Polyphenol data acquisition by PFP – QExactive HF mass spectrometry

West Coast Metabolomics Center Standard Operating Procedure | 04-04-2020
Issued: 07-26-2019; Valid from: 04-04-2020; This SOP supersedes: new
Validity area: UC Davis Genome Center, Metabolomics Core and Research Laboratories
Responsible: Arpana Vaniya; Secondary: Bryan Roberts; Approved: Oliver Fiehn

1. Instruments

  • Thermo Scientific Q Exactive mass spectrometer
  • Thermo Scientific Vanquish UHPLC Systems
  • Ultrasonicator
  • VWR VX-2500 Multi-tube vortexer
  • Eppendorf Centrifuge 5424

2. Chemicals and Consumables

3. Procedure

3.1 Pre-run procedures
3.1.1 Instrument calibration
a. Click on Tune icon. Go to File > Load tune file (i.e. Polyphenol_Tune_07122019.mstune)

b. Select appropriate polarity and click Apply.

c. Use the Thermo Scientific Pierce ESI Negative Ion Calibration Solution for negative mode calibration and Pierce™ LTQ ESI Positive Ion Calibration for positive mode calibration. Load the ready use reference standard solution into a syringe and inject directly into the ion source using syringe pump and designated peek tubing.

d. Click on syringe pump settings. Enter flow rate (µL/minutes) (i.e. 5, 10, etc), then click Apply and Start to begin the instrument calibration.

e. In (-) mode, check the profile of the calibrant and ions of m/z 265.148 (SDS), m/z 514.284 (sodium taurocholate), and Ultramark 1621. The TIC variation should be less than 15%. If the TIC variation is higher than 15%, clean the ion source and repeat the instrument calibration.

f. Once the signal intensity has stabilized, check Mass calibration (neg) and click Calibrate. The instruments should be re-calibrated weekly.

g. Repeat steps b-d for positive mode calibration.

h. In (+) mode, check the profile of the calibrant and ions of m/z 195.088 (Caffeine), m/z 524.265 (MRFA) and Ultramark 1621.

i. If the TIC variation is higher than 15%, clean the ion source and repeat the instrument calibration.

3.1.2 Column Equilibration
a. Click on Thermo Xcalibur icon

b. From the menu bar, click main menu or sequence startup

c. To take control of the UHPLC module, click Status and then Direct Control (bottom).

d. This will direct you to the pump module page.

e. Purge the system by clicking More Options. Make sure appropriate eluent is selected (i.e.A1, B1 or A2, B2). Click 100% on line A and purge for 3 minutes at 3 mL/minutes with 50:50 = (acetonitrile: water). Repeat for line B.

f. Then purge line A with mobile phase (A) and line B with mobile phase (B) for 3 minutes at 3 mL/minutes to remove acetonitrile and water.

g. Place the column with guard column into the column compartment. Equilibrate the column with mobile phase (A) and mobile phase (B) at ratio of 93:7 (H2O/ACN) (starting condition) by setting the pump flow rate at 0.1 mL/minutes; increase the flow rate to 0.4 mL/minutes gradually over 5 minutes. Leave at this flow rate for 30 minutes.

3.2 Preparation of Solutions

a. Mobile phase A (100% LC/MS grade water + 0.1% Formic Acid)

  1. Pre-rinse three times 1 L glass bottle with pure acetonitrile.
  2. Add exactly 1000 mL LC/MS grade water.
  3. Add 1 mL formic acid to the 1 L glass bottle.
  4. Sonicate for 10 minutes at room temperature.

b. Mobile phase B (100% ACN + 0.1% Formic Acid)

  1. Pre-rinse three times 1 L glass bottle with pure acetonitrile.
  2. Add exactly 1000 mL LC/MS acetonitrile.
  3. Add 1 mL formic acid to the 1 L glass bottle.
  4. Sonicate for 10 minutes at room temperature.

c. Needle wash solvent (50:50 ACN/H2O)

d. Resuspension solvent

  1. Prepare 100 mL of 7% ACN in water in a graduate flask.
  2. Aliquot 100 µL of hippuric acid-d5 iSTD (100 ppm), 100 µL of quercetin-d3 iSTD (1000 ppm), and 200 µL for all other iSTDs (100 ppm) (See Table 1 & 2) into a 20 mL volumetric flask.
  3. Bring to the final volume of the solution to 20 mL with 7% ACN in water.
  4. Sonicate for 10 minutes.
  5. Store at 4°C.

e. Preparation of samples for analysis

  1. Sonicate resuspension solvent for 5 minutes and swirl gently before use.
  2. Resuspend samples in 100 µL of the resuspension solvent depending on sample concentration.
  3. Vortex sample for 30 seconds.
  4. Sonicate samples for 5 minutes.
  5. Centrifuge for 2 minutes at 16,100 rcf.
  6. Transfer supernatant to LC-MS micro insert in an amber vial.
  7. Cap and load into auto sampler.

