What to do if my LC-QQQ Autotune Fails?

Answer

This Information Applies To: Agilent 6400 Series triple quadrupole LC/MS systems

Issue

Autotune fails to complete.

Background

Agilent 6400 LC/MS systems use an Autotune algorithm to optimize the sensitivity and mass resolution of the instrument. Sometimes when running the autotune procedure the autotune does not complete and the user is given an Autotune failed message in MassHunter. One of the main reasons for Autotune failure is a lack of sensitivity or abundance of the tuning ions. This article goes through some simple checks that a user can do to try to get the instrument to pass Autotune. 

Items Required

• Nebulizer Adjustment Fixture (p/n GT430-20470) or Nebulizer adjustment kit (p/n G1960-67470) - to check and adjust the nebulizer needle if necessary.
• Clean powder-free gloves
• Correct tuning solution for your application
  • ESI/Jetstream Tuning mix (p/n G2421-60001)
  • APCI-L Low concentration tuning mix (p/n G1969-85010)
  • Multimode MMI-L low concentration tuning mix (p/n G1969-85020)
• T6 Torx driver or 1.5 mm hex wrench.
• 1/4" x 5/16" open-end wrench (Qty 2)

Resolution / Steps to Follow

1. Check where specifically in the autotune process, the failure has occurred. At the time of Autotune failure, an error message is shown in the progress box on the bottom-right section of the Autotune window (See red box in Figure 1).
   • If you missed the error message onscreen, then open the AutotuneLogFile.csv file found in your MassHunter Tune folder. Default path is D:\MassHunter\tune\QQQ\G64xx, where xx is the last two numbers of your LC/MS model number. Scroll to the end of this excel file and the reason for the Autotune failure should be listed.
   • Autotune failures generally fall into one of the following main categories, take note of the cause of failure then move on to Step 2:
     
     1. Failure due to low abundance of one or more calibrant ions - Check that the correct tune solution is being used for the source installed on your system, and the solution is within the use by date. Ensure that sufficient volume of Tune solution is in the Tune bottle to complete the autotune procedure (15-20mL required). See the items required section for the correct tuning solution for your application, installed source, and ionization type and also check the expiration date ot the tuning solution.
     2. Incorrect m/z assignment - Can be caused by contamination of the tuning solution, calibrant delivery system or within the source or ion optics of the LC/MS. Can also be caused by an issue with the Quadrupole control electronics or can be due to a low or fluctuating abundance of tuning ions during m/z assignment.
     3. Peak width out of tolerance - Can be caused by an issue with the Quadrupole control electronics or can be due to a low or fluctuating abundance of tuning ions during m/z assignment.  
     4. Electron Multiplier Voltage (EMV) adjustment failed - Can be an indication of an Electron Multiplier at the end of its useable life. Check the EMV value on the last Autotune that passed on the instrument, and if close to or at a value of 3000 V, the EM may need replacement. This error may also be caused by low tuning ion abundance.

                                                       
        
        Figure 1 - Autotune window showing Autotune progress in the Red Box.
        

2. Check Nebulizer Spray - Proper nebulizer spray is required for good signal and is critical in Agilent Jet Stream (AJS) equipped sources. The spray pattern should be conical in shape and consistent. If the spray shape looks ok and you are getting calibrant to the nebulizer, then move to step 3.
   • To check your spray shape, go to the manual tune window and turn on the calibrant, and make sure that the LC flow is going to waste using the radio buttons in your Tune view of MassHunter. Follow the instructions in the following article to check the spray shape: How to Check and Adjust the Nebulizer Needle on Agilent LC/MS Systems.
     • If the spray shape is not conical or consistent, then this article also provides instructions on how to check and adjust your nebulizer needle to ensure efficient nebulization and ionization.
   • If the nebulizer needle adjustment looks ok, but you are not getting any spray, or the spray is not consentient:
     1. Put on powder-free nitrile gloves
     2. Remove the PEEK finger tight fitting from the top of the nebulizer (see Figure 2). Place the outlet of this capillary over a clean beaker or similar to observe the drip rate of calibrant coming out of the capillary. You should see a drop of calibration solution every 6 to 8 seconds.
     3. If no flow is present, or the flow rate is significantly lower than this, then chances are you have a blockage in your CDS system, or the calibrant bottle is not being pressurized. Please contact your local Agilent Service representative and share your findings with them. 

                             
        
        Figure 2 PEEL fitting, shown indicated by the red box, to check the drip rate of calibrant in step 2
        
        

3. If spray looks ok, then Check System Vacuum and compare the current vacuum readings to the last good autotune report on that same instrument. If both the spray shape and vacuum readings look ok, or once you have executed this step then move on to Step 4.
   
