Chloramphenicol (CAP) has previously been observed to be methylated off-line in solutions of methanol and acetonitrile before infusion in an electrospray ionization (ESI) source. Post-ESI studies revealed that de-solvation/de-clustering efficiency, a function of the tube lens voltage, defines spectral fidelity. We used structurally related compounds thiamphenicol (TPC) and florfenicol (FFC) herein to probe their behavior in the same context. TPC and FFC were dissolved in either deuterated methanol (CH3CD), deuterated-THF (d8-THF) or deuterated chloroform (CDCl3) followed by nuclear magnetic resonance (NMR) spectroscopic studies. They were also dissolved in methanol (CH3OH) and tetrahydrofuran (THF) followed by infusion into a quadrupole ion trap mass analyzer via a heated capillary and tube lens for mass spectral characteristics studies. The behavior of the charged droplets of the two analytes in the region of the tube lens was followed. NMR data showed that methylation of TPC and FFC occurred off-line in methanol but not in THF before ESI. Enhanced mass spectral signals at optimal tube lens voltages (TLVs) were seen in THF compared to methanol. In FFC, the intensity of 356 increased by 74% at optimal TLV. In TPC, the intensity of m/z 354 increased by 54% at optimal TLV. Enhanced chloride adduct ions [M+Cl]− signals were favored at optimum TLVs in THF in both instances. THF proved to be a better solvent in contrast to methanol/acetonitrile due to ease of de-solvation and de-clustering. Off-line methylation of the analytes most likely at the amide nitrogen in methanol/acetonitrile is detrimental to ESI. A THF/water solvent system is highly recommended for LC–MS analysis of phenicols.