HomeMy WebLinkAbout2020.10.02_CCO_ReviewofChemoursReportEvaluationBranched-Linear IsomersReview of Chemours Report Evaluation of the Analysis of the Branched and Linear Isomers
PMPA/PFMOPrA and PEPA/PFMOBA
Overall finding: The LC/MS/MS experiments performed for the evaluation of branched and linear
isomers PMPA/PFMOPrA and PEPA/PFMOBA should be repeated with method improvements that
better allow for better chromatographic separation and instrument detection of the four individual
analytes of interest. Method improvements suggested are for this investigation only. Routine analysis
of the isomers would not require implementation of all suggested method changes for the isomer
evaluation. However, some of the method changes may help with future routine analysis.
Monitoring of individual analytes: Two solutions containing two analytes were used in this experiment
(PMPA/PEPA and PFMOPrA/PRMOBA). One solution for each of the four analytes would provide
unambiguous data that any instrument response observed can only be coming from the single analyte in
solution.
Mass spectrometer signal for PMPA and PEPA: Several of the specific comments and recommendations
in this review are related to the amount of instrument response for each isomer. Figures 11 and 12 of
the report (below) show that under the instrument conditions used, a signal of 4 x 106 to 1 x 107 can be
achieved for PEPA and PMPA in a Site sample without saturating the instrument detector. This indicates
that global mass spectrometer settings and analyte specific parameters are providing good signal for
PMPA and PEPA in the field sample. However, similar instrument response was not achieved for the
analysis of standard material of PMPA and PEPA. In order to achieve an instrument response of about 1
x 106 for the most abundant MRM, a higher concentration stock solution for PMPA and PEPA should be
used.
Mass spectrometer signal for PFMOPrA and PFMOBA: The report indicates that there was difficulty
achieving a strong MRM signal in the mass spectrometer for PFMOPrA and PFMOBA. Supporting
information on the Wellington certificates of analysis for the two linear isomers show a good LC/MS
response for the parent ion and a strong signal for the most predominant MRM using LC/MS/MS (see
figures below). Sufficient signal is also achieved for monitoring a second MRM transition for each
analyte (see figures below). HPLC and mass spectrometer conditions under which the chromatograms in
the Wellington C of A were collected are also shown below.
The information from the Wellington Certificates of Analysis indicate that it is possible to get sufficient
response for two MRMs each for PFMOPrA and PFMOBA. The following procedures may allow for
increased signal for PFMOPrA and PFMOBA: ensuring complete solubilization of the analyte in solution
and optimizing analyte specific mass spectrometer settings (including collision energy and use of correct
masses of parent and product ion to the nearest tenth of a decimal place based on current mass
spectrometer calibration – ex. 279.0 vs 278.9 can make a difference in signal intensity).
If the above recommendations do not provide adequate signal for PFMOPrA and PFMOBA, then global
mass spectrometer settings (ex. ESI source conditions) may need to be optimized for the linear isomers.
If source parameters are different enough between the isomers such that ionization of one or the other
isomers is significantly inhibited, then two analytical methods may be needed for the purposes of this
investigation (one method with source conditions optimized for linear isomers and the second method
with source conditions optimized for branched isomers)—the same HPLC conditions would be used for
both methods.
MRM selection: Multiple MRM transitions can be monitored for each analyte once a signal of
approximately 1 x 106 is achieved for the predominant MRM. This level of instrument signal for the
predominant MRM should allow for increased signal and detection of analytes using additional MRM
transitions. Expected fragmentation patterns for each analyte is shown below:
Specifically, the following MRMs can be monitored for each analyte:
o PMPA: 229185 and 22985
o PFMOPrA: 229185 and 22985
o PEPA: 279235, 27985, and 279135
o PFMOBA: 279235, 27985, and 279135
PMPA and PFMoPrA have the same two MRM transitions. Therefore, chromatographic separation of
these two isomers is necessary for unambiguous identification. PEPA and PFMOBA have unique MRM
transitions that should provide spectral data for isomer identification even when the compounds
coelute. The unique transitions are 279135 for PEPA and 27985 for PFMOBA. The MRM transition
of 279235 is common to both isomers. When both isomers are present in solution, and particularly
when they coelute, both isomers will contribute to this signal. Chromatographic separation of PEPA and
PFMOBA, if possible, would also provide more definitive isomer identification.
