United States
Environmental Protection
Agency
Environmental Monitoring Systems
Laboratory
Las Vegas, Nevada 89193-3478
Research and Development
EPA/600/S4-90/022 Nov. 1990
EPA Project Summary
Performance Evaluation of
Particle Beam Liquid
Chromatography/Mass
Spectrometry for the
Measurement of Acid Herbicides
Chris M. Pace, Dennis A. Miller, and Mark R. Roby
Particle beam liquid chromatogra-
phy/mass spectrometry (LC/MS) was
evaluated for the measurement of acid
herbicides. An acetic acid/ammonium
acetate/methanol solvent system with a
C-8 reversed phase column gave baseline
resolution of all target analytes. Detec-
tion limits in the full scan mode were 100
ng to 500 ng for most of the target
analytes. Dalapon and dinoseb were not
detected. Response curves over the
range 200 ng to 2000 ng were non-linear
for most of the analytes. Response
factors tended to increase with Increas-
ing analyte concentration. Mass spectra
were variable and exhibited abundant
ions corresponding to "thermal" decom-
position mechanisms. Spectral appear-
ance was dependent on analyte concen-
tration, source conditions, and source
temperature. Only spectra acquired at
high concentration were library match-
able. Therefore, a rugged and reliable
method to identify and quantify acid her-
bicides in environmental samples based
on particle beam LC/MS technology does
not appear feasible at this time.
This report covers the period from
May 1,1989 to March 30,1990, and work
was completed as of June 15,1990.
This Project Summary was devel-
oped by EPA's Environmental Monitor-
Ing Systems Laboratory, Las Vegas, NV,
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering Information at
back).
Introduction
Particle beam (PB) high performance
liquid chromatography/mass spectrometry
(HPLC/MS) is a technique capable of pro-
ducing liquid chromatographic separation
and electron ionization (El) mass spectra
for polar nonvolatile and/or thermally labile
organic compounds. The PB interface is
only one of several HPLC/MS interfaces
that have been developed. The thermos-
pray (TS) HPLC/MS interface is probably
the most widely utilized today. However, the
lack of spectral information produced by
this soft ionization technique has limited the
use of TS HPLC/MS in the identification of
unknown organic compounds. The devel-
opment of the PB HPLC/MS has led to an
interface capable of removing a large por-
tion of the mobile phase from the HPLC
effluent. Once most of the mobile phase
has been removed the analyte particles
enter the mass spectrometer ion source
where they are vaporized and subsequently
ionized under electron bombardment. The
El spectra generated in such a process
contain substantial structural information.
The phenoxyacid herbicides are one
class of compounds for which an HPLC/MS
method for identification and quantification
would be very useful. Presently, samples
are analyzed for these compounds under
methods 8150 and 8151 of the SW-846.
These methods involve hydrolysis and
derivatization with diazomethane before
analysis by gas chromatography (GC) with
an electron capture detector (ECD). The
derivatization step is both time consuming
Printed on Recycled Paper
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and dangerous; a method eliminating its
use would be advantageous. Therefore, a
thermospray HPLC/MS method for the di-
rect analysis of these compounds has been
proposed (EPA/600/X-89/176 July 1989,
Liquid Chromatography/Mass Spectrometry
Performance Evaluation of Chlorinated
Herbicides and Their Esters). Likewise, this
report describes the application of PB HPLC/
MS to the analysis of phenoxyacid herbi-
cides. Factors affecting both the chroma-
tography and the response characteristics
were investigated.
Procedure
The initial chromatographic separations
were developed on a Hewlett-Packard
1090L liquid chromatograph equipped with
an autosampler and a diode array detector.
The HPLC system was controlled by a
Hewlett-Packard HPLCChemStation. The
PB HPLC/MS consisted of a 1090L liquid
chromatograph equipped with an auto in-
jector and filter photometric detector. The
HPLC was connected to a Hewlett-Packard
59980A particle beam interface. The HPLC
was controlled by a local user-interface
while the mass spectrometer was controlled
by a Hewlett-Packard 59970 MS
ChemStation.
