Mitchell A E, Jones A D, Mercer R S, Rucker R B
Department of Nutrition, University of California, Davis, California 95616-8669, USA.
Anal Biochem. 1999 May 1;269(2):317-25. doi: 10.1006/abio.1999.4039.
We describe a HPLC method coupled to electrospray ionization mass spectrometry (ESI/MS) for quantification and identification of pyrroloquinoline quinone (PQQ) and condensation products formed upon incubation of PQQ with amino acids (IPQ; imidazolopyrroloquinoline and I/OPQ/R; imidazolopyrroloquinoline with attached R-group). More importantly, using these methods we demonstrate the presence of both PQQ and IPQ in human milk in nanomolar to micromolar concentrations. PQQ was incubated with amino acids and condensation products were separated by HPLC. Fractions corresponding to each product were collected and molecular masses were determined using ESI/MS. Ala, Asp, Arg, Cys, Gly, Glu, Ser, Thr, Trp, and Tyr form IPQ upon incubation with PQQ. Yields of IPQ were low (<5%) for Asp and Glu, yet high (>60%) for Thr. In addition to IPQ, Ala, Arg, Cys, Ser, Trp, and Tyr formed IPQ/R derivatives. His, Ile, Leu, Glu, Leu, Lys, Met, and Phe form only IPQ/R derivatives. Proline did not react with PQQ. Mass spectra indicate that PQQ forms stable hydrated carbonyls and decarboxylates easily. Although mass spectra were complicated by the oxidation state of the quinone and decarboxylation of PQQ, these methods are invaluable for the rapid detection of the full range of PQQ adducts in biological matrices.
我们描述了一种高效液相色谱法（HPLC）与电喷雾电离质谱法（ESI/MS）联用的方法，用于定量和鉴定吡咯喹啉醌（PQQ）以及PQQ与氨基酸孵育后形成的缩合产物（IPQ；咪唑并吡咯喹啉和I/OPQ/R；带有连接R基团的咪唑并吡咯喹啉）。更重要的是，使用这些方法我们证明了人乳中存在纳摩尔至微摩尔浓度的PQQ和IPQ。将PQQ与氨基酸孵育，缩合产物通过HPLC分离。收集对应于每种产物的馏分，并使用ESI/MS测定分子量。丙氨酸、天冬氨酸、精氨酸、半胱氨酸、甘氨酸、谷氨酸、丝氨酸、苏氨酸、色氨酸和酪氨酸与PQQ孵育后形成IPQ。天冬氨酸和谷氨酸形成IPQ的产率较低（<5%），而苏氨酸的产率较高（>60%）。除了IPQ，丙氨酸、精氨酸、半胱氨酸、丝氨酸、色氨酸和酪氨酸还形成了IPQ/R衍生物。组氨酸、异亮氨酸、亮氨酸、谷氨酸、亮氨酸、赖氨酸、甲硫氨酸和苯丙氨酸仅形成IPQ/R衍生物。脯氨酸不与PQQ反应。质谱表明PQQ形成稳定的水合羰基且易于脱羧。尽管质谱因醌的氧化态和PQQ的脱羧而变得复杂，但这些方法对于快速检测生物基质中全范围的PQQ加合物非常重要。
