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PBDE on GC-MSMS

twitsok

Hello,

We are having issues with our PBDE method on GC-MSMS. We had a method developed using applications from Agilent for GC and MSMS conditions. When we run linearity, it seems our surrogates and/or internal standards increase in concentration as the native standard concentration increases (like a linear correlation) when they are all added at the same level. This effect occurs with the higher massed analytes, primarily Octa, Nona and Deca-BDE.

We have tried manufactured standards, making our own from serial dilution, different columns, different injection volumes and sandwiched techniques. 

The current method uses a pulsed spitless injection with gradient temperature in the MMI. When we tried a splitless technique or isocratic MMI we could not see BDE-209 (deca).

Parents
  • 0

    Hello,

    Seeing creeping internal standards means your standards may be sticking to your source.  So my first question is what is your source temperature?  We may need to increase the temp.

    A common fix to the creeping internal standard problem is to move to the 6mm or 9mm extraction lens.  I will link part numbers below.  I suggest you go to the 9mm as we have this same problem with PAHs and the 9mm fixes the issues.  However there is a tradeoff when you open the extraction lens aperture.  So there may be merit in first testing the 6mm then testing the 9mm.  The larger the aperture, the lower the resonance time for ionization.  So you may lose some sensitivity using the larger apertures.  Overall you will need to go to either the 6mm or the 9mm lens.  Its just a question of how big of a tradeoff in sensitivity you want to make.

    6mm Lens - G3870-20448 | Agilent

    9mm Lens - G3870-20449 | Agilent  

  • 0 in reply to Samuel Haddad

    Thanks for the response. 

    We actually run PAH as well and do see the same creeping on some of the heavier compounds (Dibenzopyrenes) but nothing near what we are seeing with PBDE. For the PAH we run a little hotter at 300C, the PBDE we have at 280C. I believe that I looked at the source temperature but maybe I was only evaluating sensitivity at the time. I'll have another look. 

    We just got the instrument back up and running after some downtime. I am also finding the Hepta to Deca very sensitive to a clean system (source, liner). We were lost some of the compounds in this range after the downtime.

    Any further pieces of advice or tidbits are always appreciated.

  • 0 in reply to twitsok

    Is this an HES ion source or the extractor ion source?  280° to 300° C is not that much different and you may even need to go hotter for the PAHs depending on the range. Remember there is a signfiicant boiling point depression under vacuum.  Make sure and wait long enough for thermal stability after changing temperatures, like at least an hour or two. Then tune at the new temperature as the source temperature affects the spectral tilt, the relative response for the tune ions.

    Are you sure the sensitivity to clean is to the source and not the inlet?  Inlet cleanliness affects more compounds and affects them sooner than source cleanliness does.

    Would you please share the files   acqmeth.txt and acqmethtq.pdf that are in Windows Explorer underneath your method's  XXXX.D subdirectory along with the latest autotune report?

  • 0 in reply to paulsalverda

    Sorry for the delay in response (I was away). Attached is the acq method for the GC and MS as well as the tune report. I have also included a photo of the results of cleaning the source, followed by the inlet. This is my first time attaching files so hopefully this worked. 

     

    Fullscreen 5875.acqmeth.txt Download 
                        INSTRUMENT CONTROL PARAMETERS:    GCQQQ-2
                    -----------------------------------------

   D:\MassHunter\GCMS\1\methods\EC PBDE\Validation\20240422 PBDE dMRM.M
      Mon Jul 29 11:47:00 2024

Control Information
------- -----------

Sample Inlet             : GC
Injection Source         : GC ALS
Injection Location:  Front
Mass Spectrometer        : Enabled


 No Sample Prep method has been assigned to this method.


GC
GC Summary
Run Time                                     14.417 min
Post Run Time                                0 min

Oven
Temperature
Setpoint                                     On
(Initial)                                    80 °C
Hold Time                                    1 min
Post Run                                     340 °C
Program
#1 Rate                                      40 °C/min
#1 Value                                     230 °C
#1 Hold Time                                 3 min
#2 Rate                                      30 °C/min
#2 Value                                     325 °C
#2 Hold Time                                 3.5 min


Equilibration Time                           0.5 min
Max Temperature                              350 °C
Maximum Temperature Override                 Disabled
Slow Fan                                     Disabled

