High-performance liquid chromatography/electron capture atmospheric pressure chemical ionization-mass spectrometric determination of biologically active steroids

摘要

ステロイドは生命機能維持に不可欠な生体分子であり,その生体内動態及び機能の解析は,臨床診断や新薬開発上極めて重要である.その方法論として液体クロマトグラフィー/質量分析(LC/MS)が有用と考えられるが,ステロイドのエレクトロスプレーイオン化や大気圧化学イオン化(APCI)に対するイオン化効率が必ずしも高くなく,複雑な生体試料中の微量分析では実用的な感度が得られない場合も多い.一方,電子親和性原子団を有する化合物は,APCIインターフェースにおいて電子を捕獲して効率的にイオン化され(電子捕獲APCI),また本条件下ではバックグラウンドノイズが低いことから高感度な応答が得られる.そこで著者らは,各種ステロイドの電子捕獲APCI-MS用検出指向誘導体化法を開発し,生体試料分析に適用した.すなわち,ヒト血しょう中ビタミンD_3代謝物及びラット脳内神経ステロイド定量法を開発し,後者についてはストレスによる脳内レベルの変動解析に応用した.Steroids are essential bio-molecules for the maintenance of life. Kinetic and functional analyses of steroids are necessary to elucidate the nature of the many endocrine disease processes, and thus be useful for diagnosis and treatment. High-performance liquid chromatography/mass spectrometry (LC/MS) has recently been used for steroid analysis due to its specificity and versatility. However, conventional LC/MS sometimes does not demonstrate the required sensitivity for the trace analysis of steroids in biological samples, because the ionization efficiencies of most steroids are relatively low for electrospray ionization and usual atmospheric pressure chemical ionization (APCI). On the other hand, electron-affinitive compounds are efficiently ionized by the electron capture reaction in APCI in the negative-ion mode (electron capture APCI). Therefore, in this mode, a high sensitivity can be obtained by tagging steroids with electron-affinitive (electron capturing) moieties. On this basis, we have developed detection-oriented derivatization methods for the LC/electron capture APCI-MS analysis of steroids and applied these methods to the determination of vitamin D3 metabolites in human plasma and to the analysis of changes in rat brain neurosteroid levels by acute stress.