Sep 10, 2024

Public workspaceLiquid-phase metabolomics analysis: from preparation to injection

This protocol is a draft, published without a DOI.
  • 1Oniris, INRAE, LABERCA, 44300 Nantes, France
Louise BOISSIN
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Protocol Citation: Louise BOISSIN, axel.raux, Yann Guitton, Anne-Lise ROYER 2024. Liquid-phase metabolomics analysis: from preparation to injection. Protocol exchange https://protocols.io/view/liquid-phase-metabolomics-analysis-from-preparatio-c9f5z3q6
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: In development
We are still developing and optimizing this protocol
Created: February 20, 2024
Last Modified: September 10, 2024
Protocol Integer ID: 95453
Keywords: Metabolomics, Mass spectrometry, LC-HRMS, MetaboHUB
Abstract
The purpose of this notice is to define two types of injection (RPLC and HILIC) on an LC-HRMS instrument for liquid-phase metabolomics analysis.

The steps cover the sample preparation, the quality control and the analytical condition.
Guidelines
Recommandations:

  • Perform several blank injections at the start of analysis to check for contamination;
  • Perform two injections of "Mix metabo 2" at the beginning and end of the sequence;
  • Check that there has been no drift in retention times, that intensity is good and that mass accuracy is correct;
  • Injection of 6 QC at start of sequence;
  • Injection of a QC every 5 samples;
  • Inject diluted QC (1/2, by 4 and by 8) and check that the molecules follow this decrease in concentration.

Materials
Solvents :

  • Methanol (LC-MS, Honeywell) - CAS 67-56-1
  • Chloroform (ANALAR Normapur)- CAS 67-66-3
  • Water (LC-MS, ThermoFisher Scientific) - CAS 7732-18-5
  • MTBE - CAS 1634-04-4
  • Acetonitrile (LC-MS, Carlo Erba) - CAS 75-05-8
  • Ammonium acetate (Supelco) - CAS 631-61-8

Calibrants :

  • Thermo Solution: ESI+ (Reference: 88322)
  • Thermo Solution: ESI- (Reference : 88324)
Safety warnings
EPI
Before start
  • To avoid any degradation of samples, the use of plastic materials should be strictly limited. A temperature close to zero must be maintained throughout the process;
  • The protocols are presented for guidance only, and it will be necessary, in the context of a new project, to carry out pre-tests in order to assess the proportions of matrix and solvents. Similarly, MS and gradient parameters can be adjusted according to matrix richness;
  • Ready-to-use column: reconditioning of the column after each injection sequence.
I. Sample preparation
I. Sample preparation
For serum, cell culture or plasma
Attention: The use of filter cones is essential for matrix sampling

Triphasic preparation: Blight and Dyer

  • Thaw samples previously stored at Temperature-20 °C or Temperature-80 °C on ice
  • After thawing, vortex each sample for Duration00:00:20
  • RemoveAmount30 µL of matrix and transfer to a Amount5 mL glass tube
  • Add Amount190 µL of cold methanol (LC-MS) and vortex for Duration00:00:20
  • Add Amount390 µL of cold chloroform solution (ANALAR Normapur) and vortex for Duration00:00:20
  • Add Amount120 µL water (LC-MS) and vortex for Duration00:00:20
  • Centrifuge tubes for Duration00:20:00 at Centrifigation450 x g, 4°C
  • After centrifugation, removeAmount190 µL of the methanolic phase and transfer to an HPLC vial
  • Evaporate vials under nitrogen flow at Temperature25 °C
  • Store vials at Temperature-20 °C
21m 20s
Triphasic preparation: MTBE

