I am currently working with collaborators in our metabolomics core to replicate the method in Zhang et al, Biochem Pharmacol, 2011 to detect nucleotide pools in infected cells treated with nucleotide analogs like acyclovir. The methods section in this paper uses TCA to precipitate the proteins/etc out of solution and then neutralizes the supernatant with a trioctylamine/trichloro-trifluoro-ethane solution. The paper also implies that the sample is injected directly onto the column in that solution. The TCA and trioctylamine shouldn’t be a problem, but our collaborators in the metabolomics core are a little wary about the trichloro-trifluoro-ethane.
Previously, the core used an additive called 1,1,1,3,3,3-hexafluoroisopropanol (HFIP, another fluoro- compound) in their HPLC solvents to help sharpen chromatographic peaks for oligonucleotide analysis. It worked great; peaks were narrow and easily detected in the negative polarity mode on the mass spectrometer. However, HFIP was very sticky and contaminated the tubing, injector valves, and inside the mass spec for a long time leading to poor results in subsequent unrelated experiments when running in positive polarity mode. The core needed to completely disassemble their mass spec and HPLC system to remove all of the HFIP residue.
I am wondering if using trichloro-trifluoro-ethane for this assay would cause a similar issue. Ideally, we would perform the assay as published to avoid any need to tweak/redevelop method prep. Has anyone detected any similar contamination issues on your instruments following use of trichloro-trifluoro-thane? Alternatively, does anyone have a different method to resolve nucleic acid pools using HPLC/MS?
Thanks for your input everyone!
Plenty of experience using hexafluoroisopropanol (HFIP) and other fluoro additives in samples and mobile phase. When used in sample solution, usually not a problem (you are only injecting a tiny % in a few uL of solution), but when mixed in mobile phase and (see below for more info) you will want to avoid it IN ALL HPLC methods, esp those that have an inline vacuum degasser installed as it may damage and contaminate the entire flow path of the instrument.
As a general rule, we try to never use fluorinated solvents or additives in our systems as they tend to bind to everything (contamination of flow path, column, detector, tubing, modules etc) causing months of downtime and $$$$ cleaning. *Most vacuum degasser modules that use "Teflon AF" membranes specifically state to not use them with ANY fluorinated compounds (that includes trichloro-trifluoro-ethane too).
We recommend using a weaker, non-fluorinated ion-pairing agent (if needed as it is best to try and develop a method that does not need one). *Most of our clients dedicate an entire LC-MS system for use with samples that are likely to contaminate the LC-MS system esp for metabolomics (this preserves the other instruments for use with all of the other samples). Columns too should always be labeled after use with any ion-pairing agents and never used on other methods (dedicated to specific samples only as the agent changes the surface chemistry of the support so the column can not be used for other samples).
The HPLC mobile phase will still need to be degassed for use and we recommend two different methods to accomplish this: (1) Continuously sparged ultra high purity helium through the liquids, at very low pressure (usually 2 psi) to degas them OR (2) use a far more rugged inline vacuum degasser which uses Teflon (not Teflon AF) membranes for degassing. IOW: By-pass or remove the degasser you have with the Teflon AF membranes and replace it with a standalone degasser such as the Agilent G1322A degasser (This degasser can be used with most organic, aqueous and ion-pairing reagents/additives without being damaged, BUT you will still have contamination of your flow path if use any type of ion-pairing agent, including Fl versions. but the degasser will not be damaged).
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