Another possibility with instrumentational analysis of A domains is the perhaps obvious ability to detect adenylated intermediates directly. This relies on the leakiness of A domains, which might allow the substrate-AMP complex to dissociate from the active site after activation, which is not the case for all enzymes. Indeed, in practice, not all substrate-AMP intermediates can be detected in reaction solutions. However, in a reasonable number of cases, high performance liquid chromatography (HPLC) or liquid chromatography coupled to mass spectrometry (LC-MS) can be used to detect AMP-bound substrates directly. To its credit, this method does allow for kinetic profiling of adenylation reactions. By invoking tandem MS and a similar workflow to that of the Ppant ejection assay, the multiplexed hydroxamate assay (HAMA) was developed. In this workflow, A domains are incubated with a defined pool of substrates before addition of a hydroxamate acceptor, which converts the substrate-AMP to a hydroxamic acid conjugate detectable by LC-MS 2 . The initial report on HAMA highlighted the importance of analyzing A domains exposed to a substrate pool, which more accurately recapitulates the competition conditions experienced in vivo and therefore deduces more precise kinetic constants such as kcat/KM. Unfortunately, hydroxamate quantification by LC-MS 2 requires the generation of synthetic standards for calibration and optimization, which is not chemically possible for all proteinogenic and nonproteinogenic building blocks. Further, the study notes that detection of some hydroxamates is complicated by either isobaric, coeluting compounds present in the assay mixture or deuterium labelling required to differentiate between enantiomeric pairs of certain amino acids.