Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, and Global COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Nov 1;879(29):3220-8. doi: 10.1016/j.jchromb.2011.02.016. Epub 2011 Feb 13.
The resolution of free DL-amino acids in human nail was carried out by combination of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] derivatives and UPLC-ESI-TOF-MS. The reaction of the reagent with amino acids effectively proceeds at 55 °C for 20 min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers. Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H(2)O and CH(3)CN containing 0.1% formic acid (HCOOH) or 5 mM CH(3)COONH(4) and CH(3)CN) using a reversed-phase ACQUITY UPLC™ BEH C(18) (1.7 μm, 100 mm × 2.1 mm i.d.) column and sensitively detected by TOF-MS. The detection limits (S/N=3) of the TOF-MS were 1.0-750 fmol, respectively. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 6-aminohexanoic acid, versus the injected amounts of each amino acid (r(2)>0.996), and the intra-day and inter-day assay precisions were less than 8.93%. The derivatives of the free DL-amino acids in human nail were successfully identified by the proposed procedure. As we know, for the first time, these five kinds of D-amino acids, which were D-Ala, D-Val, D-Pro, D-Ile and D-Leu, were found from human nail samples. Fifteen kinds of L-amino acids were also recognized from human nails. Using these methods, the amounts of DL-amino acids in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from diabetic patients to those from healthy volunteers, there is no significant difference in the content of the L-amino acids in the nails. However, a statistically significant (P<0.01) correlation was observed between the D/L-amino acid concentration ratios (Ala, Val, Ile, Leu). Therefore, because the proposed method provides a good mass accuracy and the trace detection of the DL-amino acids in human nails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients.
采用 R(-)-4-(3-异硫氰酸基吡咯烷-1-基)-7-(N,N-二甲基氨基磺酰基)-2,1,3-苯并恶二唑[R(-)-DBD-PyNCS]衍生物和 UPLC-ESI-TOF-MS 联合测定人指甲中游离 DL-氨基酸的分辨率。在 55°C 下，当存在 1%三乙胺（TEA）时，试剂与氨基酸的反应能有效地进行 20min，以生成相应的非对映异构体。所得衍生物的每一对都可以通过梯度程序（含有 0.1%甲酸（HCOOH）的 H2O 和 CH3CN 的混合物或 5mM CH3COONH4 和 CH3CN）有效地分离，使用反相 ACQUITY UPLC™BEH C18（1.7μm，100mm×2.1mm id）柱，并通过 TOF-MS 进行灵敏检测。TOF-MS 的检测限（S/N=3）分别为 1.0-750fmol。通过绘制相对于内标（IS），即 6-氨基己酸的分析物峰面积比与每种氨基酸的进样量之间的校准曲线，可以得到良好的线性关系（r2>0.996），日内和日间测定精密度均小于 8.93%。通过所提出的程序成功鉴定了人指甲中游离 DL-氨基酸的衍生物。据我们所知，这是首次从人指甲样本中发现五种 D-氨基酸，即 D-Ala、D-Val、D-Pro、D-Ile 和 D-Leu。还从人指甲中鉴定出 15 种 L-氨基酸。使用这些方法，测定了健康志愿者和糖尿病患者指甲中的 DL-氨基酸含量。当比较糖尿病患者和健康志愿者的指标时，指甲中 L-氨基酸的含量没有显著差异。然而，在 D/L-氨基酸浓度比（Ala、Val、Ile、Leu）之间观察到具有统计学意义的（P<0.01）相关性。因此，由于所提出的方法提供了良好的质量精度和对人指甲中 DL-氨基酸的痕量检测，因此该分析技术可以成为一种非侵入性技术，以协助诊断和评估糖尿病患者的疾病活动。
