Oct 05, 2022
High Performance Liquid Chromatography (HPLC) and sample preparation
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. High Performance Liquid Chromatography (HPLC) and sample preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm2ko5g3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70860
Keywords: ASAPCRN
Abstract
HPLC for metabolites in mDA neurons
Pre-sample prep
Pre-sample prep
Prior to harvesting mDA neurons, all cells were incubated in phenol-free medium for 24 hours with and without the presence of 80 micromolar (µM) L-Dopa (Sigma).
Extracellular metabolite sample prep
Extracellular metabolite sample prep
For extracellular metabolites, media after 24 hours was mixed 1:1 in 0.8 Molarity (M) ice cold perchloric acid.
Samples were incubated on ice for 00:10:00 , followed by centrifugation at 12000 x g, 4°C, 00:05:00 .
15m
The supernatant was removed and frozen in dry ice for HPLC analysis.
Intracellular metabolite sample prep
Intracellular metabolite sample prep
25m
25m
For intracellular metabolites, after 24 hours, the cells were removed and pelleted by centrifugation at 1200 x g, 4°C, 00:05:00 .
5m
The cells were washed once in PBS and lysed on ice using lysis buffer (10mM Tris (pH 7.4), 1mM EDTA, 320mM sucrose in HPLC grade water).
Lysate was mixed with 1:1 in 0.8 Molarity (M) ice cold perchloric acid and incubated on ice for 00:10:00 .
10m
The samples were centrifuged at 12000 x g, 4°C, 00:10:00 and the supernatant was harvested and frozen for HPLC analysis.
10m
HPLC
HPLC
Quantification of neurometabolites (DOPAC, 3-OMD, 5-HIAA, HVA and dopamine) was carried out using reverse phase HPLC and an electrochemical detector
The mobile phase (flow rate 1.5ml/min) contained 16% methanol, 20mM sodium acetate trihydrate, 12.5mM citric acid monohydrate, 3.35mM 1-octanesulfonic acid, 0.1mM EDTA disodium and adjusted to pH 3.45 with 12 M hydrochloric acid (HCl).
The stationary phase was maintained at 27 °C .
The detector electrode was set at 450mV and screening electrode at 50mV.
50µl of sample was injected and calculated against a 500nM external standard solution of the 5 compounds of interest made in HPLC grade water acidified with 12M HCl.
Peak areas were quantified with EZChrom Elite™ chromatography data system software, version 3.1.7 (JASCO UK Ltd., Great Dunmow, UK).