   
   • If the Rough vacuum is too good (i.e. lower Torr value than normal), then the Ion capillary might be blocked.
     1. Instructions to remove the ion capillary for cleaning can be found here for standard 6400 series instruments, and here or LC/MS system equipped with a gate valve for ion capillary removal without venting the instrument.
   • If the Rough Vacuum is not good enough (i.e., higher Torr value than normal), it could indicate one of the following: Gross vacuum leak, low oil level (check MS40+ oil level here), insufficient drying gas flow or Temperature, or a defective rough pump.
     1. Gather up all observations, plus the last good checktune and autotune reports generated by the instrument.
     2. Contact your local Agilent Service Representative for further assistance.
   • If the High Vacuum is too good (lower Torr value than normal), it could indicate Insufficient Collision Cell Gas Flow.
     1. Turn off the collision cell gas using the collision cell gas check box in the Manual tune window.
     2. Wait for the new high vacuum reading to stabilize and record this value.
     3. Then turn the collision cell back on again, wait for the high vacuum to stabilize again, and record the High Vacuum reading again.
     4. Contact your local Agilent Service Representative and have these values ready to provide to the Service Agent for further assistance.
   • If the High Vacuum is not good enough (higher Torr value than normal), it could indicate either too much collision cell gas flow, a vacuum leak, or a failing turbo pump.
     1. Compare vacuum readings with Collison cell gas on and off.
     2. Contact your local Agilent Service Representative and have your observations of any potential leaks and the vacuum values ready to provide to the Service Agent for further assistance.
        
4. Compare current tune settings with the last good autotune report to make sure that nothing has been changed in the current tune file that would cause issues with tuning. If there are no major differences to tune parameters that are obvious, especially with the values for source temperature, gas flows, and voltage settings, then move on to Step 5.
    
5. Try to load the last Autotune that passed to check for an issue or corruption with the current tune file.
   1. Click the Manual Tune Tab of the MassHunter Tune view.
   2. Click the Files Subtab and then click the Load button to bring up a list of available tunes to load.
   3. Choose the last known good autotune on the system when the instrument tuned successfully. Autotune files are in the following format showing the date and time of when the Autotune completed (Autotune_<date>_< atunes_backup _<date>_<time>.TUNE.XML)
   4. Load this last good autotune file into memory, and then click Autotune to start a new Autotune process using the last good Autotune as the starting parameters.
   5. Observe the Autotune process, if it fails again then check the new reason for failure and compare it to the old reason for Autotune failure. Move on to Step 6
      
6. Try to perform an autotune in one polarity mode only - Most LC-QQQ are set to autotune in both positive and Negative polarity modes.
   1. Try an Autotune in either positive or Negative mode only (Figure 2). It is useful to know if only one polarity mode is failing. For example, positive mode can be failing due to contamination, interference and/or ion suppression that may not be seen in Negative mode. 
   2. Record which mode passes or fails, then move onto Step 7.
      
7. Try an Autotune starting from factory defaults.
   1.  Click the Autotune Tab in the MassHunter Tune view.
   2. Check the box next to Start from factory defaults (Figure 3)
   3. Click the Autotune button to start an Autotune using factory default parameters.
   4. If this Autotune also fails, move onto Step 8.
      
8. Acquire MS1 and MS2 scans while infusing calibrant for further problem analysis.
   1. Maximize your MassHunter window and then click the Manual Tune tab in the MassHunter Tune view. (Figure 4)
   2. Click the MS1 Subtab.
   3. Under Scan Type, select MS1 Scan.
   4. In the MS1 parameters section, make sure that the scan range is set to the full mass range available for your LC/TQ model. Usually scanning from 50-2250 m/z is sufficient (Figure 4)
   5. Turn on calibrant (check box) and check that the LC flow is going to waste (setting LC > Waste)
   6. Click Apply to start the MS1 scan. Wait a couple of minutes for the scan signal to stabilize, then take a screenshot of the entire window showing results and MS1 tab parameters.
   7. Repeat steps 5a-f on the MS2 Scan Subtab to run an MS2 Scan and take a screenshot of the result.
   8. Send both screenshots and any other observations and vacuum readings to your local Agilent Service Representative for further assistance.  

            

Figure 3 - To start an Autotune from factory default parameters, check the circled Start from factory defaults box and then click the Autotune button.

      

Figure 4 - Screenshot of the Manual Tune tab showing the MS1 subtab settings (shown in red section).

Learn how to effectively tune and calibrate your Agilent 6400 series LC-QQQ instrument: LCMS-6400-1120sV2 - Tune and Calibrate Agilent 6400 Series Triple Quadrupole LC/MS with MassHunter Acquisition (Ver. 10.0) e-learning course available from Agilent education