  • Thaw samples previously stored at Temperature-20 °C or Temperature-80 °C on ice
  • After thawing, vortex each sample for Duration00:00:20
  • Place Amount30 µL of matrix and Amount220 µL of cold methanol (LC-MS) in a Amount5 mL glass tube
  • Vortex the tube for Duration00:00:20
  • Add Amount750 µL of MTBE (LC-MS) and vortex for Duration00:00:20
  • Place samples at Temperature4 °C and vortex every Duration00:02:00 for Duration00:06:00
  • Add Amount188 µL of water (LC-MS), vortex for Duration00:00:20
  • Centrifuge tubes for Duration00:20:00 atCentrifigation450 x g
  • Collect Amount190 µL of aqueous phase. Evaporate vials to dryness under nitrogen flow at Temperature25 °C
  • Store vials at Temperature-20 °C

29m 20s
Methanol extraction

  • Thaw samples previously stored at Temperature-20 °C or Temperature-80 °C on ice
  • After thawing, vortex each sample for Duration00:00:20
  • Amount400 µL matrix + Amount1400 µL MeOH in an eppendorf tube
  • Vortex forDuration00:00:15
  • Centrifuge at room temperature at Centrifigation4.5 rpm for Duration00:20:00
  • Collect Amount370 µL of the methanolic fraction and place in a vial without insert
  • Evaporate to dryness under nitrogen at Temperature25 °C

20m 35s
For urine matrix
Mesuring pH and urine density

  • Thaw in iceTemperature0 °C samples previously stored at Temperature-20 °C or TemperatureOn ice
  • Take an aliquot of Amount1 mL in a Amount5 mL glass tube
  • Vortexing
  • Centrifuge at Centrifigation750 x g, 4°C , at least 5 min
  • The pH is measured using pH paper. The pH value can provide information to explain why a sample may be atypical
  • The urine specific gravity value is used to normalize the sample. It is measured using a refractometer. Zero measurement is carried out before each use and every 30 samples measured. Milli-Q water is placed on the refractometer's measuring cell, the "Zero" button is pressed and the value of 1,000 is displayed. Between each measurement, rinse the cell with Milli-Q water, then wipe clean with paper. To obtain good measurements, make sure the cell is sufficiently covered with liquid (approximative Amount200 µL of liquid are required to cover the cell).

Normalization

The normalization presented here is a urinary density normalization. The Excel model "normalization by urine density" can be used to perform all normalization calculations. The dilution rate depends on the type of sample analyzed (human urine, pig urine, etc.)
In the Excel "Urine Density Normalization", in order to evaluate the dilution ratio, a standard curve is created by measuring the urine density of the urine with the highest urine density at different dilution ratios. Normalization is then performed for each sample. Randomly check the new urine specific gravity value of a few samples.
The excel file is available on LABERCA's "GED".

CITATION
Jacob CC, Dervilly-Pinel G, Deceuninck Y, Gicquiau A, Chevillon P, Bonneau M, Le Bizec B (2017). Urinary signature of pig carcasses with boar taint by liquid chromatography-high-resolution mass spectrometry..
LINK
https://doi.org/10.1080/19440049.2016.1265152


Filtration and centrifugation

  • Collect Amount500 µL of urine and place in an Eppendorf tube fitted with a 10kDa filter
  • Centrifuge at Centrifigation13000 x g for about Duration00:30:00 at about Temperature5 °C
  • Remove the filter and store the filtrate at Temperature4 °C

30m
Preparing samples for injection
For RPLC analysis

  • If samples have been frozen, thaw in melting ice
  • Transfer samples to Amount100 µL of LC-MS-grade H2O-ACN (95:5) and vortex for Duration00:00:20
  • If necessary, centrifuge samples for Duration00:10:00 at Centrifigation450 x g and Temperature4 °C
  • Transfer to a vial with insert

10m 20s
For HILIC analysis

  • If samples have been frozen, thaw on ice
  • Transfer samples to Amount100 µL H2O/ACN mixture (10:90) and vortex for Duration00:00:20
  • If necessary, centrifuge samples for Duration00:10:00 at Centrifigation450 x g and Temperature4 °C
  • Transfer to a vial with insert

