Consumables


  • Agilent Zorbax SB-Aq RRHT 2.1 x 100mm 1.8-Micron (P.N. 828700-914)
  • Agilent InfinityLab Deactivator Additive (P.N. 5191-4506)
  • Rather than a guard column we use an inline 0.3um Frit on our LC to prevent precipitate from hitting our analytical column. Each lab may already be using a different vender specific frit within their LC system but if they are not, the part number for the frit holder and 5 pack of frits that we use is: Agilent 1290 Infinity II in-line filter, 0.3um, 2mm ID, SST. max 1300 bar (P.N. 5067-6189)
  • Water (Burdick and Jackson; Cat. 365-4)
  • Acetonitrile optima LCMS grade (Fisher; Cat No. A955-4)
  • Formic acid, 99+% 10X1ML 10/PK (Fisher; Cat No. PI28905)

Procedure for LCMS analysis using SBAq C18 column

1. Determine whether the instrument is operating at optimal performance prior to running samples.

  • Reference ions
    • Different reference ions are used for positive mode and negative mode. To ensure good instrument sensitivity, take note of the intensity of the reference ions. This will vary based on MS instrument type (6520 Q-TOF, 6210 ESI-TOF, 6550 ion funnel Q-TOF, 6560 ion mobility Q-TOF 6545 Q-TOF) but the range should be between 1.5M and 2.0M counts on the 6545. See the respective MS instrument manual for more precise and comprehensive parameters.
  • QC
    • Inter-project QC: Inject an instrument blank, followed by an instrument QC (preferably the NIST QC). Observe the total ion chromatogram and compare this QC to a previously run QC to monitor any retention time and signal intensity variations
    • Intra-project QC: Bracket all analytical samples with a QC sample of relevant matrix (preferably a pooled sample) to monitor the instrument variability throughout the run and between batches.
  • Column pressure
    • Column pressure will vary based on whether the column is new or old. An older column which is starting to go bad may have a high back pressure and if so, will need to be discarded. A typical pressure range for the SBAQ analytical column using the method in this SOP is 110 and 320 bar.

2. Column selection

  • If performing method development, or doing a small pilot project (<10 samples), an older HPLC column with <100 injections may suffice
  • If performing larger metabolomics studies (>100 samples), multiple brand new columns are needed
  • The columns used to analyze the aqueous fraction following Captiva-based extraction is as follows:

3. Buffer preparation for SBAQ LC-MS

  • Buffer A: Water with 0.1% formic acid and 0.1% Agilent Deactivator Reagent
    1. In a 1000mL bottle, add 1000mL water
    2. Add 1mL of formic acid and 1mL Agilent Deactivator Reagent to the water and invert a few times to mix. Note: We use the 1mL formic acid ampules.
  • Buffer B: Acetonitrile 0.1% formic acid and 0.1% Agilent Deactivator Reagent
    1. In a 1000mL bottle, add 1000mL acetonitrile
    2. Add 1mL of formic acid and 1mL Agilent Deactivator Reagent to the water and invert a few times to mix. Note: We use the 1mL formic acid ampules.
  • Buffer and needle wash preparation notes:a. Ensure that your buffer bottles are placed in the correct location (A1, A2, B1, B2) based on instrument method.
    b. Always purge the system with new buffer prior to starting the worklist to ensure there are no air bubbles.
    c. After connecting your analytical and guard columns, ensure that there are no leaks. Look at the pressure on the Acquisition software, and use a kimwipe to check for any ‘wetness’ around the fittings.
    d. Make sure that you have sufficient needle wash (50% methanol in water for SBAQ) and that the tubing is all the way in the bottom of the bottle.
    e. Samples: To prevent ‘no signal injections’, check each vial prior to putting in autosampler tray, to ensure no air bubbles are present in vial. Flick once or twice if necessary.

4. Reference solution preparation for instrument analysis
Make sure that there is enough TOF reference mix in the reference bottle so you do not run out halfway through your run, especially when running a method overnight. Also make sure that all tubing is secured and goes all the way to the bottom of the reference bottle. (Sometimes it gets knocked out)

Prepare this large batch:

  • Make Reference Solution in 1000mL solvent bottle that is only used for mass spectrometry solvents using the following volumes of each reagent:
    • 2.2mL purine
    • 0.88mL HP-0921
    • 1045mL acetonitrile
    • 55mL water
  • Typical reference masses when running in positive mode: 121.0509, 922.0098
  • Typical reference masses when running in negative mode: 112.9856, 966.0007
    An alternative for 112.9856 is 119.03632This bottle is stored in the refrigerator. An aliquot is poured into a smaller (usually a 100mL) LC-MS bottle attached to the instrument.

5. Instrument parameters for SBAQ LC-MS

a. The parameters below are built into the method but will vary for each instrument, particularly the ion Funnel instruments such as the 6550Q-TOF. Please consult the instrument manual for its recommended source parameters.

Positive ionization mode Negative ionization mode
m/z range: 75 – 1700 m/z m/z range:75 – 1700 m/z
Scan rate: 2 spectra/s Scan rate: 2 spectra/s
Gas temperature: 300°C Gas temperature: 300°C
Gas flow: 12 L/min Gas flow: 12 L/min
Nebulizer: 35 psi Nebulizer: 35 psi
Sheath Gas Temp 275°C Sheath Gas Temp 275°C
Sheath Gas Flow 12 L/min Sheath Gas Flow 12 L/min
Vcap: 3500V Vcap: 4000V
Nozzle Voltage 250V Nozzle Voltage 1000V
Fragmentor: 100V Fragmentor: 100V
Skimmer: 65V Skimmer: 65V
Reference masses: 121.0508; 922.0098 Reference masses: 112.9856; 966.0007

b. The gradient elution chromatographic parameters below remain the same for each instrument to ensure reproducibility of the TICs.
Buffer A: Water with 0.1% formic acid and 0.1% Agilent Deactivator reagent
Buffer B: Acetonitrile with 0.1% formic acid and 0.1% Agilent Deactivator reagent

Time %B
0.0 min 1.80
3.0 min 1.80
10.00 min 54.00
15.00 min 90.00
20.00 min 90.00
20.10 min 1.80
25.00 min 1.80

c. Injection volume: 2µL; this is standard for plasma and food but may have to be increased for cells, BAL fluid, EBC or other dilute sample types.
d. Flow rate: 250µL/min
e. Maximum pressure: 600 bar
f. Column temperature: 30°C
g. Autosampler tray temperature: 4°C

6. Sample randomization and wordlist setup on the instrument

Sample Vial Position
Instrument Blank Vial 1
Instrument Blank Vial 1
Instrument Blank Vial 1
Prep Blank P1-A1
Prep Blank P1-A1
Prep Blank P1-A1
QC Vial 2
QC Vial 2
QC Vial 2
QC Vial 2
Instrument Blank Vial 1
1
2
3
4
5
6
7
8
9
10
QC Vial 2
Instrument Blank Vial 1
11
12
13
14
15
16
17
18
19
20
QC Vial 2
Instrument Blank Vial 1

a. Instrument and Wordlist Standby Scripts
Navigate to worklist parameters, click on ‘Acquisition cleanup’, and select the ‘AllPumpsOff’ script.