Chromatographic separation: Retention times of isomers run by the Table 3 and Table 6 method in the
report are as follows:
PMPA: 6.3 min (Table 3), 6.5 min (Table 6)
PFMOPrA: 6.8 min (Table 3), 6.3 min (Table 6)
PEPA: 8 min (Table 3), 7.13 min (Table 6)
PFMOBA: 8 min (Table 3), 7.14 min (Table 6)
Once sufficient signal is obtained for each analyte in multiple MRM transitions, chromatographic
separation of the isomer pairs should be optimized, with baseline resolution of the isomers preferred.
Separation of PMPA and PFMoPrA is the most important because both share the same two MRM
transitions and therefore are not able to be distinguished spectrally. The Table 3 and Table 6 methods
do not provide baseline resolution between these isomers. Identification of this isomer pair cannot be
made using retention times alone because the isomers are not well enough resolved and retention
times can shift, particularly in different sample matrices. Three chromatographic methods that may
provide better separation (from Strynar et al 2015, EPA Method 533, and Song et al 2018) are shown
below along with representative chromatograms. The Strynar et al 2015 and Song et al 2018 HPLC
conditions in particular show chromatographic separation for both isomer pairs.
Strynar et al 2015 – the second chromatogram shows likely PFMOPrA and PMPA separation
EPA Method 533 – Peaks 4 and 9 are PFMOPrA and PFMOBA
Song et al 2018 – Second and third chromatograms in the surface water portion of figure below show
separation of PFMOPrA/PMPA and PFMOBA/PEPA using an Acquity HSS PFP HPLC Column
The above chromatographic methods are examples that may be used. Any chromatographic method
that provides baseline resolution of PMPA and PFMOPrA would be sufficient. Resolution of PEPA and
PFMOBA is desired, however this isomer pair may be more difficult to resolve chromatographically and
has unique MRMs that can be used for identification.
Surrogates: After MRM signal and chromatographic separation have been optimized and PMPA, PEPA,
PFMOPrA, and PFMOBA have each been run with the optimized method, a surrogate can be introduced
into the method. The surrogate serves as a reference point to monitor any retention time shifts that
occur in samples. Good surrogate candidates are perfluoropentanoic acid (PFPeA) or labeled
perfluoropentanoic acid (13C5- PFPeA) because these compounds elute at a retention time in between
that of the isomer pairs (see figure below- PFMOPrA, PFPeA, 13C5- PFPeA, and PFMOBA are analytes
4,5,6, and 9 in the chromatogram, respectively).
Surrogate should be added to each individual analyte standard solution and analyzed by LC/MS/MS to
establish a relative retention time for each analyte. Environmental samples analyzed should have
surrogate added prior to analysis to provide additional confidence in the identification of isomers using
retention time information. Surrogates should be optimized in the mass spectrometer to determine two
suitable MRMs transitions (PFPeA has a transition of 263219 and 13C5- PFPeA has a transition of
268223 in EPA Method 533).
Environmental Samples: Environmental samples should be freshly collected, preserved, and stored
refrigerated within the holding time of the method rather than from archived samples. Analysis for all
analytes should occur at the same time using the following MRMs for each analyte:
o PMPA: 229185 and 22985
o PFMOPrA: 229185 and 22985
o PEPA: 279235, 27985, and 279135
o PFMOBA: 279235, 27985, and 279135
The chromatographic method should achieve as much separation as possible between PMPA and
PFMOPrA. A surrogate should be added to samples in order to better track shifts in retention times that
may occur in environmental samples analyzed by LC/MS/MS.
