Phthalylglycyl Chloride as a Derivatization Agent for UHPLC-MS/MS Determination of Adrenaline, Dopamine and Octopamine in Urine

Abstract

Dopamine, adrenaline and octopamine are small polar molecules that play a vital role in regulatory systems. In this paper, phthalylglycyl chloride was proposed as a derivatization agent for octopamine, adrenaline and dopamine determination in urine for the first time. The derivatization procedure facilitated the use of reversed-phase liquid chromatography with positive electrospray ionization-high-resolution mass spectrometry. An LC-HRMS method was developed that provided quantification limits of 5 ng/mL and detection limits of 1.5 ng/mL for all analytes. The 95-97% yield of derivates was observed after a 10 min derivatization with phthalylglycyl chloride at pH 6.5 and 30 °C. The proposed method was successfully applied to the analysis of human urine samples. The obtained results were compared with those of conventional derivatization procedures with 9-fluorenyl-methoxycarbonyl chloride and dansyl chloride.

Keywords: adrenaline; catecholamines; derivatization; dopamine; octopamine; phthalylglycyl chloride.

Figure 1

Scheme of catecholamines derivatization with phthalylglycyl chloride (pathway 1—dopamine, pathway 2—octopamine, pathway 4—adrenaline) and pathway 3—gabapentin.

Figure 2

Scheme of N-phthaloylglycine chloride synthesis.

Figure 3

Optimization of derivatization temperature with PG-Cl at 100 ng/mL dopamine, octopamine, adrenaline and 50 ng/mL gabapentin.

Figure 4

Optimization of derivatization reaction time with PG-Cl at 100 ng/mL dopamine, octopamine, adrenaline and 50 ng/mL gabapentin.

Figure 5

Possible fragmentation method of the most abundant product ion (m/z 160.0393) for investigated analytes on dopamine example.

Figure 6

LC-HRMS chromatogram of catecholamines and gabapentin derivatives in synthetic urine at quality control (QC) low (10 ng/mL) concentration: 1—PG-octopamine, 2—PG-adrenaline, 3—PG-dopamine, 4—PG-gabapentin (IS).