Calibration curve from previous working method not linear on new GC/MS

I am trying to validate our methamphetamine quantitation method on our new GC/MS.  We have been using this method successfully for years on 7 other GC/MS instruments, but our new instrument does not produce a linear curve.  Does anyone have any ideas on what could be causing our curve to look like this?

  • I looked up the highest split ratio available to use, 1038:1.  The R^2 of the curve is now 0.99929.  Besides decreasing the injection volume, are there any other settings I can change on the instrument?  I don't want to use all of my helium.  Thanks for any tips!

  • Please upload the file under your method subdirectory called   acqmeth.txt .  That file contains the method parameters.  Any other information that will help would be appreciated - desired calibration range, sample preparation used, solvent injected, liner installed, etc.

    What is the model number of your older GCMS systems?  Your new one?  What software and versions on the older and the newer?

  •                        INSTRUMENT CONTROL PARAMETERS:    Havok
          Tue Sep 19 14:51:19 2023
    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 Summary
    Run Time                                     4 min
    Post Run Time                                0 min
    Setpoint                                     On
    (Initial)                                    150 °C
    Hold Time                                    0.5 min
    Post Run                                     0 °C
    #1 Rate                                      25 °C/min
    #1 Value                                     220 °C
    #1 Hold Time                                 0.7 min
    Equilibration Time                           0.5 min
    Max Temperature                              325 °C
    Maximum Temperature Override                 Disabled
    Slow Fan                                     Disabled
    Front Injector
    Syringe Size                                 5 μL
    Injection Volume                             0.5 μL
    Solvent A Washes (PreInj)                    1
    Solvent A Washes (PostInj)                   3
    Solvent A Volume                             4 μL
    Solvent B Washes (PreInj)                    1
    Solvent B Washes (PostInj)                   3
    Solvent B Volume                             4 μL
    Sample Washes                                1
    Sample Wash Volume                           4 μL
    Sample Pumps                                 3
    Dwell Time (PreInj)                          0 min
    Dwell Time (PostInj)                         0 min
    Solvent Wash Draw Speed                      150 μL/min
    Solvent Wash Dispense Speed                  1000 μL/min
    Sample Wash Draw Speed                       150 μL/min
    Sample Wash Dispense Speed                   1000 μL/min
    Injection Dispense Speed                     3000 μL/min
    Viscosity Delay                              1 sec
    Sample Depth                                 0 mm
    Injection Type                               Standard
    L1 Airgap                                    0.1 μL
    Solvent Wash Mode                            A, B
    Sample Overlap
    Mode                                         Sample overlap is not enabled
    ALS Errors                                   Pause for user interaction
    Front SS Inlet He
    Mode                                         Split
    Heater                                       On    250 °C
    Pressure                                     On    16.008 psi
    Total Flow                                   On    1249.8 mL/min
    Septum Purge Flow                            On    3 mL/min
    Pre-Run Flow Test                            Off
    Gas Saver                                    On    15 mL/min after 0.5 min
    Split Ratio                                  1038 :1
    Split Flow                                   1245.6 mL/min
    Liner                                        A Liner has not been selected.
    Column #1
    Setpoint                                     On
    (Initial)                                    1.2 mL/min
    Post Run                                     0.57353 mL/min
    Column Information                           Agilent 13323-1628655
    Description                                  Rxi-1ms
    Temperature Range                            -60 °C—325 °C (350 °C)
    Dimensions                                   30 m x 250 μm x 0.25 μm (Uncalibrated)
    Heater                                       Oven
    Column lock                                  Unlocked
    In                                           Front SS Inlet He
    Out                                          MSD 
    (Initial)                                    150 °C
    Pressure                                     16.008 psi
    Flow                                         1.2 mL/min
    Average Velocity                             41.683 cm/sec
    Holdup Time                                  1.1995 min
    Control Mode                                 Constant Flow
    Column Outlet Pressure                       0 psi
    Detector Evaluation
    Perform Detector Evaluation Test             Off
    MSD Transfer Line
    Setpoint                                     On
    (Initial)                                    260 °C
    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
    MS Information
    -- -----------
    General Information
    ------- -----------
    Acquisition Mode         : SIM
    Solvent Delay (minutes)  : 1.5
    Tune file                : D:\MassHunter\GCMS\1\5977\etune.u
    EM Setting mode Delta    : 0.000000
    Number of SIM Groups     : 1
    Run Time (if MS only)    : 10 minutes 
    [SIM Parameters]
    Group 1               Group ID                  : 1
    Resolution                : 1
    Group Start Time          : 1.5
    Number of Ions            : 4
    Dwell In Group            :(  Mass,  Dwell) (  Mass,  Dwell)  (  Mass,  Dwell) 
                               (  58.00,50  )  (  91.00,50  )  (  168.00,50  ) 
                               (  169.00,50  ) 
    MS Source                   : 230 C   maximum 250 C
    MS Quad                     : 150 C   maximum 200 C
    Timed Events
    ----- ------
    Number Events= 0
                         END OF MS ACQUISITION PARAMETERS
                            TUNE PARAMETERS for SN: US2312MA29
     Trace Ion Detection is OFF.
        34.593   :   EMISSION     
        70.007   :   ENERGY       
         0.503   :   REPELLER     
        89.822   :   IONFOCUS     
        27.717   :   ENTRANCE_LENS
       928.951   :   EMVOLTS      
                                  929  : Actual EMV 
                                   0.18   : GAIN FACTOR 
      1640.000   :   AMUGAIN      
       133.938   :   AMUOFFSET    
         1.000   :   FILAMENT     
         0.000   :   DCPOLARITY   
         6.495   :   ENTLENSOFFSET
        10.002   :   Ion_Body     
         0.303   :   EXTLENS      
      -470.000   :  MASSGAIN        
       -23.000   :  MASSOFFSET      
                            END OF TUNE PARAMETERS
                            END OF INSTRUMENT CONTROL PARAMETERS

