Troubleshooting LC/TQ Contamination Issues

Answer

This Information Applies To the Following Products: All Agilent iFunnel and Agilent Jet Stream (AJS) 6400 Series LC/MS triple quadrupole systems, Agilent 6460A/C, 6470A, 6490A, and 6495A/B/C

Issue:

As LC Triple Quadrupole Mass Spectrometers (LC/TQ) have continued to become more sensitive to analytes, the capability to detect contaminants has also increased. The advent of Agilent Jet Stream ion source and iFunnel technologies starting with the Agilent 6490A, have contributed with the increasing sensitivity of the systems. It becomes more important to make sure that the entire flow path is clean. This article gives some specific examples of common contaminants found in LC/TQ systems and some tips on how to prevent and remove these types of contamination.

Note: It is uncommon to see all three contaminant classes described in the following sections, in the same instrument. However it is not uncommon to see one or two of these contaminant classes in combination in the same LC/TQ.

Steps to follow. 

To compare your LC/TQ instrument to the following examples, please implement MS2 scan data collection (also known as Solvent Blank Scan - no injection) to help identify background or contaminant issues. To ensure an accurate comparison, the chromatography (including gradient) and MS methods (including delta EMV) remain the same as the acquisition method you are currently using for your application.

1. Create a solvent blank method by following these steps
   1. Save your current method with a different method name. Click Method > Save As, then input a suitable method name or add Solvent Blank Scan to the end of the method name.
   2. Set the acquisition mode in all segments to MS2Scan.
   3. Save the method.
   4. Run a solvent blank using this method.
        
2. Compare Total Ion Chromatogram (TIC) in your solvent blank from step 1 to historical tune data of your instrument, including the checkout data produced at instrument installation.
   • Identify the main contaminant ions in the TIC data to try to identify potential common contaminants as per the examples in the following sections.
     
     
3. Isolate potentially contaminated solvents by changing channel compositions as well as the solvents themselves. Isolate hardware (HPLC modules, LC analytical columns, etc.) by changing plumbing to bypass potential sources of contamination. Use a process of elimination to try to identify the source of these contaminants.
   
   
4. Next, collect actual MS2 scan data using an LC gradient of 50:50 water(0.1% formic acid, FA): acetonitrile(0.1% FA) and compare main contaminant ions to the following examples. 

Inorganic Clusters - Example of Acetonitrile-Cu clusters

• In this example, the TIC signal is above 5 x 10⁸. As shown in Figure 1, m/z 145.0 has a height of 5673131. There are several classes of contaminants present; but, always address base peaks first. 

Figure 1: MS2 Scan showing contamination in the low mass range. 

• Inorganic acetonitrile-Cu clusters are present with 145 and 147 m/z as markers and causes ion suppression. When in doubt to the type of contaminant, apply collision energy (in either product ion scan or MRM modes for LC/TQ) to see product ions to determine structure.
  
  

Figure 2: Zoom of MS2 Scan showing a cluster of ions around m/z 145

Solution

• Flush HPLC with EDTA solution (10 mM in water); Divert flow away from MS to waste. Use fresh Acetonitrile if necessary.

Polyethylene Glycol (PEGs) [C₂H₄O]_nH2O·K

• In this example, the TIC abundance is around 1.5 x 10⁸ (see Figure 3). The prominent base peaks are Polyethylene Glycol or PEGs with markers as 173.1, 195.1, 217.1, 261.1, 305.1, etc. (+22 / +44 m/z apart). These ions cause high background and ion suppression at these levels. (See Figure 4)

Figure 3: Total Ion Chromatogram (TIC) showing abundance in the  1 - 1.7x 10⁸ range.

Figure 4: MS2 Scan showing the major PEG contamination ions

Solution

• Remove analytical column.
• Flush aqueous flow path with aqueous mobile phase containing no modifiers and at a high temperature (100C). This is only effective if the source of PEGs has been identified and removed from the flow path.

Possible Causes

• Contaminants found in solvents, samples made/frozen in bad solvents, solvent bottles (if plastic), solvent bottle caps, and solvent bottle cap adhesive tape.
• Identified in some packing material of LC analytical columns.  In this case, "wet" the column longer in 100% methanol.  

Triton and Triton Reduced (Detergent) [C₁₅H₃₀O][C₂H_4O]_n

• Now the background is almost clean after EDTA and aqueous flushing; but TIC is still around 1 x 10⁸. 
• Base peak is now 293.2 m/z and a marker for Triton or detergent. Interestingly enough, both inorganic and PEGs clusters also caused Triton ion suppression (See Figure 5).

Figure 5: MS2 Scan showing a reduction in contamination ions after EDTA and Aqueous flushing. Triton ions can still be seen.

Solution

• Wash bottles without detergent and dry in muffle furnace.
• Can use acid etch and rinse thoroughly with aqueous followed by organic solvents (but not as effective as muffle furnace drying).
  
  

Possible Causes

• Contaminants found in glassware washed with detergent.

Learn how to effectively troubleshoot your Agilent 6400 Series LC/MSD system LCMS-6400-1201s - Essential Housekeeping Tasks and Best Practices for Agilent 6400 Series LC/TQ e-learning course on Agilent University