Report figures: This section contains specific comments about figures contained in the report.
Figure 3 has no explanation regarding the hump on the back side of the peak in the
229185 transition. The 22985 may have more signal with a more concentrated
standard.
Figure 4 PFMOPrA signal for both MRMs likely will be better with mass spectrometer
optimized conditions
Figure 5 has no explanation of peak at 7.336 minutes. More signal from a more
concentrated standard would allow for observations in MRM 22985 as well
Figure 6 signal likely will be better with optimized mass spectrometer conditions
Figure 7 zooming to a narrower scale on the y axis for the 27985 transition would
allow the viewer to see whether there is any signal in the 102 to 103 range. The
transition 279135 should also be monitored
Figure 8 expected retention time is at 8 minutes. Please expand the chromatogram in
this area to see any 102 to 103 intensity peaks present there. Signal is present at 8
minutes for the 27985. Please adjust the range of the y-axis to better show this
response. The transition 279135 should also be monitored
Figure 9 PEPA data should be collected at the MRMs 279235, 27985, and 279135
in a higher concentration standard (to obtain a signal of 1 x 106 response for the MRM
shown)
Figure 10 PFMOBA data should be collected at the MRMs 279235, 27985, and
279135 with optimized mass spectrometer conditions and a more concentrated
standard solution
Figure 11 Demonstrates good signal for PMPA in 229185 transition. Should also
monitor 22985 transition. Chromatographic resolution of isomers needed.
Figure 12 Figure 11 Demonstrates good signal for PMPA in 279235 transition. Should
also monitor 27985 and 279135 transitions. Chromatographic resolution of
isomers needed.
Each analyte should be monitored using the Table 3 and Table 6 methods, even when weak
signal was observed previously because this is work is investigative in nature and the
analytes may show up unexpectedly.
Comments on Report Conclusions:
Conclusion stated in Isomer Report: Additionally, both the lack of peak broadening or
shoulder peaks for PMPA and PEPA and lack of spectral response characteristic of
PFMOPrA and PFMOBA under Table 6 conditions indicate that Chemours’s PMPA and
PEPA standards consist primarily or entirely of the branched isomer.
o Reviewer Responses:
o Figure 3 does show a shoulder on the peak for PMPA in the 229185 transition.
o Figure 5 additionally shows a peak at 7.336 minutes in the 229185 transition
for PMPA.
o There is not enough signal in the MRM transitions for either PFMOPrA or
PFMOBA to assess spectral response. Method optimization (mass spectrometer
conditions) for these compounds would likely allow for increased signal and
collection of data for these compounds, from which then conclusions can be
drawn. Two different mass spectrometer methods may need to be run, one
each with MS conditions optimized for linear and branched isomers.
o The branched isomer is likely to be the predominant form of the isomers;
however more data needs to be collected, especially regarding the linear
isomers to support this statement and the purity assessment.
Conclusion stated in Isomer Report: Both the Table 3 and Table 6 Methods are likely to
be able to resolve the PMPA/PFMoPrA and PEPA/PFMOBA isomer pairs when
chromatographic and spectral data are considered together.
Reviewer Responses:
o PEPA/PFMOBA cannot be resolved with the chromatographic and spectral data
collected in this report. The compounds are not chromatographically resolved
using either the Table 3 or Table 6 method. The MRM transitions that could
identify the compounds are either not monitored (279135 for PEPA) or
appear to not be optimized (27985, PFMOBA). The MRM transition
(279235) is used but is common to both PEPA and PFMOBA and cannot be
used to distinguish between the two isomers.
o PMPA/PFMoPrA cannot be resolved with the chromatographic and spectral data
collected in this report. These isomers share the same two MRM transitions
(229185 and 22985) and cannot be distinguished spectrally when the peaks
coelute. Section 3.2 of the report further states the following: The peak width
for PMPA in both methods is wide, and the peak for PFMOPrA overlaps,
indicating that PMPA and PFMOPrA will not likely be fully chromatographically
resolved in both are present in a sample.