ALS
Front Injector
Syringe Size                                 10 μL
Injection Volume                             2 μL
Solvent A Washes (PreInj)                    0
Solvent A Washes (PostInj)                   5
Solvent A Volume                             8 μL
Solvent B Washes (PreInj)                    1
Solvent B Washes (PostInj)                   5
Solvent B Volume                             8 μL
Sample Washes                                0
Sample Wash Volume                           1 μL
Sample Pumps                                 2
Dwell Time (PreInj)                          0 min
Dwell Time (PostInj)                         0 min
Solvent Wash Draw Speed                      300 μL/min
Solvent Wash Dispense Speed                  3000 μL/min
Sample Wash Draw Speed                       300 μL/min
Sample Wash Dispense Speed                   3000 μL/min
Injection Dispense Speed                     6000 μL/min
Viscosity Delay                              2 sec
Sample Depth                                 Disabled
Injection Type                               Standard
L1 Airgap                                    0.2 μL
Solvent Wash Mode                            A, B

Sample Overlap
Mode                                         Sample overlap is not enabled

ALS Errors                                   Pause for user interaction

Front MM Inlet He
Front MM Inlet He Temperature
Setpoint                                     On
(Initial)                                    100 °C
Hold Time                                    0.2 min
Program
#1 Rate                                      900 °C/min
#1 Value                                     330 °C
#1 Hold Time                                 3 min


Mode                                         Pulsed Splitless
Pressure                                     On    15.407 psi
Total Flow                                   On    54 mL/min
Septum Purge Flow                            On    3 mL/min
Septum Purge Flow Mode                       Switched
Pre-Run Flow Test                            Off
Gas Saver                                    On    15 mL/min after 3 min
Injection Pulse Pressure                     50 psi Until 1.5 min
Purge Flow to Split Vent                     50 mL/min at 2.5 min
Cryo                                         On
Cryo Type                                    N2
Fast Cooldown                                On
Cryo Use Temperature                         125 °C
Timeout Detection                            On    10 min
Fault Detection                              On
Liner                                        Agilent 5190-4006: 200 μL (Splitless, dimpled, Ultra Inert)

Back SS Inlet He
Mode                                         Split
Heater                                       Off
Pressure                                     Off
Total Flow                                   Off
Septum Purge Flow                            Off
Pre-Run Flow Test                            Off
Liner                                        A Liner has not been selected.

Column
Column #1
Flow
Setpoint                                     On
(Initial)                                    1 mL/min
Post Run                                     1.2 mL/min

Column Information                           Agilent 19091A-008: ECU01
Description                                  Ultra 1
Temperature Range                            -60 °C—325 °C (350 °C)
Dimensions                                   17 m x 200 μm x 0.11 μm (Uncalibrated)
Main Segment Heated By                       Oven
Segment inSeg                                Heated By Oven: 1.5 m x 180 μm x 0 μm
Column lock                                  Unlocked
In                                           Front MM Inlet He
Out                                          MSD 
(Initial)                                    80 °C
Pressure                                     15.407 psi
Flow                                         1 mL/min
Average Velocity                             48.102 cm/sec
Holdup Time                                  0.641 min
Control Mode                                 Constant Flow

Column Outlet Pressure                       0 psi

Detector Evaluation
Perform Detector Evaluation Test             Off
Signal Selected                              No Signal Selected
Checkout Sample                              None

MSD Transfer Line
Temperature
Setpoint                                     On
(Initial)                                    325 °C


Collision Cell/Aux
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Quench Gas He                                On    4 mL/min
Collision Gas N2                             On    1.5 mL/min

PSD 1
Pressure
Setpoint                                     Off
(Initial)                                    3.2897 psi
Post Run                                     50 psi

PSD Purge                                    Off

Signals
Signal #1: 
Description                                  None

Signal #2: 
Description                                  None

Signal #3: 
Description                                  None

Signal #4: 
Description                                  None

Signal #5: 
Description                                  None

Signal #6: 
Description                                  None

Signal #7: 
Description                                  None

Signal #8: 
Description                                  None






                      END OF INSTRUMENT CONTROL PARAMETERS
                      ------------------------------------

Reply
  • 0 in reply to paulsalverda

    Sorry for the delay in response (I was away). Attached is the acq method for the GC and MS as well as the tune report. I have also included a photo of the results of cleaning the source, followed by the inlet. This is my first time attaching files so hopefully this worked. 

