The use of online heart‐cutting high‐performance liquid chromatography coupled with linear ion trap mass spectrometry in the identification of impurities in vidarabine monophosphate

Abstract

It is difficult to identify unknown impurities in nucleotide analogues by mass spectrometry because mass-spectrometry-incompatible mobile phases need to be used to separate the major ingredient from impurities. In this study, vidarabine monophosphate was selected, and unknown impurities were identified by online heart-cutting two-dimensional high-performance liquid chromatography and linear ion trap mass spectrometry. The one-dimensional reversed-phase column was filled with a mobile phase containing nonvolatile salt. In two-dimensional high-performance liquid chromatography, we used an Acclaim Q1 column with volatile salt, and the detection wavelength was 260 nm. The mass spectrum was scanned in positive- and negative-ion mode. The online heart-cutting and online demineralization technique ensured that the mobile phase was compatible with mass spectrometry; seven impurities were identified by MS and MS fragments. The mass fragmentation patterns of these impurities were investigated. The two isomers were semiprepared and complemented by nuclear magnetic resonance. The results were further compared with those of normal-phase high-performance liquid chromatography with mass spectrometry. The online heart-cutting two-dimensional high-performance liquid chromatography with mass spectrometry was superior in identifying more impurities. The method solves the problem of incompatibility between the mobile phase and mass spectrometry, so it is suitable for identifying unknown impurities. This method may also be used for investigating impurities in other nucleotide analogues.

Current developments in LC-MS for pharmaceutical analysis

“…In heart-cutting mode, the effluent of a first-dimension separation is only collected in a region of particular interest (e.g., where compounds are co-eluting) and re-injected on a seconddimension column for further separation. Typical applications of heart-cutting 2D-LC in pharmaceutical analysis involve the isolation of impurities from the active pharmaceutical ingredient (API) or an interfering matrix in the first dimension, the combination of achiral and chiral columns for the separation of enantiomers in the second dimension, and online desalting of peaks eluting in MSincompatible mobile phases in the first dimension 83,[85][86][87] .…”

Residue measurement of pendimethalin in tobacco by using heart‐cutting two dimensional liquid chromatography coupled with tandem mass spectrometry

“…Compared to the widely used 1D chromatographic methods, 2D‐LC greatly improved the peak capacity. By coupling two different columns, heart‐cutting 2D‐LC method could remove interference compounds effectively [19‐21]. In our work, a heart‐cutting RP/RP–2D‐LC–MS/MS method was developed for the quantification of pendimethalin.…”

Silver ion chromatography for peak resolution enhancement: Application to the preparative separation of two sesquiterpenes using online heart-cutting LC-LC technique

“…Notably, heart-cutting two-dimensional liquid chromatography (LC-LC) offers the probability to improve these drawbacks based on a tandem combination of two independent liquid phase separation systems [8][9][10][11].…”