Cyanoacetohydrazide as a Novel Derivatization Agent for the Determination of UHPLC-HRMS Steroids in Urine

Abstract

The possibility of cyanoacetohydrazide usage as a novel derivatizing agent is demonstrated in the presented article, and a comparison with hydroxylamine as the most commonly used reagent is provided. Optimal conditions for steroid derivatization with cyanoacetohydrazide are provided. According to the collected data, the maximum yield of derivatives was observed at pH 2.8 within 70 min at 40 °C with 5 ng/mL limit of detection for all investigated analytes. It was shown that cyanoacetohydrazide derivatives produces both syn- and anti-forms as well as hydroxylamine, and their ratios were evaluated and shown in presented work. An efficiency enchantment from two to up to five times was achieved with a novel derivatization reagent. Its applicability for qualitative analysis of steroids in urine was presented at real samples. Additionally, the reproducible fragmentation of the derivatizing agent in collision-induced dissociation offers opportunities for simplified non-targeted steroidomic screening. Furthermore, cyanoacetohydrazide increases ionization efficiency in positive mode, which can eliminate the need for redundant high-resolution instrument runs required for both positive and negative mode analyses.

Keywords: LC-HRMS; cyanoacetohydrazide; derivatization; non-targeted screening; steroidomics; steroids; testosterone.

Figure 1

Optimization of the pH for derivatization with cyanoacetohydrazide.

Figure 2

Optimization of the derivatization temperature.

Figure 3

Optimization of the reaction time.

Figure 4

Extracted ion chromatogram of CAH derivatives of steroidal hormones mixture at 10 ng/mL (1—CAH-cortisol; 2—CAH-cortisone; 3—CAH-11α-hydroxyprogesterone; 4—CAH-estrone; 5—CAH-testosterone; 6—CAH-methyltestosterone; 7—CAH-progesterone).

Figure 5

MS/MS spectra of CAH derivative of 11α-hydroxyprogesterone obtained at 15 eV collision energy.