Determination of Phosphoethanolamine in Urine with HPLC-ICPMS/MS Using 1,2-Hexanediol as a Chromatographic Eluent

Abstract

The importance of element-selective detection with inductively coupled plasma mass spectrometry (ICPMS) has been significantly increased in recent years following the introduction of tandem ICPMS (ICPMS/MS), which unlocked access to nonmetal speciation analysis. However, nonmetals are ubiquitous, and the feasibility of nonmetal speciation analysis in matrices with complex metabolomes is yet to be demonstrated. Herein, we report the first phosphorous speciation study by HPLC-ICPMS/MS in a human sample, namely, urine, involving the determination of the natural metabolite and biomarker phosphoethanolamine. A simple one-step derivatization procedure was employed to enable the separation of the target compound from the hydrophilic phosphorous metabolome in urine. The challenge of eluting the hydrophobic derivative under ICPMS-compatible chromatographic conditions was addressed by employing hexanediol, a novel chromatographic eluent recently described in our previous work but has not yet been exploited in a real-world application. The developed method features fast chromatographic separation (<5 min), no need for an isotopically labeled internal standard, and an instrumental LOD of 0.5 μg P L–1. The method was evaluated for recovery (90–110%), repeatability (RSD ±5%), and linearity (r 2 = 0.9998). The method accuracy was thoroughly examined by comparing with an independently developed method based on HPLC-ESIMS/MS without derivatization, where agreement was found within ±5–20%. An application is presented to gain first insight into the variability in the human excretion of phosphoethanolamine, which is key for the interpretation of its levels as a biomarker, by repeated urine collection from a group of volunteers over 4 weeks.

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

“…Whilst phosphoethanolamine (PEt) has been identified as a potential urine biomarker for hypophosphatasia, complexity of the phosphorous metabolome renders speciation analysis challenging. Lajin et al 220 described an HPLC-ICP-MS method to quantify phosphoethanolamine in human urine following derivatisation with fluorenylmethyloxycarbonyl chloride (fmoc-cl). Fast chromatographic separation (<4 min) of fmoc-PEt was achieved using a C18 column with an isocratic gradient of 9% 1,2 hexanediol (v/v) and 0.1% CH 3 COOH (v/v).…”

Atomic spectrometry update: review of advances in elemental speciation

“…Air PM 2.5 and PM 10 material collected on Teon lters in an urban area in Amman, Jordan was examined 92 for the element content by PIXE, and the speciation was determined by XANES. The measurements were made at the IAEA X-ray spectrometry endstation at the XRF beamline of Elettra-Sincrotrone Trieste, Trieste, Italy, where XANES spectra, generated with grazing incidence geometry (to maximise the footprint of the beam) at the Pb L 3 -edge (13 035 eV), were collected by a Si (111) monochromator in uorescence mode. The XANES spectra were normalised with respect to the EXAFS part of the spectrum using the ATHENA soware package and then analysed through linear combination tting to spectra of the available reference lead compounds (Pb metal, PbO, PbO 2 , Pb 3 O 4 , PbS and PbSO 4 ).…”

“…Continuing on their studies of new developments in HPLC separations with ICP-MS detection , the research group of Goessler, based in Graz, has developed a simple one-step derivatisation procedure to enable the separation of the metabolite and biomarker phosphoethanolamine from the hydrophilic P-containing metabolome present in urine. 111 In particular, phosphoethanolamine has been reported as a reliable biomarker with consistently elevated urinary levels in hypophosphatasia, an inherited metabolic bone disorder. It was possible to elute the hydrophobic derivative using 1,2-hexanediol, a novel chromatographic eluent developed previously by the group and discussed in the 2023 ASU review.…”