Tredget E E, Falk N, Scott P G, Hogg A M, Burke J F
Department of Surgery, University of Alberta, Edmonton, Canada.
Anal Biochem. 1990 Nov 1;190(2):259-65. doi: 10.1016/0003-2697(90)90190-k.
Derivatization of 4-hydroxyproline (Hyp) in collagen using trifluoroacetylation and methanol esterification produces two derivatives when analyzed by gas chromatography/mass spectrometry (GC/MS). The diacyl derivative N,O-bis(trifluoroacetyl)-4-hydroxy-L-proline methyl ester (N,O-TFA-Hyp) formed in this manner has a shorter retention time and different fragmentation pattern by GC/MS as compared to the slower eluting monoacetylated species N-trifluoroacetyl-4-hydroxy-L-proline methyl ester (N-TFA-Hyp). By selected ion monitoring of the appropriate ions of either N,O-TFA-Hyp (m/z 164, 278) or N-TFA-Hyp (m/z 164, 182) efficient quantitation of Hyp in collagen is possible within the broad range of 5-1000 ng with a lower limit of detection of 0.5 ng per injection. Measurement of 18O2 incorporation into collagen is possible by selected ion monitoring of the m/z 182 ion formed only from the monoacetylated derivative, N-TFA-Hyp, produced by methanol solvolysis of the N,O-TFA-Hyp derivative, as proposed herein.
使用三氟乙酰化和甲醇酯化对胶原蛋白中的4-羟基脯氨酸（Hyp）进行衍生化处理后，通过气相色谱/质谱联用仪（GC/MS）分析会产生两种衍生物。以这种方式形成的二酰基衍生物N,O-双（三氟乙酰基）-4-羟基-L-脯氨酸甲酯（N,O-TFA-Hyp）与洗脱速度较慢的单乙酰化产物N-三氟乙酰基-4-羟基-L-脯氨酸甲酯（N-TFA-Hyp）相比，在GC/MS中有较短的保留时间和不同的碎片模式。通过对N,O-TFA-Hyp（m/z 164、278）或N-TFA-Hyp（m/z 164、182）的适当离子进行选择离子监测，可以在5-1000 ng的宽范围内对胶原蛋白中的Hyp进行有效定量，每次进样的检测下限为0.5 ng。如本文所提出的，通过对仅由N,O-TFA-Hyp衍生物的甲醇溶剂解产生的单乙酰化衍生物N-TFA-Hyp形成的m/z 182离子进行选择离子监测，可以测量18O2掺入胶原蛋白的情况。