Conclusion stated in Isomer Report: Review of a Site sample containing PMPA and PEPA
and analyzed by the Table 6 method shows PFMOPrA and PFMOBA are unlikely to be
present, as indicated by the lack of peak broadening or peak shoulders.
Reviewer Responses:
o PFMOBA elutes at the same retention time PEPA and would not likely be
present as a peak shoulder, but within the center of the PEPA peak.
o The MRM transition monitored (279235) is common to both PFMOBA and
PEPA and would therefore not distinguish between the two isomers.
Additionally, the signal for PFMOBA in this MRM does not appear to be
optimized. Additional MRMs unique to PEPA (279135) and PFMOBA
(27985) under optimized MS conditions would provide data for isomer
identification. Two analytical methods may have to be run – one each
containing optimized MS parameters for linear and branched isomers.
o PFMOPrA and PMPA may not be chromatographically resolved in the Table 6
method and have the same MRM transitions (22985 and 229185). Analysis
of a sample with baseline resolution of the isomer pair would provide more
definitive results.
Proposed Path Forward: Repeat the experiments as outlined below (additional information about each
aspect is covered in a previous section of this document):
o Step 1: Optimize the method
o Prepare four individual analyte standard solutions, one each for PFMOPrA, PMPA,
PFMOBA, and PEPA
o Ensure that PFMOPrA and PFMOBA standards are completely solubilized in the standard
solution
o Ensure analyte specific mass spectrometer settings are optimized for PFMOPrA and
PFMOBA.
o Optimize global mass spectrometer settings for PFMOPrA and PFMOBA (if these
parameters are significantly different between linear and branched isomers may need
one method each for linear and branched isomers-one each with optimized ESI source
conditions)
o Run each analyte standard by LC/MS/MS using full scan mode over the range 50 to 300
m/z in order to confirm correct selection of MRM transitions for each analyte and
provide the full scan chromatograms
o Optimize MRM signal for predominant transition so that the response is approximately
1 x 106 using an existing chromatography method. Once this is achieved, add in
additional MRMs for each analyte
o Use a different chromatographic method to baseline resolve PFMOPrA and PMPA.
Ideally PFMOBA and PEPA would have better separation than in the Table 3 and Table 6
methods, but separation is less important for these isomers.
o Run each of the four individual analyte standard solutions using additional MRMs,
optimized mass spectrometer settings for the linear isomers, and a chromatography
method that baseline resolves PFMOPrA and PMPA
o Perform LC/MS/MS analysis of two solutions, one containing PFMOPrA and PMPA and
the other containing PFMOBA and PEPA to show resolution of isomer pairs together in a
solution
o Step 2: Add in surrogates
o Prepare a standard solution of surrogate standard
o Optimize two MRMs for the surrogate
o Add surrogate to an aliquot of each standard solution (one each PFMOPrA, PMPA,
PFMOBA, and PEPA)
o Run each of the four individual analyte standard solutions containing surrogate using
additional MRMs, optimized mass spectrometer settings for the linear isomers and
surrogate, and a chromatography method that baseline resolves PFMOPrA and PMPA
o Establish relative retention times for each analyte to the surrogate
o Step 3: Analyze Environmental/Site Samples
o Collect samples fresh, add appropriate preservative, store refrigerated
o Add surrogate to sample
o Analyze within refrigerated holding time for sample
o Perform analysis for all compounds at once using the optimized LC/MS/MS method
containing additional MRMs and chromatography method capable of baseline resolving
PFMOPrA and PMPA
o Any quantitation of linear isomers should be done with linear isomer calibration
standards
o Any quantitation of branched isomers should be done with branched calibration
standards