     

    Fullscreen 5875.acqmeth.txt Download 
                        INSTRUMENT CONTROL PARAMETERS:    GCQQQ-2
                    -----------------------------------------

   D:\MassHunter\GCMS\1\methods\EC PBDE\Validation\20240422 PBDE dMRM.M
      Mon Jul 29 11:47:00 2024

Control Information
------- -----------

Sample Inlet             : GC
Injection Source         : GC ALS
Injection Location:  Front
Mass Spectrometer        : Enabled


 No Sample Prep method has been assigned to this method.


GC
GC Summary
Run Time                                     14.417 min
Post Run Time                                0 min

Oven
Temperature
Setpoint                                     On
(Initial)                                    80 °C
Hold Time                                    1 min
Post Run                                     340 °C
Program
#1 Rate                                      40 °C/min
#1 Value                                     230 °C
#1 Hold Time                                 3 min
#2 Rate                                      30 °C/min
#2 Value                                     325 °C
#2 Hold Time                                 3.5 min


Equilibration Time                           0.5 min
Max Temperature                              350 °C
Maximum Temperature Override                 Disabled
Slow Fan                                     Disabled

ALS
Front Injector
Syringe Size                                 10 μL
Injection Volume                             2 μL
Solvent A Washes (PreInj)                    0
Solvent A Washes (PostInj)                   5
Solvent A Volume                             8 μL
Solvent B Washes (PreInj)                    1
Solvent B Washes (PostInj)                   5
Solvent B Volume                             8 μL
Sample Washes                                0
Sample Wash Volume                           1 μL
Sample Pumps                                 2
Dwell Time (PreInj)                          0 min
Dwell Time (PostInj)                         0 min
Solvent Wash Draw Speed                      300 μL/min
Solvent Wash Dispense Speed                  3000 μL/min
Sample Wash Draw Speed                       300 μL/min
Sample Wash Dispense Speed                   3000 μL/min
Injection Dispense Speed                     6000 μL/min
Viscosity Delay                              2 sec
Sample Depth                                 Disabled
Injection Type                               Standard
L1 Airgap                                    0.2 μL
Solvent Wash Mode                            A, B

Sample Overlap
Mode                                         Sample overlap is not enabled

ALS Errors                                   Pause for user interaction

Front MM Inlet He
Front MM Inlet He Temperature
Setpoint                                     On
(Initial)                                    100 °C
Hold Time                                    0.2 min
Program
#1 Rate                                      900 °C/min
#1 Value                                     330 °C
#1 Hold Time                                 3 min


Mode                                         Pulsed Splitless
Pressure                                     On    15.407 psi
Total Flow                                   On    54 mL/min
Septum Purge Flow                            On    3 mL/min
Septum Purge Flow Mode                       Switched
Pre-Run Flow Test                            Off
Gas Saver                                    On    15 mL/min after 3 min
Injection Pulse Pressure                     50 psi Until 1.5 min
Purge Flow to Split Vent                     50 mL/min at 2.5 min
Cryo                                         On
Cryo Type                                    N2
Fast Cooldown                                On
Cryo Use Temperature                         125 °C
Timeout Detection                            On    10 min
Fault Detection                              On
Liner                                        Agilent 5190-4006: 200 μL (Splitless, dimpled, Ultra Inert)

Back SS Inlet He
Mode                                         Split
Heater                                       Off
Pressure                                     Off
Total Flow                                   Off
Septum Purge Flow                            Off
Pre-Run Flow Test                            Off
Liner                                        A Liner has not been selected.

Column
Column #1
Flow
Setpoint                                     On
(Initial)                                    1 mL/min
Post Run                                     1.2 mL/min

Column Information                           Agilent 19091A-008: ECU01
Description                                  Ultra 1
Temperature Range                            -60 °C—325 °C (350 °C)
Dimensions                                   17 m x 200 μm x 0.11 μm (Uncalibrated)
Main Segment Heated By                       Oven
Segment inSeg                                Heated By Oven: 1.5 m x 180 μm x 0 μm
Column lock                                  Unlocked
In                                           Front MM Inlet He
Out                                          MSD 
(Initial)                                    80 °C
Pressure                                     15.407 psi
Flow                                         1 mL/min
Average Velocity                             48.102 cm/sec
Holdup Time                                  0.641 min
Control Mode                                 Constant Flow

Column Outlet Pressure                       0 psi

Detector Evaluation
Perform Detector Evaluation Test             Off
Signal Selected                              No Signal Selected
Checkout Sample                              None

MSD Transfer Line
Temperature
Setpoint                                     On
(Initial)                                    325 °C


