Nov 21, 2024

Public workspaceLiquid Chromatography High-Resolution Mass Spectrometry metabolomics analysis: from preparation to injection

 Forked from Liquid-phase metabolomics analysis: from preparation to injection
DOI
dx.doi.org/10.17504/protocols.io.5jyl82odrl2w/v1
  • 1Oniris, INRAE, LABERCA, 44300 Nantes, France
Yann GUITTON
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82odrl2w/v1
External link: https://www.laberca.org/en/analytical-platform/presentation
Protocol Citation: Louise BOISSIN, axel.raux, Yann Guitton, Anne-Lise ROYER 2024. Liquid Chromatography High-Resolution Mass Spectrometry metabolomics analysis: from preparation to injection. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82odrl2w/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: In development
We are still developing and optimizing this protocol
Created: September 11, 2024
Last Modified: November 21, 2024
Protocol Integer ID: 107328
Keywords: Metabolomics, Mass spectrometry, LC-HRMS, MetaboHUB
Abstract
The purpose of this notice is to define two types of metabolomics analysis on an LC-HRMS instrument (Orbitrap or ToF) with either polar (HILIC column) or non-polar (RPLC column).

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

The protocol enables samples to be obtained which can then be statistically reprocessed according to the metabolomics workflow.
Image Attribution
MELISA platform
Guidelines
Recommandations:

  • Perform several blank injections at the start of analysis to check for contamination;
  • Perform a system suitability test (SST) with two injections of the standard mix ("Mix metabo 2") at the beginning and end of the sequence;
  • Before launching the samples injection check with "Mix Metabo 2" profiles 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;
  • Inject diluted QC (1/2, by 4 and by 8) and check that the molecules follow this decrease in concentration;
  • Injection of a QC every 5 samples.

Materials
Calibrants:

  • Thermo Solution: ESI+ (Reference: 88322)
  • Thermo Solution: ESI- (Reference : 88324)

Pipettes and tips:

  • Filter tips
  • P20, P200 and P1000 pipettes

Tubing:

  • Centrifuge tube
  • HPLC glass vials with 200 µL insert
  • 5 mL glass tubes (except for urine preparation)
  • 10 kDa filters centrifuge tubes (only for urine preparation)

Equipment lab:

  • Centrifuge for tube, vials or eppendorf
  • Nitrogen evaporator with racks for HPLC vials and tubes
  • Refractometer (only for urine preparation)
  • Universal Indicator Paper Strips for pH 1-14 (only for urine preparation)

Instruments:

  • LC-HRMS : Mass spectrometer QExactive - Orbitrap - Thermo Fisher
  • LC-HRMS : Mass spectrometer Exactive - Orbitrap - Thermo Fisher
  • LC-QTOF : Mass spectrometer Synapt Acquity - TOF - Waters
Protocol materials
ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #366919-1L
Step 1.1
ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5
In 26 steps
ReagentMTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML
Step 1.2
ReagentEtOH 100% at -20ºC
Step 4.1
ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
In 5 steps
ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998
In 16 steps
ReagentAcetic acid glacial ≥99.7% ACSVWR InternationalCatalog #36289.K3
In 2 steps
ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5
In 7 steps
ReagentAcetic acidFisher Scientific
In 2 steps
Safety warnings
Wear your Personal Protective Equipment (PPE) at least a lab coat, protective eyewear, gloves, long pants and closed toe shoes
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.


Note
ACN and methanol solvents are HPLC grade, Milli-Q grade water and LC-MS grade isopropanol and chloroform.

I. Sample preparation
I. Sample preparation
For serum, plasma or cell culture media
Triphasic preparation: Bligh and Dyer


Attention: The use of filter tips is essential for matrix sampling

  • 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 coldReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 and vortex for Duration00:00:20
  • Add Amount390 µL of cold ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #366919-1L and vortex for Duration00:00:20
  • Add Amount120 µL ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 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 (with 200 µL insert)
  • Evaporate vials under nitrogen flow at Temperature25 °C
  • Store vials at Temperature-20 °C
Equipment
HERAEUS Multifuge x3 FR
NAME
Centrifuge
TYPE
Thermo Fisher Scientific
BRAND
HERAEUS Multifuge x3 FR
SKU

Equipment
Nitrogen evaporator
NAME
Nitrogen evaporator with racks for HPLC vials and tubes
TYPE
Liebisch Labortechnix
BRAND
-
SKU

Equipment
HPLC transparent vial with insert 0.2mL
NAME
32x11.65mm
TYPE
Thermo
BRAND
-
SKU

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 ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 in a Amount5 mL glass tube
  • Vortex the tube for Duration00:00:20
  • Add Amount750 µL of ReagentMTBE: Sigma Chromasolv 99.8% for HPLC 100mL (smallest available) (34875-100mL)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34875-100ML 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 ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 , 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

Equipment
HERAEUS Multifuge x3 FR
NAME
Centrifuge
TYPE
Thermo Fisher Scientific
BRAND
HERAEUS Multifuge x3 FR
SKU

