Simultaneous Degradation Study of Isomers in Human Plasma by HPLC-MS/MS and Application of LEDA Algorithm for Their Characterization

Abstract

This paper proposes a tandem mass spectrometry (MS/MS) approach in isomer recognition by playing in the “energetic dimension” of the experiment. The chromatographic set up (HPLC) was tuned to minimize the run time, without requiring high efficiency or resolution between the isomers. Then, the MS/MS properties were explored to solve the signal assignment by performing a series of energy resolved experiments in order to optimize the parameters, and by applying an interesting post-processing data elaboration tool (LEDA). The reliability of the new approach was evaluated, determining the accuracy and precision of the quantitative results through analysis of the isomer mixture solutions. Next, the proposed method was applied in a chemical stability study of human plasma samples through the simultaneous addition of a pair of isomers. In the studied case, only one of the isomers suffered of enzymatic hydrolysis; therefore, the influence of the stable isomer on the degradation rate of the other was verified. In order to monitor this process correctly, it must be possible to distinguish each isomer present in the sample, quantify it, and plot its degradation profile. The reported results demonstrated the effectiveness of the LEDA algorithm in separating the isomers, without chromatographic resolution, and monitoring their behavior in human plasma samples.

Removal of isobaric interference using pseudo‐multiple reaction monitoring and energy‐resolved mass spectrometry for the isotope dilution quantification of a tryptic peptide

“…9,[15][16][17] Crotti et al have shown that accurate quantification of co-eluted isomers can be achieved by multilinear regression of ERMS. 9,[18][19][20][21] Complementary with the latter approach, Memboeuf et al have shown that ERMS and, more specifically, the survival yield (SY) plots can be used as a tool for detecting iso-baric/meric contamination. 15 Typically, SY curves of pure compounds are sigmoidal.…”

“…Although other techniques have been developed to analyse iso‐baric/meric mixtures, such as the kinetic method 7–10 and, more recently, ion mobility mass spectrometry (MS), 9,11–14 energy‐resolved mass spectrometry (ERMS) can also provide reliable results for quantitative analysis in the presence of iso‐meric/baric interferences 9,15–17 . Crotti et al have shown that accurate quantification of co‐eluted isomers can be achieved by multilinear regression of ERMS 9,18–21 . Complementary with the latter approach, Memboeuf et al have shown that ERMS and, more specifically, the survival yield (SY) plots can be used as a tool for detecting iso‐baric/meric contamination 15 .…”

Isomers Recognition in HPLC-MS/MS Analysis of Human Plasma Samples by Using an Ion Trap Supported by a Linear Equations-Based Algorithm

“…Then, the same HPLC conditions can be used with a large number of analytes without worrying about their separation, and the LEDA elaboration of the MS/MS data assures the signal distinction of the compounds. The reliability of this procedure allowed us to carry out a kinetic study on the enzymatic hydrolysis of a pair of isomers present simultaneously in plasma samples, evaluating their different degradation profiles [ 22 ].…”

Very Low-Pressure CID Experiments: High Energy Transfer and Fragmentation Pattern at the Single Collision Regime

“…Increasing the collision energy increases the internal energy of the colliding ions, and that in turn increases the degree of fragmentation, changes the ratio of various fragment ion abundances, and may also change fragmentation routes. Varying and optimizing the collision energy has various practical applications, such as determination of binding energies [ 8 , 9 ], differentiation of isomers [ 10 , 11 , 12 ], characterization of oligonucleotides [ 13 ] and small molecules [ 14 , 15 , 16 , 17 ], and various proteomic applications [ 18 , 19 , 20 ] including quantitation [ 21 ].…”