Collision Cell/Aux
Pressure
Setpoint                                     Off
(Initial)                                    10 psi
Post Run                                     10 psi

Quench Gas He                                On    4 mL/min
Collision Gas N2                             On    1.5 mL/min

PSD 1
Pressure
Setpoint                                     Off
(Initial)                                    3.2897 psi
Post Run                                     50 psi

PSD Purge                                    Off

Signals
Signal #1: 
Description                                  None

Signal #2: 
Description                                  None

Signal #3: 
Description                                  None

Signal #4: 
Description                                  None

Signal #5: 
Description                                  None

Signal #6: 
Description                                  None

Signal #7: 
Description                                  None

Signal #8: 
Description                                  None






                      END OF INSTRUMENT CONTROL PARAMETERS
                      ------------------------------------

Children
  • 0 in reply to twitsok

    The tune report looks good.  

    If you have enough sample volume I'd recommend at least some sample washes. I'd increase to 6 sample pumps to help get a representative sample in the syringe.

    Please turn your gas saver up to 20 ml/min- or even 30 ml/min.  15 ml/min may allow some back diffusion of air into the inlet subsystem and harm your column.

    That is an interesting method with some good choices.  Without experimentation or research, I'd suggest that the oven initial time may need to be longer than 1 minute, or the initial temp lower than 80, or both to get better concentration of the sample at the head of the 1.5m restrictor/column.   I like longer oven equilibration time for better reproducibility, but it may not make a difference with this method.  I wrote that before I looked at : application-pbde-nbfr-in-soil-7000c-gc-ms-5994-0195en-agilent.pdf .... and your method is quite close to that one.  I'm glad you are not using the column flows they chose, though. 

    Are the lower mass analytes fine?  Are the peak shapes of the higher mass analytes normal - they're just bigger than expected? 

  • 0 in reply to paulsalverda

    The lower masses are fine and all peak shapes look nice.

    I can give those suggestions a try when I get things back up and running. I am experiencing a sensitivity loss and need to figure out if it is standards, or the new column I installed.

  • 0 in reply to twitsok

    If the lower mass peaks are fine...it's not a mass spec problem.  OK, it's 99.9% that it's not a mass spec problem as long as the temperatures are correct. 

    Are the higher mass peaks reproducibly higher?  Maybe make a new batch of standards...taking all the care required. The person I know running PBDEs says that most issues he sees have come back to standards. 

  • 0 in reply to paulsalverda

    Thanks again. 

    We resolved the issue of sensitivity, a bad column (although brand new). The patterns of the PBDEs weren't what they should be. After much testing we concluded the column has a defect. I have now installed a different column that we had on hand that is different than the previous but still one suggested from an Agilent application. 

    With the mass-labelled standards, I did get a chance to try upping the source to 325C and didn't have any luck (even with this other column - I did test on both). I can give a try to the injection parameters next week (I tested it on the "bad" column with no success).

    I did try to inject a standard with ONLY mass-labelled vs a low standard and found that it looks like enhancement happens even with a minor amount of native added. It isn't very noticeable with the lower congeners but definitely when they get higher, around the Hepta - Deca.

    When we run a full calibration there is a slight increase in area counts for the lower masses but nothing like the high ones. The low masses it can only be noticed between the low and high standard but with higher congeners, it is a positive linear response between all the calibration points.

    The peak shapes all look great with baseline resolution for everything in the test mix.

  • 0 in reply to twitsok

    I did a few more experiments today with no success.

    Decreased the time on the pulsed pressure program and the oven temperature to allow more equilibration time

    Tried the source at a hotter temperature (325C) - Last week

    Started using a new column (HP-5MS vs HP-Ultra1) - Last week

    Tried to use the back inlet of the 8890 and did a spitless injection (isocratic temperature)

    There is still the difference in the high congeners (Hepta-Deca) with all these tests for the mass-labelled analytes, increasing as the concentration of the native increases. The mid and low ones seem fine still, reproducible.

  • 0 in reply to twitsok

    Two different type inlets are the same. The early eluters are good. The method looks reasonable. The tunes are good.   I'm sorry, but I think we've exhausted about all we can do here on this forum.  The next step would be to contact Agilent (Contact Us)  and setup a consultation with someone who can look directly at your sample preparation, methods, and the data on your system.    There is every chance that this is normal for these compounds, too, and someone who knows that would be the best person to answer.

    Thank you for your patience and persistence.

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