HPLC flow rates of either 0.25 mL/min
or 0.4 mL/min were used. Several mobile
phases and HPLC columns were consid-
ered. Routine PB parameters were:
nebulizer setting of 12, nebulization helium
pressure of 30-50 psi, desolvation chamber
temperature of 45° to 55°C, and PB probe
distance to the source of 0.5 mm. The PB
desolvation chamber vacuum pressure was
estimated at 200 torr by the instrument
manufacturer. The pressure in the first
stage of the momentum separator was
typically 10 torr and that of the second stage
was typically 0.5 torr as measured by
Hastings-Raydist gauges. The mass
spectrometer ion source for these studies
was slightly modified. First, a stainless
steel plug was inserted into the GC inlet of
the source. Second, the particle beam inlet
was drilled to a larger diameter by the in-
strument manufacturer. Except for one set
of experiments the ion source was operated
at 250°C. A typical MS operating pressure
of 1.2 x 10'5 torr was measured by a Bayard-
Alpert ion gauge tube. The mass spec-
trometer was run in the El mode (except for
one experiment) with a filament emission
current of 300 u,A and an electron energy of
70 eV. The MS electron multiplier was a
Galileo channeltron and was typically op-
erated at 2200 V. The MS system was
tuned to maximize the m/z 219 ion of PFTBA
introduced through a reservoir on the PB
transfer tube.
The acid herbicide standards were pure
compounds (>97%) obtained from the U.S.
EPA Repository (Research Triangle Park,
North Carolina). The compounds were sub-
sequently diluted with acetonitrile.
Results and Discussion
Before any mass spectrometric work
was carried out, experiments were per-
formed on an HPLC/U V system to optimize
the chromatographic separations for the
phenoxyacid herbicides. After such a sepa-
ration was identified, these conditions were
tried on the HPLC coupled with the PB
interface. The system was further modified
and the final chromatographic conditions
that were used for all subsequent studies
are listed in Table 1.
The flow through the column had been
0.4 mL/min, but because of the high initial
aqueous content of the mobile phase a
substantial loss of sensitivity for the acid
herbicides through the PB interface was
observed. To compensate for this loss of
sensitivity, the flow rate was reduced to 0.25
mL/min and the nebulizer helium pressure
was increased to 50 psi.
The limits of detection for the phenoxy-
acid herbicides are listed in Table 2. These
data were estimated from a full scan total
ion chromatogram at a concentration near
these limits. The individual peaks were ex-
trapolated to give a signal to noise ratio of
three. The addition of ammonium acetate to
the mobile phase appears to slightly im-
prove the limits of detection for these com-
pounds.
Table 1. Chromatographic Conditions for the PB HPLC/MS System
Column: Spherisorb S3C-8 5 u,m 2 x 100 mm
Flow: 0.25 mL/min Temperature: 50°C
Gradient:
Time(min)
~0
2
25
1% Acetic Acid + 0.01 M NH4QAc
75%
75%
40%
CH3OH(l%HOAc)
25%
25%
60%
Table 2. Estimated Limits of Detection
Compound
Dalapon
Dicamba
2,4-D
MCPA
2,4,5-T
Dichloroprop
MCPP
Si I vex
2,4-D B
Dinoseb
2,4-D butoxy ethyl ester
2,4,5-T butoxy ethyl ester
Limit of Detection (ng)
ND
520
130
260
320
130
400
130
120
>2500
50
50
ND Not detected
Response curves were prepared for
each analyte over a range of 200 ng to 2000
ng. Dicamba and MCPP exhibited near
linear response over this range, but the
response to silvex and 2,4-DB was distinctly
nonlinear. Response to the other analytes
was intermediate with relative standard
deviations ranging from 15 to 25 percent.
Examination of the response curve data
reveals two trends. First, response curves
for most of the target analytes were
nonlinear over the range 200 ng to 2000 ng.
Secondly, response factors tend to increase
with increasing analyte amount.
