Structural analysis of echinocandins via high-performance liquid chromatography-quadrupole/time-of-flight-tandem mass spectrometry

Abstract

Herein, the structures of echinocandins were analyzed by high-performance liquid chromatography-quadrupole/time-of-flight-tandem mass spectrometry (HPLC-Q/TOF-MS/MS). The echinocandin components were separated by using a Diamonsil Plus C18 column (5 μm, 4.6 mm × 250 mm), with 0.1 % formic acid in water as mobile phase A and 0.1 % formic acid in acetonitrile as mobile phase B under gradient elution. A systematic strategy for the structural characterization of echinocandin B, which is the main component of echicandins, is presented based on the accurate molecular mass and diagnostic ions obtained from both adduct ions [M+H]⁺ and [M+Na]⁺ as precursor ions. In the three fragmentation pathways obtained from the precursor ion of [M+H]⁺, echinocandins and its fragment ions produced continuous dehydrated ions ([M-nH₂O + H]⁺, n = 1, 2, 3, 4). The N-terminal fatty acyl (FA) connecting to the ornithine derivative transferred to the amino group of the proline derivative during fragmentation. Different characteristic ions, depending on the number of hydroxyls (0, 1, 2) on the ornithine derivative of different echinocandin analog. Moreover, the threonine residues underwent the neutral consecutive loss of acetaldehyde from the [M+Na]⁺ precursor ion. The [M+H]⁺ and [M+Na]⁺ echinocandin precursor ions respectively lost 1-(4-hydroxyphenyl)ethane-1,2-diol and 2-hydroxy-2-(4-hydroxyphenyl)acetaldehyde from the homotyrosine residues to generate different fragmentation ions. Thirteen echinocandin analogs were identified, including echinocandin C, echinocandin D, and echinocandin B variants with different FA and amino acid compositions.