Reference standards of 1'-hydroxy-midazolam (1'-OH-MDZ) and 4-hydroxy-midazolam (4-OH-MDZ) are commercially available. To explore the feasibility of identifying MDZ metabolites using infrared ion spectroscopy and quantum-chemical calculations, we recorded IR spectra of the protonated ions ([M+H] + , m/z 342) of the two references. Figure compares their IR spectra, showing that they are similar, but also present clear and distinct differences. Note for instance the doublets around 1100 cm -1 and 1150 cm -1 in the 1'-OH-MDZ spectrum, the band around 650 cm -1 in the 4-OH-MDZ spectrum and the very weak band around 900 cm -1 in the 4-OH-MDZ spectrum, which is redshifted compared to the corresponding peak in the 1'-OH-MDZ spectrum. Of special interest is the weak band just above 1700 cm -1 in the 4-OH-MDZ spectrum, which is attributed to a C=O stretch vibration. The structure of 4-OH-MDZ (inlayed in Figure ) appears not to include a C=O moiety, so that we rationalize the observed C=O stretch by enol-to-amide tautomerization (see Figure ). Likely, a small fraction of the ion population is in this tautomeric form. An analogous tautomerization is not possible for 1'-OH-MDZ, making the C=O stretch band very diagnostic in the differentiation of the two isomers.