10m 20s
II. Quality control
II. Quality control
Preparing the "Mix Metabo 2"
A solution of standard compounds used in metabolomics, called "Mix Metabo 2" at 1, 5 or 10 ng/µL. This solution can be used as an internal standard, but also as a standard at the start of a sequence to validate instrumental performance.
This mix contains the following 4 standard compounds, available from Sigma-Aldrich and Cluzeau Info Labo (CIL): L-leucine-5,5,5-d3 (CAS: 87828-86-2), L-tryptophan-2,3,3-d3 (CAS: 133519-78-5), Indole-2,4,5,6,7-d5-3-acetic acid (CAS: 76937-78-5), 1,14-tetradecanedioic-d24 acid (CAS: 130348-88-8).

In a 15mL amber vial, add the following volumes of each standard at 1mg/mL to obtain the metabo 2 mix solution at 10 ng/µL:

NameSolventVolume introduce
L-tryptophan-2,3,3-d3H2O:EtOH (25:75), 1 mg/mL100 µL
L-leucine-5,5,5-d3H2O:EtOH (75:25), 1 mg/mL100 µL
Indole-2,4,5,6,7-d5-3-acetic acidH2O:EtOH (25:75), 1 mg/mL100 µL
1,14-tetradecanedioic-d24 acidH2O:EtOH (25:75), 1 mg/mL100 µL
Preparation of stock solutions

Make up to Amount10 mL with LCMS water/HPLC ethanol solution (25:75, (v:v)).

Preparing Mix Metabo 2 as the standard for the start of the sequence:

In a vial with insert, take Amount100 µL of the mix, evaporate under nitrogen at Temperature25 °C . Then transfer to Amount100 µL of LC-MS-grade Acetonitrile (ACN)/Water (5:95, (v:v)) for reverse-phase analysis, or Amount100 µL of LC-MS-grade ACN/H2O (90:10) for HILIC analysis.
QC

The QC corresponds to the sample pool, and can be carried out at different stages of the manipulation. If the matrix is in liquid form, it is preferable to carry out the QC before handling, and to treat the QC pool as a sample.

In the case of a solid matrix, QC pooling can be carried out when the organic phase is sampled.

Caution: Allow for a sufficiently large volume of QC to be injected throughout the sequence, and plan for possible re-injections.
Blank
A "handling blank" can be carried out to assess the contamination caused by the handling. The handling blank follows the same protocol as that for the samples, the only difference being that the matrix is replaced by water.
Preparing the mobile phase (PM)
Preparation of mobiles phases:

  • For RPLC analysis: H2O/ACN (95/5)
  • For HILIC analysis: H2O/ACN (10/90)

III. Analytical conditions
III. Analytical conditions
Instrument set-up

Create a randomized injection sequence incorporating QC injection every five samples.

Typical sequence :

. 3*Phase Mobile (PM)
. 2* Mix metabo 2
. White handling
. 6* QC
. 5* samples
. 1 QC
. 5* samples
...
. QC
. Metabo 2 mix
. 3* PM

Instruments used :
  • LC-HRMS : Mass spectrometer QExactive - Orbitrap - Thermo Fisher
  • LC-HRMS : Mass spectrometer Exactive - Orbitrap - Thermo Fisher
  • LC-QTOF : Mass spectrometer Synapt Acquity - TOF - Waters

LC-HRMS instrument : LC-QExactive
RPLC analysis

Solvents:

  • Solvent A: Amount1 L H2O, Amount1 mL glacial acetic acid
  • Solvent B: Amount1 L ACN, Amount1 mL glacial acetic acid
  • Washing solutions depending on equipment type

Gradient:

This is a typical gradient, which can be re-evaluated depending on the type of analysis performed.

