人体头发由于其采样无创性、时序性、长效性和易于保存等优点，在疾病的长期监测、疾病进程和发病机制研究方面具有独特优势，越来越受到研究者的关注。已有头发代谢组学技术应用到疾病的诊断和发病机制的研究中，但相关研究报道还不多，因此，建立灵敏、可靠的头发代谢组学分析技术，对于拓展头发在疾病研究中的作用有着十分重要的意义。

本论文以头发为研究对象，重点突破头发前处理技术，充分富集不同类型内源性化合物，建立涵盖脂质代谢、激素代谢网络核心代谢物的头发靶向脂质代谢组学分析技术，并应用于罕见的库欣病临床头发样本，准确、高效地分析头发的代谢物轮廓，探索与疾病相关的潜在生物标志物，为临床诊断和预后评价提供无创的监测指标信息。

针对人体头发中的脂质化合物，优化了头发洗涤试剂、提取方法和片段长度，建立了简便易行的前处理方法：采用植物香波-正己烷对头发进行洗涤，晾干后剪碎成5mm片段，采用氯仿-甲醇浸提法，从30mg头发中同时提取鞘脂、甘油酯和甘油磷脂类化合物，采用高效液相色谱-三重四极杆质谱联用分析技术靶向表征头发中鞘脂类化合物，并对检测到的鞘脂类化合物进行定量分析；采用高效液相色谱-高分辨质谱联用技术对头发甘油酯和甘油磷脂类化合物进行表征，并对检测到的脂类化合物同时进行定量分析。对所建立的方法进行了方法验证，对于鞘脂化合物,线性范围为0.50-160 pmol/mg或0.05-16 pmol/mg，线性相关系数r值均大于0.99；定量限（LOQ）为0.05-0.50 pmol/mg，检测限（LOD）为0.0125 pmol/mg；日内精密度均小于14.9%，日间精密度均小于16.7%，准确度在80.2-119%之间，提取回收率在79.6 -104%之间，基质效应在80.5-119%之间。对于甘油酯和甘油磷脂化合物, 线性范围为0.50-100 pmol/mg，线性相关系数r值均大于0.99；定量限（LOQ）为0.50 pmol/mg，检测限（LOD）为0.125-0.25 pmol/mg；日内精密度均小于20.1%，日间精密度均小于17.3%，准确度在80.0-120%之间，提取回收率在80.7-99.9%之间，基质效应在86.9-118%之间，验证结果表明所建立分析方法能够满足人体头发中脂质类化合物分析要求。将所建立的方法应用于不同性别的人体头发样本，成功表征了32种鞘脂类化合物、72种甘油脂类化合物和2种甘油磷脂类化合物，并测定了含量，发现了性别相关的29种差异性化合物，并探究了头发发干至发梢方向各类内源性脂质化合物含量的变化趋势。

针对人体头发中的激素化合物，优化了洗涤试剂、提取方法和片段长度，建立了简单易行的前处理方法：采用植物香波-正己烷对头发进行洗涤，晾干后，剪碎成5mm片段，采用甲醇18小时浸提法，从30mg头发中提取激素化合物。优化了课题组已建立的血浆激素超高效液相色谱-三重四极杆质谱联用分析方法，增加激素化合物的分析通量由20种至28种。对所建立的激素分析方法进行了方法验证，线性范围为0.25-100 pg/mg或0.05-20 pg/mg，线性相关系数r值均大于0.99；定量限（LOQ）为0.05-0.25 pg/mg，检测限（LOD）为0.05-0.1 pg/mg；日内精密度均小于17.9%，日间精密度均小于16.5%，准确度在79.5-115%之间，提取回收率在77.1-106%之间，基质效应除醛固酮（127-134%）和17-OH PROG（67.6-72.%）外，其余化合物在81.5 % - 119 %之间，方法验证结果表明所建立方法能够满足人体头发中激素类化合物的表征和定量分析要求。将所建立的方法应用于不同性别的人体头发样本，成功表征了17种激素化合物，并测定了含量，发现了性别相关的5种差异性化合物，并探究了头发发干至发梢方向激素化合物含量的变化趋势。

本论文将所建立的头发脂质组学分析技术应用于库欣病人与非库欣病对照组临床头发样本，在库欣病人头发中检测到17种激素、33种鞘脂、100种甘油酯、4种磷脂，按照投影中的变量重要性值（VIP）大于1，并同时满足T检验具有显著性差异的标准，共找到表征库欣病人与非库欣病对照组差异的40种潜在生物标志物，包括6种激素，3种鞘脂、31种甘油酯。库欣病头发脂质组学研究结果为今后深入研究该病的临床诊断新指标提供了有价值的数据，有可能对现有临床诊断模式起到互补作用，为临床有效治疗和预后判断提供无创的监测指标。

Hair has unique advantages in long-term disease monitoring, disease process research and pathogenesis research since it is noninvasive, time sequential and easy storing compared to other bio-specimens. Thus researchers have paid more and more attention on it. There are a little reports of hair metabolomics on diagnosis and pathogenesis of the disease. The sensitive and reliable hair metabolomics analysis has significant importance for the expandence of hair application in disease research.