Equipment
Nitrogen evaporator
NAME
Nitrogen evaporator with racks for HPLC vials and tubes
TYPE
Liebisch Labortechnix
BRAND
-
SKU

Equipment
HPLC vial without insert - screw vial , 1.5mL
NAME
10 mm clear WO
TYPE
Thermo Fisher Scientific
BRAND
-
SKU

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 ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 in a centrifuge 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

Equipment
Mini-centrifuge
NAME
Centrifuge
TYPE
Fisher
BRAND
S67601B
SKU
https://www.fishersci.com/shop/products/fisherbrand-standard-mini-centrifuge-standard-mini-centrifuge/s67601b
LINK
Any standard mini centrifuge with adapters for different tube sizes will suffice
SPECIFICATIONS

Equipment
Nitrogen evaporator
NAME
Nitrogen evaporator with racks for HPLC vials and tubes
TYPE
Liebisch Labortechnix
BRAND
-
SKU

Equipment
Centrifuge Tube
NAME
1.5mL
TYPE
Fisher Scientific
BRAND
05-408-129
SKU
https://www.fishersci.com/shop/products/fisherbrand-premium-microcentrifuge-tubes-1-5ml-natural-1-5ml-o-d-x-l-10-8-x-40-6mm/05408129
LINK

Equipment
HPLC vial without insert - screw vial , 1.5mL
NAME
10 mm clear WO
TYPE
Thermo Fisher Scientific
BRAND
-
SKU

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 strips. 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. ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 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 ,then wipe clean with paper. To obtain good measurements, make sure the cell is sufficiently covered with liquid (approximative Amount200 µL liquid are required to cover the cell).
Equipment
HERAEUS Multifuge x3 FR
NAME
Centrifuge
TYPE
Thermo Fisher Scientific
BRAND
HERAEUS Multifuge x3 FR
SKU

Equipment
MQuant Supelco pH indicator strips
NAME
Universal Indicator Paper Strips for pH 1-14
TYPE
Supelco
BRAND
1.09535.0001
SKU


Equipment
AR200 Automatic Digital Refractometer
NAME
Digital Refractometer
TYPE
Reichert
BRAND
13950000
SKU
https://www.reichertai.com/products/handheld-digital/ar200-automatic-digital-refractometer/
LINK


Filtration and centrifugation

  • Collect Amount500 µL of urine and place in an centrifugation 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
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 Quality Management System.

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


Preparing samples for injection
For RPLC analysis

  • If samples have been frozen, thaw in melting ice
  • Add Amount10 µL of ISTD "Mix Metabo 2" mix into a clean HPLC vial with 200 µL insert
  • Evaporate to dryness under nitrogen flow at Temperature25 °C
  • Transfert Amount100 µL of sample extract into the HPLC vial insert and vortex for Duration00:00:20 (Note: If necessary, centrifuge samples for Duration00:10:00 at Centrifigation450 x g and Temperature4 °C before transfer to the HPLC vial with insert)

Note
Tips: Prepare the Mix Metabo 2 SST at the same time (see 4.1).

Depending on the available sample extract really available adjust the transfered sample extract amount in order to keep a constant ISTD dilution.

If you have to dilute your sample extracts then do it with ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 mixture (95:5) and vortex for Duration00:00:20 .



Equipment
HERAEUS Multifuge x3 FR
NAME
Centrifuge
TYPE
Thermo Fisher Scientific
BRAND
HERAEUS Multifuge x3 FR
SKU

Equipment
Nitrogen evaporator
NAME
Nitrogen evaporator with racks for HPLC vials and tubes
TYPE
Liebisch Labortechnix
BRAND
-
SKU

Equipment
HPLC transparent vial with insert 0.2mL
NAME
32x11.65mm
TYPE
Thermo
BRAND
-
SKU

10m 20s
For HILIC analysis

  • If samples have been frozen, thaw in melting ice
  • Add Amount10 µL of ISTD "Mix Metabo 2" mix into a clean HPLC vial with 200 µL insert
  • Evaporate to dryness under nitrogen flow at Temperature25 °C
  • Transfert Amount100 µL of sample extract into the HPLC vial insert and vortex for Duration00:00:20 (Note: If necessary, centrifuge samples for Duration00:10:00 at Centrifigation450 x g and Temperature4 °C before transfer to the HPLC vial with insert)

Note
Tips: Prepare the Mix Metabo 2 SST at the same time (see 4.1).

Depending on the available sample extract really available adjust the transfered sample extract amount in order to keep a constant ISTD dilution.

If you have to dilute your sample extracts then do it with ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 mixture (10:90) and vortex for Duration00:00:20 .



Equipment
HERAEUS Multifuge x3 FR
NAME
Centrifuge
TYPE
Thermo Fisher Scientific
BRAND
HERAEUS Multifuge x3 FR
SKU

Equipment
HPLC transparent vial with insert 0.2mL
NAME
32x11.65mm
TYPE
Thermo
BRAND
-
SKU

10m 20s
II. Quality control
II. Quality control
Preparation of System Suitability and Internal Standard "Mix Metabo 2"
A solution of standard compounds used in metabolomics, called "Mix Metabo 2" at 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 LC-MS ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentEtOH 100% at -20ºCContributed by users (25:75, (v:v)).