3.3 Run Sequence Setup
a. From the menu bar, click sequence startup.
b. Create run list in Excel spreadsheet and save as .csv.
c. From the menu bar, choose File > Import sequence > .csv file
d. After running the pre-run sequence (3 x blank injection), inject the following:
i) Method Blank
ii) BioRec EDTA plasma / NIST SRM3291
iii) Pool sales
iv) 10 samples
v) Repeat steps I-iv

TABLE 1
Internal Standards for Positive Mode

Name Conc. in Resuspension Solvent MS1 (m/z RT (min)
Hippuric acid-d5 iSTD 0.5 185.097 0.76
Caffeine-d9 iSTD 1 204.144 0.77
(−)Epigallocatechin Gallate-d3/d4 iSTD 1 462.111 1.44
trans-Cinnamic Acid -d5 iSTD 1 154.091 3.01
trans-Resveratrol-d4 iSTD 1 233.111 3.03
Daidzein-d4 iSTD 1 259.09 3.16
Quercetin-d3 iSTD 5 306.069 3.92
Genistein-d4 iSTD 1 275.085 4.32
Apigenin-d5 iSTD 1 276.092 4.62
2-hydroxyfluorene-d9 iSTD 1 192.136 5.39
CUDA iSTD 1 341.28 5.71
Reserpine-d9 iSTD 1 618.337 7.58

TABLE 2
Internal Standards for Negative Mode

Name Conc. in Resuspension Solvent MS1 (m/z) RT (min)
Hippuric acid-d5 iSTD 0.5 183.081 0.76
(−)Epigallocatechin Gallate-d3/d4 iSTD 1 460.095 1.37
trans-Cinnamic Acid -d5 iSTD 1 152.075 2.95
trans-Resveratrol-d4 iSTD 1 231.095 2.97
Daidzein-d4 iSTD 1 257.075 3.11
Quercetin-d3 iSTD 5 304.053 3.86
Genistein-d4 iSTD 1 273.07 4.27
Apigenin-d5 iSTD 1 274.076 4.59
2-hydroxyfluorene-d9 iSTD 1 190.121 5.38
CUDA iSTD 1 339.265 5.71
Betulinic acid-d3 iSTD 1 458.372 6.18
Reserpine-d9 iSTD 1 616.322 7.54

3.4 Reverse Phase Data Acquisition Method
a. The methods for reverse phase analysis are located under the folder C:\Xcalibur\methods\2019\CORE\Polyphenol

Positive ion mode: RP_pos_CE203040_A2B2_10min
Negative ion mode: RP_neg_CE203040_A2B2_10min
Polarity switching mode: RP_pos_neg_CE203040_A2B2_10min

b. The acquisition method for the reverse phase analysis method is as shown below:

3.4.1 Positive Ion Mode
i) Pump

ii) Sampler Module

iii) Column Compartment

iv) System

v) Startup Shutdown

The MS conditions are the following:

3.4.2 Negative Ion Mode
The parameters that vary from the positive mode are the following:

3.5 Column Cleaning and Storage
Use this procedure to avoid precipitation mobile phase additives on the column and in the system. Clean column before storage.

Column Cleaning:
a. Flush column with 93:7 (H2O/ACN) by setting the pump flow rate to 0.1 mL/minutes and increase the flow rate to 0.4 mL/minutes over 5 minutes; keep column at this flow rate for 5 minutes.
b. Flush column with 75:25 (H2O/ACN) at 0.4 mL/minutes for 5 minutes.
c. Flush column with 50:50 (H2O/ACN) at 0.4 mL/minutes for 5 minutes.
d. Flush column with 5:95 (H2O/ACN) at 0.4 mL/minutes for 5 minutes.

Column Storage:
a. Flush column with 35:65 (H2O/ACN) at 0.4 mL/minutes for 5 minutes.
b. Remove the column from the system.
c. Store the column in the box until the next batch analysis.

3.6 Additional Column Care Information
Maximum Backpressure: 15,000 psi (1,034 bar) Maximum Temperature: 60°C
Maximum pH: 8 and above
Column volume: 0.10 mL

Column Installation:
a. Install Guard Holder and Cartridge to column.
b. Install the column to LC making sure that the arrow is in the direction of flow. Flush column with 60:40 (ACN/H2O) at 0.1 mL/min then increase to 0.2 mL/min for 10 minutes. Collect solvent in a small beaker.
c. Stop flow and wipe outlet and remove any particulates before connecting to MS.
d. After connecting tubing to ion source, run the column at 60:40 (ACN/H2O) at 0.2 mL/min for 5 minutes (this is a 10 column volumes) while monitoring the backpressure. Note: A steady pressure should indicate a constant flow while pressure fluctuation will indicate air in the system.
e. Column is ready to equilibrate to starting conditions (see step 3.1.1 g)
f. Keep a log of initial pressure at starting conditions and keep record of number of injections.

Join the Discussion