Optimization of micro‐HPLC peak focusing for the detection and quantification of low hepcidin concentrations

Abstract

Micro-high-performance liquid chromatography is a miniaturized, economic and ecological chromatographic system allowing the use of reduced size chromatographic columns. Coupled with electrospray ionization tandem mass spectrometry, this technique can be used to detect and quantify low concentrations of peptides. In this study, hepcidin was used as the model compound and analysed using octadecylsilica stationary phase by means of a gradient elution mode at a flow rate of 4 μL/min. Several parameters were studied to optimize peak focusing. Using the methodology of experimental design, the mobile-phase gradient conditions and the sample composition were optimized in order to maximize the sensitivity and minimize retention time. Stability of the target peptide in solution was also demonstrated.

Microflow Liquid Chromatography Coupled to Mass Spectrometry—An Approach to Significantly Increase Sensitivity, Decrease Matrix Effects, and Reduce Organic Solvent Usage in Pesticide Residue Analysis

“…This graphic also presents XIC chromatograms obtained by HPLC at a flow rate of 400 μL per minute. Better results, in terms of sensitivity, compared with classical HPLC, have already been reported in other applications. , Since the Guidance document SANCO/12571/2013 also recommends LC-MS performance requirements, a mixture of pesticides and internal standards was used to assess the stability and repeatability of retention times and peak areas. Mixtures containing 90 pesticides in a solvent and in the selected matrices (within a 5 μg kg –1 to 400 μg kg –1 concentration range) were analyzed over 3 days to assess the stability of chromatographic conditions, and thus the repeatability of retention times.…”

“…23, 26 Since Guidance document 275 SANCO/12571/2013 24 also recommends LC-MS performance requirements, a mixture of pesticides 276…”

“…LC-MS/MS analysis 26 : the acquisition of two SRM transitions, the retention time (a tolerance of ±0.2 178 min) and SRM ratio compliance (the relationship between the abundance of transitions selected for 179…”

“…This manuscript reports a new pesticide residues analysis method employing a microflow-liquid 26 chromatography system coupled to a triple quadrupole mass spectrometer (microflow-LC-ESI-QqQ-27 MS). This uses an electrospray ionization source with a narrow tip emitter to generate smaller 28 droplets.…”

“…The most intense SRM 1 transition was selected for quantitation. Identification was based on the EU guideline for LC−MS/MS analysis: 26 the acquisition of two SRM transitions, the retention time (a tolerance of ±0.2 min), and SRM ratio compliance (the relationship between the abundance of transitions selected for identification and for quantification, SRM 2 /SRM 1 with a tolerance of ±30%). Analyst software 1.6.2 was used for data acquisition and processing.…”