One of the critical steps during LC-MS/MS hormone analyses that affects the sensitivity of the assay is the ionization process. Enhancing ionization efficiencies by the addition of supercharging reagents might be one way to improve sensitivity and reduce the limit of quantification (LOQ). Therefore, we investigated whether the addition of the supercharging reagents m-nitrobenzyl alcohol (m-NBA), sulfolane, propylene carbonate, and o-nitroanisole (o-NA) increased ionization efficiency and improved assay LOQ of insulin, oxytocin, sex steroids, and corticosteroids in test solutions. Additionally, the influence of the supercharging reagents was tested in serum samples after sample pretreatment to determine whether ionization would be enhanced similarly in routine analyses and, subsequently, lead to improved sensitivity. The screening experiments showed that the impact of the supercharging reagents varied for each hormone; although the addition of m-NBA increased the signal of all hormones, the other reagents only enhanced ionization efficiencies for some hormones. While the addition of 0.05 v/v% m-NBA and 0.05 v/v% o-NA did result in an increase in peak area in both test solutions and serum samples, it did not significantly improve the signal-to-noise ratio, as a simultaneous increase in noise was observed. In conclusion, even though supercharging reagents can enhance ionization efficiencies of hormones significantly, the addition of these reagents does not result in an improved LOQ for hormone measurements with LC-MS/MS.
Keywords: Electrospray ionization; Hormones; Ionization efficiency; LC-MS/MS; Limit of quantification; Supercharging.
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