    New model: 8890 / 5977C Column: Restek Rsi-1ms

    Software: Mass Hunter Workstation 10.2.524.1 (G6845AA) and Chemstation G1701FA, but all data processing is in Excel

    Old models: 8890 / 5977B, 6890/ 5973, 7890A / 5975C, 6890 / 5973N  Same method in use for 10 years on multiple instruments, multiple labs.  Varity of software versions, but all using Chemstation

  • 4 mg/ml = 4,000 µg/ml.

    One Agilent app notes calls out methamphetamine: "The 5 μg/mL original standards were directly used for injection, and were diluted 10 times with blank solvent to 500 ng/mL solution. "    5990-7596EN.pdf (

    Another:  "TIC of amphetamine mixture at 250 μg/mL (split 20:1 for on‑column concentration of 12.5 μg/mL)" application-drugs-of-abuse-gc-mass-spectrometry-5994-1012en-agilent.pdf

    Since you are splitting so much, the highest I've ever seen, the easiest guess is inlet or source overload or both.  

    Try injecting even less sample. You're only injecting 0.5 µL now, but try 0.1 µL. The peaks will still be too big, so dilute 1:100 and change your split ratio to something like 100:1 or less..  Just because the split ratio can be entered does not mean that it is useful.  The system may be able to get 1245.6 ml/min of split flow through, but the effectiveness, linearity, and reproducibility of that much flow in the liner are very suspect. 

    15 ml/min in gas saver mode may be entered, but that is not the safest for your column. That flow is so low that there is the possibility of oxygen migration into the column through diffusion in O rings and even back diffusion through the vents. Please increase this to 20 ml/min or even 30 ml/min. 

    That second app note says that the 9mm draw out lens was installed in the MS. This changes the dynamics in the ionization chamber and reduces linearity problems at very high concentrations/too much sample on column/ too much sample into the ion source. G3870-20449 | Agilent   That would be worth a try.  The tuning might be slightly different, and you do give up some ultimate sensitivity, but it does not look like sensitivity is a problem. 

    All of that stuff you inject has to go somewhere, so it's in the split vent line and the split vent trap.  What goes into the column will be contaminating the ion source, source radiator, and manifold, and over time everything inside the vacuum chamber will need to be cleaned.   You want to inject the smallest volume of the lowest concentration of the cleanest samples you can and still see the lowest standard concentration of your peaks of interest.  The smallest peaks should be very small - in the low thousands of counts, maybe 3x to 4x the background noise.  The cleaner you keep the system - the less you contaminate the system with samples - the longer the interval will be between maintenance and the lower the number of consumable parts replaced.

Was this helpful?