| Diabetes has become a worldwide epidemic in the 21st century.It is the third most serious chronic non-communicable disease that threatens human health after tumor and cardiovascular and cerebrovascular diseases.It has become a common public health problem faced by all countries in the world.Precision medicine has become an integral part of China’s strategic emerging industries.Compared with traditional medicine,precision medicine has the advantages of precision and convenience,and has become one of the focuses of medical research today.Insulin and blood lactate are two important diagnostic markers related to diabetes.Determination of insulin level can reflect the insulin secretion of diabetics and is an important index for diabetes classification.Diabetic lactic acidosis is an acute complication of diabetes.Serious lactic acidosis can lead to life-threatening dangers such as blood pressure drop,dehydration,unconsciousness,shock,etc.Metrology is an activity to achieve unit unity and accurate and reliable measurement value.Throμgh the combination and application of metrology,a high-accuracy diagnostic marker measurement method can be established and used in the development of reference materials.The reference materials are used as the carrier of quantity value for transmission to achieve the purpose of accurate detection of relevant indicators,thereby contributing to the realization of precision medicine for diabetes and effectively protecting the health of the public.Drawing on the idea of metrology,this paper mainly studies the high-accuracy measurement methods of human insulin and blood lactate,two diabetes-related in-vitro diagnostic markers,and develops insulin and blood lactate reference materials.1.A high-performance liquid chromatography-circular dichroism（LC-CD）method for accurate quantification of derivatized amino acids was established,and the separation and simultaneous quantification of valine,phenylalanine and leucine were achieved.The error of the measurement results of the three amino acids was-1.42%～2.46%;the relative standard deviation was 0.99%～1.25%;the detection limits were 6.73μg/g、24.67μg/g and 27.28μg/g;the quantification limits were 6.91μg/g、24.91μg/g and 27.51μg/g.The established method was applied to the determination of pure human insulin and the development of reference material.Raw materials were identified by de-novo sequencing using mass spectrometry and Edman degradation,and also by UV and IR spectroscopy.Mass balance（MB）and amino acid analysis-based isotope dilution mass spectrometry（AAA-IDMS）methods were used for assignment of certified value,which was（0.857±0.024）g/g.At the same time,the newly established LC-CD method was used for validation of the reference material,and the measured result was highly consistent with those obtained by the classical MB and AAA-IDMS methods,which proved the accuracy and effectiveness of the method and the certified value of the reference material.The developed human insulin reference material can be used as the traceability standard for serum insulin measurement,as well as for quality control in the development of biopharmaceuticals and in-vitro diagnostic products,providing technical support for accurate diagnosis and effective treatment of diabetes.2.Two methods for accurate quantification of blood lactate based on gas chromatography-isotope dilution mass spectrometry（GC-IDMS）and high performance liquid chromatography-isotope dilution tandem mass spectrometry（HPLC-IDMS/MS）were established.The method performance was evaluated and the recoveries of GC-IDMS and HPLC-IDMS/MS were 97.02%～104.45%and 98.84%～101.48%;respectively.The relative standard deviation was 0.47%～1.38%and 0.30%～1.10%,the detection limits were 3.8×10-5 mmol/L and4.8×10-4 mmol/L,the quantification limits were 1.3×10-4 mmol/L and 1.6×10-3mmol/L,respectively.The established methods were applied to determine the blood lactate levels of samples,and to develope two-level of blood lactate reference materials.The results obtanied by GC-IDMS and HPLC-IDMS/MS were highly consistent.The average results were used for the assessment of certified valuse of blood lactate reference materials,which were（2.33±0.06）mmol/L and（20.32±0.18）mmol/L,respectively.The reference materials can be used for establishement of traceability of blood lactate measurement,for the development of blood lactate test reagents,and for the verification of method performance.So as to ensure the accuracy of lactate test results in diabetic patients and avoid the occurrence of acute and critical diseases such as lactic acidosis,a diabetic complication.The developed reference material has been used in the calibration of glucose lactate analyzers in many medical laboratories and third-party medical laboratories.At the same time,the reference material can also be used to guide the scientific training of athletes,thereby improving the performance of competitive sports.During the preparations for the 24th Beijing Winter Olympics,the developed reference materials were used in the training support of the Chinese Winter Olympics training team（freestyle ski slalom chase team）,and achieved satisfactory results. |
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