Time (min)A (%)B (%)
0955
2.4955
4.57525
112575
140100
16.50100
19955
25955
Elution gradient




Wash solution:

Rinse bottle N°1 H2O LC-MS; rinse bottle N°2 H2O-MeOH or H2O-ACN (20 :80).
Q-Exactive: H2O-MeOH or H2O-ACN (95:5). ACN and methanol solvents are HPLC grade, Milli-Q grade water and LC-MS grade isopropanol

Column:

The column used is a Hypersil Gold C18 (1.9µm, 100mm*2.1mm), Thermo Scientific. This column is used with a pre-column whose filter is changed regularly. Flow rate is 0.4 mL/min, oven temperature Temperature35 °C .
The Hypersil Gold C18 column is reconditioned before storage in ACN/H2O (80:20).

Tune parameters:

These are typical parameters, which can be re-evaluated according to the type of analysis performed.

AB
Sheath gas (L/h)55
Auxiliary gas (L/h)10
Sweep gas (L/h)0
Capillary temperature (°C)350
Capillary voltage (V)(+): 30 ; (-): -30
Tube lens voltage (V)(+): 100 ; (-): -80
Injection on Thermo brand instruments, for both ionization modes



HILIC analysis

Solvents:

  • Solvent A: Amount990 mL H2O, Amount10 mL 1000 mM ammonium acetate
  • Solvent B: Amount990 mL ACN, Amount10 mL 1000 mM ammonium acetate
  • Washing solutions depending on equipment type

Gradient:

This is an example of a gradient, which can be re-evaluated depending on the type of analysis performed.


Time (min)A (%)B (%)
0595
2595
52080
124060
146040
166040
18595
28595
Elution gradient




Wash solution:

Rinse bottle N°1 H2O LC-MS; rinse bottle N°2 H2O-MeOH or H2O-ACN (20 :80).
Q-Exactive: H2O-MeOH or H2O-ACN (95:5). ACN and methanol solvents are HPLC grade, Milli-Q grade water and LC-MS grade isopropanol

Column:

The column used is a SeQuant ZIC HILIC (5µm, 100mm*2.1mm), Merck KGaA. This column is used with a pre-column whose filter is changed regularly. Flow rate is 0.25 mL/min, oven temperature 25°C.
The ZIC HILIC column is reconditioned before storage in ACN-H2O (80:20).

Tune parameters:

These are typical parameters, which can be re-evaluated according to the type of analysis performed.

AB
Sheath gas (L/h)55
Auxiliary gas (L/h)10
Sweep gas (L/h)0
Capillary temperature (°C)350
Capillary voltage (V)(+): 30 ; (-): -30
Tube lens voltage (V)(+): 100 ; (-): -80
Injection on Thermo brand instruments, for both ionization modes


AB
Source temperature (°C)120 to 150
Sampling cone (V)30 to 40
Source offset (V)80
Source gas flow (mL/min)50
Desolvatation temperature (°C)650
Cone gas flow (L/h)400
Desolvatation gas flow (L/h)800
Injection on Synapt (Waters) type instruments, in positive ionization mode

Remark:

The use of solvents containing salts can lead to clogging of the tubing. To avoid this effect, perform several injections at the end of the sequence without heating the probe (HESI = 50°C). In addition, recondition the column with salt-free solvents, taking care that the reconditioning solvents also pass into the source and not into the waste garbage can.

Calibration
Thermo Solution: ESI+ (Reference : 88322) et ESI- (Reference : 88324)
Calibration to be carried out each time you start using the project
Cone and capillary cleaning according to instrument
Acquisition software
  • QExactive: Xcalibur (version 4.4) and processing: Chromeleon
  • Exactive: Xcalibur (version) and processing: Aria

Citations
Step 2.2
Jacob CC, Dervilly-Pinel G, Deceuninck Y, Gicquiau A, Chevillon P, Bonneau M, Le Bizec B. Urinary signature of pig carcasses with boar taint by liquid chromatography-high-resolution mass spectrometry.
https://doi.org/10.1080/19440049.2016.1265152