This thesis focus on hair metabolomics. Hair pre-treatment method were carefully optimized to achieve extraction of different types of endogenous compounds. The hair target lipidomics covering core metabolites of lipid and steroid metabolism network were established based on previous study. The established method was applied to profile the hair metabolites of rare Cushing disease for exploring the potential biomarkers associated with the disease, providing noninvasive monitoring indicators for clinical diagnosis and prognosis evaluation.

The washing solvent, extraction method and the length of the hair fragment were optimized for the lipid compounds in human hair. A simple hair pretreatment method was established as following: the hair was washed with the shampoo-n-hexane method, then cut into 5 mm pieces. Sphingolipid, glyceride and phospholipid compounds were extracted using chloroform-methanol extraction method from 30mg hair. The high performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to profile hair sphingolipids and quantify the detected sphingolipids in hair. High performance liquid chromatography coupled with high resolution mass spectrometry was used to profile hair triglycerides and phospholipids and quantify the detected triglycerides and phospholipids in hair. Method validation was carried out. For sphingolipids, the linear range of standard substances were 0.50-160 pmol/mg and 0.05-16 pmol/mgwith r>0.99. LOQ were 0.50 pmol/mg and LOD were 0.125-0.25 pmol/mg. The intra-precision was less than 14.9% and inter-precision was less than 16.7%. The accuracy was between 80.2-119%. The extraction recovery of each standard substance at each concentration level was between 79.6 -104%. The matrix effect was between 80.5-119%. For glyceride and phospholipid, the linear range of standard substances were 0.50-100 pmol/mg with r>0.99. LOQ were 0.50 pmol/mg and LOD for these compounds were 0.125-0.25 pmol/mg. The intra-precision was less than 20.1% and inter-precision was less than 17.3%. The accuracy is between 80.0-120%. The extraction recovery of each standard substance at each concentration level was between 80.7-99.9%. The matrix effect was between 86.9-118%. The results showed that the method meet the requirements of characterization and determination of lipid compounds in human hair. The established method was successfully applied to hair samples of male and female. 32 sphingolipids, 72 glycerides and 2 phospholipids in hair were successfully profiled and quatified and 29 compounds showed sex-related significant differences. In addition, the lipid concentration trends were explored from the proximal to more distal hair segments.

The washing solvent, extraction method and the length of the hair fragment were optimized for the steroid compounds in human hair. A simple hair pretreatment method was established as following: he hair was washed with the shampoo-n-hexane method, then cut into 5 mm pieces. Steroid compounds from 30mg hair were extracted using methanol (18h). The ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry was used to profile hair steroids and quantify the detected steroids in hair. The previous established ultra-high performance liquid chromatography triple quadrupole mass spectrometry method for plasma steroids was optimized, increasing the throughput of the steroids compounds from 20 to 28. Method validation was performed. The linear range of standard substances were 0.25-100 pg/mg and 0.05-20 pg/mgwith r>0.99. LOQ were 0.05-0.25 pg/mg and LOD for these compounds were 0.05-0.1 pg/mg. The intra-precision was less than 17.9% and inter-precision was less than 16.5%. The accuracy is between 79.5-115%. The extraction recovery of each standard substance at each concentration level was between 77.1-106 %. The matrix effect was between 81.5 % and 119 % except for corticosterone (127-134%) and 17-OHPROG (67.6 -72.5%). The results showed that the method meet the requirements of characterization and determination of steroids in human hair. The established method was successfully applied to hair samples of male and female. 17 steroids in hair were successfully profiled and quatified and 5 compounds showed sex-related significant differences. In addition, the steroid concentration trends were analyzed from the proximal to more distal hair segments.

In the thesis, the established method was applied to hair samples of Cushing disease patients group and control group. 17 steroids, 33 sphingolipids, 100 glycerides and 4 phospholipids were detected. According to the standard of variable importance value (VIP) >1 and significant difference in T test, 40 potential biomarkers were found to present different status of Cushing disease group and control group, including 6 steroids, 3 sphingolipids and 31 glycerides. The result of hair metabolomics of Cushing disease provided valuable data for the future study in the new indicators of clinical diagnosis of Cushing disease and it may be complementary to the clinical diagnosis mode and provide noninvasive monitoring indicators for cinical effective treatment and prognosis.