Comparison of amino acid derivatization reagents for LC–ESI-MS analysis. Introducing a novel phosphazene-based derivatization reagent

Amino acid analysis with high performance liquid chromatography with electrospray ionization mass spectrometry (LC–ESI-MS) is an emerging method. For more sensitive analysis, derivatization is used and next to commercially available derivatization reagents such as dansyl chloride (DNS), 9-fluorenylmethyl chloroformate (FMOC-Cl) and diethyl ethoxymethylenemalonate (DEEMM), new derivatization reagents are designed specially for LC–ESI-MS, like p-N,N,N-trimethylammonioanilyl N′-hydroxysuccinimidyl carbamate iodide (TAHS) which provides very low limits of detection. In this work, a novel phosphazene based derivatization reagent (FOSF) that provides comparable limits of quantitation (LoQ) to TAHS is introduced. Moreover, a thorough comparison between FOSF, TAHS, DNS, FMOC-Cl and DEEMM is carried out for 7 different amino acids – Arg, Asp, Gly, β-Ala, Pro, Trp and Phe. This is a first time that thorough comparison is carried out on the same instrument for amino acid derivatization reagents. Results on the same instrument for five amino acid derivatization reagents show that novel reagents are sensitive with LoQ values around 80 fmol but have disadvantages such as problematic chromatographic separation. Next to novel reagents, DEEMM offers very good LoQ-s (average of 150 fmol) and wide dynamic linear range.

► TAHS and FOSF provide poorer chromatographic separation and smaller dynamic linear range. ► TAHS and FOSF provide lower LoD/LoQ values than DNS, FMOC-Cl and DEEMM. ► DNS, FMOC-Cl and DEEMM have good chromatographic separation and wide dynamic linear range. ► DEEMM is most optimal, with good LoQ values, chromatographic separation and wide linear range.