González-López Nicolás Mateo, Guerra-Acero-Turizo Luisa María, Blanco-Medina Isabella, Barragán-Cárdenas Andrea Carolina, Ramírez-Celis David Augusto, Martínez-Ramírez Jorge Ariel, Fierro-Medina Ricardo, García-Castañeda Javier Eduardo, Rivera-Monroy Zuly Jenny
Pharmacy Department, Universidad Nacional de Colombia, Bogotá, Colombia.
Biotechnology Institute, Universidad Nacional de Colombia, Bogotá, Colombia.
Biomed Chromatogr. 2023 Dec;37(12):e5741. doi: 10.1002/bmc.5741. Epub 2023 Sep 9.
Matrix effect and sample pretreatment significantly affect the percentage recovery of peptides in biological matrices, affecting the method robustness and accuracy. To counteract this effect, an internal standard (IS) is used; however, in most cases this is not available, which limits the analytical method. It is important to identify short peptides that can be used as ISs in the quantification of peptides in biological matrices. In this study, doping peptides GHRP-4, GHRP-5, GHRP-6, Sermorelin (1-11), Sermorelin (13-20) and Sermorelin (22-29) were synthesized using solid-phase peptide synthesis. Treatment with human blood, trypsin and chymotrypsin was used to determine the stability of the peptides. Products were evaluated using the high-performance liquid chromatography-diode array detector (HPLC-DAD) method. The analytical methodology and sample pretreatment were effective for the analysis of these molecules. A unique profile related to protein binding and enzymatic stability of each peptide was established. GHRP-4, GHRP-6 and Sermorelin (22-29) can be considered as in-house ISs as they were stable to enzyme and blood treatment and can be used for the quantification of peptides in biological samples. Peptides GHRP-6 and Sermorelin (22-29) were used to analyse a dimeric peptide ( [F] LfcinB (20-30) ) in four different matrices to test these peptides as in-house IS.
基质效应和样品预处理显著影响生物基质中肽段的回收率，进而影响方法的稳健性和准确性。为抵消这种效应，需使用内标（IS）；然而，在大多数情况下无法获得内标，这限制了分析方法。识别可作为生物基质中肽段定量内标的短肽很重要。在本研究中，采用固相肽合成法合成了掺杂肽GHRP-4、GHRP-5、GHRP-6、生长激素释放肽（1-11）、生长激素释放肽（13-20）和生长激素释放肽（22-29）。用人血、胰蛋白酶和胰凝乳蛋白酶处理以确定肽段的稳定性。使用高效液相色谱-二极管阵列检测器（HPLC-DAD）法对产物进行评估。该分析方法和样品预处理对这些分子的分析有效。建立了与每种肽段的蛋白质结合和酶稳定性相关的独特图谱。GHRP-4、GHRP-6和生长激素释放肽（22-29）可被视为内部内标，因为它们对酶和血液处理稳定，可用于生物样品中肽段的定量。使用肽GHRP-6和生长激素释放肽（22-29）在四种不同基质中分析一种二聚体肽（[F]LfcinB（20-30）），以测试这些肽作为内部内标的性能。
