Mass spectrometry plays a crucial role for qualitative and quantitative analysis in various scientific fields. For LC-MS, ESI is the most frequently used ionization technique. However, ionization processes are still not fully understood, and ion enhancement or ion suppression effects can usually be found during ESI. In the current work, adduct formation by coeluting (endogenous) compounds was observed for the diuretic hydrochlorothiazide during negative mode ionization, decreasing the corresponding deprotonated hydrochlorothiazide signal. After identification of the corresponding adduct partners, being hippuric acid and indoxyl sulfate, systematic investigations on this formation were conducted using different concentrations of both compounds. Additionally, APCI and different ESI sources, as well as different mobile phase compositions, were tested. It was shown that the adduct formation was only found by ESI and was independent of vendor-specific source design. The adduct formation was also independent of the composition of the mobile phases and was found to be dependent on the concentration of the coeluting compounds. An over-additive effect on hydrochlorothiazide signal loss was found if hippuric acid and indoxyl sulfate adducts were formed. Finally, adduct formation was studied for other structural related and non-structural related compounds. For compounds with a high(er) structural similarity to hydrochlorothiazide (namely: bendroflumethiazide, buthiazide, cyclopenthiazide, and dorzolamide), adduct formation was observed. This study provided new insights into the ESI process. Graphical Abstract

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