TITLE: Liquid-phase metabolomics analysis: from preparation to injection
AUTHORS: Louise BOISSIN, axel.raux, Yann Guitton, Anne-Lise ROYER

[DESCRIPTION]
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.

[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.

[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.


[STEPS]
SECTION: I. Sample preparation
1. For serum, cell culture or plasma 
SECTION: II. Quality control
4. Preparing the "Mix Metabo 2"
SECTION: I. Sample preparation
1.1. Attention: The use of filter cones is essential for matrix sampling

Triphasic preparation: Blight and Dyer

Thaw samples previously stored at -20 °C or -80 °C on ice
After thawing, vortex each sample for 20 s
Remove30 µL of matrix and transfer to a 5 mL glass tube
Add 190 µL of cold methanol (LC-MS) and vortex for 20 s
Add 390 µLof cold chloroform solution (ANALAR Normapur) and vortex for 20 s
Add 120 µLwater (LC-MS) and vortex for 20 s
Centrifuge tubes for 20 min at 450 x g, 4 °C
After centrifugation, remove190 µL of the methanolic phase and transfer to an HPLC vial
Evaporate vials under nitrogen flow at 25 °C
Store vials at -20 °C
SECTION: III. Analytical conditions
8. 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

SECTION: I. Sample preparation
1.2. Triphasic preparation: MTBE

Thaw samples previously stored at -20 °C or -80 °C on ice
After thawing, vortex each sample for 20 s
Place 30 µL of matrix and 220 µL of cold methanol (LC-MS) in a 5 mL glass tube
Vortex the tube for 20 s
Add 750 µL of MTBE (LC-MS) and vortex for 20 s
Place samples at 4 °C and vortex every 2 min for 6 min
Add 188 µL of water (LC-MS), vortex for 20 s
Centrifuge tubes for 20 min at450 x g
Collect 190 µL of aqueous phase. Evaporate vials to dryness under nitrogen flow at 25 °C
Store vials at -20 °C

SECTION: I. Sample preparation
1.3. Methanol extraction

Thaw samples previously stored at -20 °C or -80 °C on ice
After thawing, vortex each sample for 20 s
400 µL matrix + 1400 µLMeOH in an eppendorf tube
Vortex for15 s
Centrifuge at room temperature at 4.5 rpm for 20 min
Collect 370 µLof the methanolic fraction and place in a vial without insert
Evaporate to dryness under nitrogen at 25 °C

SECTION: I. Sample preparation
2. For urine matrix
SECTION: I. Sample preparation
2.1. Mesuring pH and urine density

Thaw in ice°C  samples previously stored at -20 °C or on ice 
Take an aliquot of 1 mL  in a 5 mL glass tube 
Vortexing 
Centrifuge at 750 x g, 4 °C 
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 200 µLof liquid are required to cover the cell). 

SECTION: I. Sample preparation
2.2. 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".

 


SECTION: I. Sample preparation
2.3. Filtration and centrifugation

Collect 500 µL of urine and place in an Eppendorf tube fitted with a 10kDa filter
Centrifuge at 13000 x g for about 30 min at about 5 °C
Remove the filter and store the filtrate at 4 °C

SECTION: I. Sample preparation
3. Preparing samples for injection
SECTION: I. Sample preparation
3.1. For RPLC analysis

If samples have been frozen, thaw in melting ice
Transfer samples to 100 µL of LC-MS-grade H2O-ACN (95:5) and vortex for 20 s
If necessary, centrifuge samples for 10 min at 450 x g and 4 °C
Transfer to a vial with insert

SECTION: II. Quality control
4.1. 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: 

 

Make up to 10 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 100 µLof the mix, evaporate under nitrogen at 25 °C. Then transfer to 100 µLof LC-MS-grade Acetonitrile (ACN)/Water (5:95, (v:v)) for reverse-phase analysis, or 100 µLof LC-MS-grade ACN/H2O (90:10) for HILIC analysis.  
SECTION: II. Quality control
7. Preparing the mobile phase (PM)
SECTION: II. Quality control
7.1. Preparation of mobiles phases:

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

SECTION: III. Analytical conditions
9. LC-HRMS instrument : LC-QExactive
SECTION: III. Analytical conditions
9.1. RPLC analysis 

Solvents:

Solvent A: 1 LH2O, 1 mLglacial acetic acid
Solvent B: 1 L ACN, 1 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.

 


 

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 35 °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.

 



SECTION: III. Analytical conditions
9.2. HILIC analysis 

Solvents:

Solvent A: 990 mL H2O, 10 mL 1000 mM ammonium acetate
Solvent B: 990 mL ACN, 10 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.


 


 

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.

 


 

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.

SECTION: II. Quality control
5. QC 
SECTION: II. Quality control
6. Blank
SECTION: II. Quality control
5.1. 
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.
SECTION: II. Quality control
6.1. 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. 
SECTION: I. Sample preparation
3.2. For HILIC analysis

If samples have been frozen, thaw on ice
Transfer samples to 100 µL H2O/ACN mixture (10:90) and vortex for 20 s
If necessary, centrifuge samples for 10 min at 450 x g and 4 °C
Transfer to a vial with insert

SECTION: III. Analytical conditions
10. Calibration
SECTION: III. Analytical conditions
10.1. 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
SECTION: III. Analytical conditions
11. Acquisition software
SECTION: III. Analytical conditions
11.1. QExactive: Xcalibur (version 4.4) and processing: Chromeleon
Exactive: Xcalibur (version) and processing: Aria