Preparing Mix Metabo 2 for System Suitability Test run before starting of the sample analysis sequence:

In an HPLC vial with insert, take Amount100 µL of the Mix Metabo 2 mix at 10 ng/uL (or less if your instrument is sensitive enought), evaporate under nitrogen at Temperature25 °C . Then add into the HPLC vial Amount100 µL of ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 /ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 (5:95, (v:v)) for RPLC analysis, or Amount100 µL of ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 /ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 (90:10, (v:v)) for HILIC analysis.
This SST vial will be used to qualify the LC-HRMS system before any injections and after each batch analysis.


Equipment
HPLC transparent vial with insert 0.2mL
NAME
32x11.65mm
TYPE
Thermo
BRAND
-
SKU



Preparation of QC pool

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.
QC pool dilutions by 2, by 4 and by 8 have to be prepared.
  • TransfertAmount100 µL of sample extract in a centrifuge tube
  • AddAmount100 µL of the dilution solvant either ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 mixture (95:5) for RPLC or ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 mixture (10:90) for HILIC then vortex for Duration00:00:20
  • Transfert 100 µL of solution into a clean HPLC vial with insert and label it as "dil2 QC pool"
  • Repeat these steps to create "dil4 QC pool" and "dil8 QC pool" vials
These vials will be used to facilitate data filtration during data analysis (e.g. search for matrix effect)


Equipment
HPLC transparent vial with insert 0.2mL
NAME
32x11.65mm
TYPE
Thermo
BRAND
-
SKU

20s
Blank
A "handling blank" have to be carried out to assess the contamination caused by the sample 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 mixture (95:5) with 0.1% Acetic Acid
  • Solvent A: Amount1 L ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 , Amount1 mL ReagentAcetic acidFisher Scientific
  • Solvent B: Amount1 L ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 , Amount1 mL ReagentAcetic acidFisher Scientific

For HILIC analysis: H2O/ACN mixture (10:90) with 10mM ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
  • Solvent A: Amount990 mL ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 , Amount10 mL 1000 mM ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
  • Solvent B: Amount990 mL ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 , Amount10 mL 1000 mM ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G



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 (SST)
. 1* Procedural Blank
. 6* QC
. 3* dil8 QC pool
. 3* dil4 QC pool
. 3* dil2 QC pool
. 5* samples
. 1 QC
. 5* samples
...
. QC
. QC with DDA or DIA MS/MS method (Positive mode)
. QC with DDA or DIA MS/MS method (Negative mode)
. Mix Metabo 2 (SST)
. 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



Note
Check PM and SST injections results before going further. Look at the control chart available.


CITATION
Broadhurst D, Goodacre R, Reinke SN, Kuligowski J, Wilson ID, Lewis MR, Dunn WB (2018). Guidelines and considerations for the use of system suitability and quality control samples in mass spectrometry assays applied in untargeted clinical metabolomic studies..
LINK
https://doi.org/10.1007/s11306-018-1367-3

Equipment
LC-HRMS
NAME
QExactive Mass spectrometer (MS) Orbitrap
TYPE
Thermo Fisher
BRAND
-
SKU

Equipment
LC-HRMS
NAME
Exactive Mass Spectrometer (MS) Orbitrap
TYPE
Thermo Fisher
BRAND
-
SKU

Equipment
LC-QTOF
NAME
Mass spectrometer Synapt Acquity
TYPE
Waters
BRAND
-
SKU

Analyze
LC-HRMS instrument : LC-QExactive
RPLC analysis

Solvents:

  • Solvent A: Amount1 L ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 , Amount1 mL ReagentAcetic acid glacial ≥99.7% ACSVWR InternationalCatalog #36289.K3
  • Solvent B: Amount1 L ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 , Amount1 mL ReagentAcetic acid glacial ≥99.7% ACSVWR InternationalCatalog #36289.K3
  • 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: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 ;
  • Rinse bottle N°2: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 or ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 (20:80).

Q-Exactive: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 or ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 (95:5).


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 ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 /ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 (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 ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 , Amount10 mL 1000 mM ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
  • Solvent B: Amount990 mL ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 , Amount10 mL 1000 mM ReagentAmmonium AcetateMerck MilliporeSigma (Sigma-Aldrich)Catalog #A1542-500G
  • 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: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 ;
  • Rinse bottle N°2: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 or ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 (20 :80).

Q-Exactive: ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentMethanol LC-MS grade B&J BrandVWR InternationalCatalog #BJLC230-2.5 or ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 /ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 (95:5).

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 Temperature25 °C .
The ZIC HILIC column is reconditioned before storage in ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34998 /ReagentWater LC-MS grade B&J BrandVWR InternationalCatalog #BJLC365-2.5 (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


Note
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 = Temperature50 °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 Chromeleon for HPLC
  • Exactive: Xcalibur (version 2.1) and Aria for HPLC

Citations
Step 2.3
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