组人群血清Hcy、半胱氨酸（cysteine,Cys）及甲硫氨酸（methionine,Met）水平。检测采用HPLC-MS/MS系统，使用色谱柱[（2.1×100.0） mm,2.1μm]分离，柱温40℃，流动相为0.2%甲酸水溶液和纯乙腈，梯度洗脱，并应用线性、定量下限、精密度、正确度、基质效应、样本稳定性、残留、干扰及稀释一致性等参数进行方法的性能验证。结果 共411例CON人群及CKD患者入选该研究，CON组103例，年龄（50.01±12.41）岁，男性58例，女性45例，CKD组308例，年龄（54.44±15.38）岁，男性172例，女性136例；Hcy、Cys、Met的线性范围分别是1～100、5～500、2～200μmol/L；定量下限分别是1、5、2μmol/L；批内、批间变异系数（coefficient of variation,CV）均<±15%；回收率分别是97%～105%、93%～111%、102%～109%;1∶1混合样本的Hcy、Cys、Met测定值比血清基质样本及纯溶液样本测定值的均值低1.45%、3.52%、2.18%；血清Hcy、Cys、Met的浓度含量在7 d内以及样本处理后24 h内与首次测定结果的相对偏差均<±15%；该方法残留较少，且不受溶血、黄疸和脂血的干扰；稀释5倍、10倍和20倍血清样品中的Hcy、Cys及Met测定值CV均小于15%，且回收率均在85%～115%以内；与CON组相比，CKD组血清Hcy及Cys水平显著升高（P<0.001）。结论 本研究建立的血清Hcy谱HPLC-MS/MS检测方法性能良好，可用于检测CKD患者血清Hcy谱的含量。更多还原

【Abstract】 Objective To establish a method for quantitative detection of serum homocysteine(Hcy) profile by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) and its clinical application in chronic kidney disease(CKD). Methods This study enrolled individuals initially presenting to the Second Hospital of Hebei Medical University between January 2024 and January 2025, including 308 patients with a clinical diagnosis of CKD(CKD group) and 103 healthy individuals undergoing physical examinations(control group, CON). Serum levels of Hcy, cysteine(Cys) and methionine(Met) were measured and compared between the two groups. The detection was carried out using a HPLC-MS/MS system. A chromatographic column [(2.1×100.0) mm, 2.1 μm] was used for separation at a column temperature of 40℃. The mobile phase was a 0.2% formic acid aqueous solution and pure acetonitrile, with gradient elution. The performance of the method was verified by applying parameters such as linear range, lower limit of quantification(LLOQ), precision, accuracy, matrix effect, sample stability, residue, interference and dilution consistency. Results A total of 411 people, including healthy controls and patients with CKD, were enrolled in this study. There were 103 patients in the CON group, with an age of(50.01±12.41) years, including 58 males and 45 females. There were 308 patients in the CKD group, with an age of(54.44±15.38) years, including 172 males and 136 females. The linear ranges of Hcy, Cys and Met were 1―100 μmol/L, 5―500 μmol/L and 2―200 μmol/L, respectively. The LLOQ was 1 μmol/L, 5 μmol/L, and 2 μmol/L, respectively. The coefficients of variation(CV) within and between batches were within ±15% and the recovery rates were 97%―105%, 93%―111% and 102%―109%, respectively. Compared with the mean of mixed serum samples and pure solution samples, the measured values of mixed samples were 1.45%, 3.52% and 2.18% lower in concentration. The relative deviation between the concentration of serum Hcy, Cys and Met within 7 d after detection and 24 h after the initial measurement were within ±15%. The method exhibited low residue in detecting Hcy metabolite profiles and remained unaffected by hemolysis, jaundice and lipemia. The CV of Hcy, Cys and Met in serum samples diluted 5, 10 and 20 times were all less than 15% and the recovery rates were between 85%―115%. Compared with the CON group, the levels of serum Hcy and Cys in the CKD group were significantly increased(all P<0.001). Conclusion The HPLC-MS/MS method for detecting serum Hcy profile established in this study demonstrated favorable performance and can be reliably utilized to quantify serum Hcy profile in CKD patients.更多还原