The single day, single concentration
level (1000 ng) reproducibility was gener-
ally less than 10 percent for the phenoxyacid
herbicides. However, the variation in day
to day response at a single level (1000 ng)
was between 43 and 51 percent. These
data were recorded after every attempt was
made to return the instrument to the same
operating condition each day.
An experiment was designed to exam-
ine matrix effects on phenoxyacid herbicide
response. Instrument response to a mixed
herbicide standard solution (200 ng/^L) was
compared to the response observed for
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acid herbicides in a soil extract and a soil
extract containing phenoliccompounds. The
results of this experiment indicate the matrix
components from the extract and the soil
extract plus phenols do not affect the re-
sponse characteristics by a significant
amount.
There has been evidence of variation
in the quality of the El spectra from the PB
introduction of the phenoxyacid herbicides.
Several ions which appear in the spectra of
these compounds jnder certain conditions
are characteristic of thermal decomposition
in the ion source. Elxperiments designed to
study this phenomenon indicate that the
particle beam spectra of the phenoxyacid
herbicides are variable and depend on
source cleanliness, quality of the water in
the mobile phase, source temperature, and
analyte concentration. Preliminary results
suggest a surface catalyzed decomposition
may be occurring inside the ion source.
Conclusions and
Recommendations
A rugged and reliable method to iden-
tify and quantify acid herbicides in environ-
mental samples based on PB LC/MS tech-
nology does not appear feasible at this time.
A number of fundamental issues need to be
addressed before a reliable method can be
developed. Among these issues are poor
sensitivity, non-linear response, response
drift, and variation in spectral quality.
Based on SW-846 extraction proce-
dures and assuming 10 nl_ injection vol-
umes, the observed detection limits for the
analytes except dalapon and dinoseb which
were not detected result in method detec-
tion limits of 50 to 250 u,g/L These values
are comparable to Method 8150 detection
limits for MC.PA and MCPP, but are con-
siderably higher than the method detection
limits for the remaining acid herbicides.
Selected ion monitoring of the acid herbi-
cides improved detection limits by 10 to 50
times, but identification based on full scan
mass spectra is lost. Some improvement in
detection limits may be achieved by sum-
ming over all isotopic ions associated with
the base peak ratherthan quantifying on the
base peak alone. Further enhancements
may be achieved by incorporating special-
ized injection techniques. One of these
techniques, well suited to liquid chromatog-
raphy, is on column preconcentration used
in conjunction with a switching valve. In
addition, larger sample size or more ex-
tensive concentration may be incorporated
into the sample preparation to improve
overall method detection limits.
Response curves for most of the acid
herbicides exhibited non-linear behavior
over the range 200 ng to 2000 ng. Re-
sponse factors tended to increase with in-
creasing concentration. Inaccuracy result-
ing from inappropriate calibration may be
minimized by operating over a narrower
calibration range. For these compounds,
linear calibration models may be used pro-
vided the calibration range is less than a
factor of 10. For an extended range, a non-
linear calibration must be employed to
maintain acceptable accuracy.
Spectral quality was found to be de-
pendant on the amount of analyte reaching
the ion source of the mass spectrometer. At
high levels of sample (2u,g), the El mass
spectra are of library matchable quality.
However, at lower sample amounts the
mass spectra show an increase in the rela-
tive abundance of thermal decomposition
ions. Investigation into the cause of thermal
decomposition ions leads us to the hy-
pothesis that these ions are formed through
interactions of analyte particles with the ion
source surface resulting in decomposition
to a more thermally stable and volatile
species followed by desorption and subse-
quent ionization. The extent to which these
events occur are variable; depending on
analyte concentration, source temperature,
and source cleanliness, and may be the
principal cause of non-linear response over
an extended range and response variation
at low concentrations.
Chris M. Pace, Dennis A. Miller and Mark R. Roby are with Environmental Programs,
Lockheed Engineering & Sciences Company, Las Vegas, NV89114.
LD. Betowski is the EPA Project Officer (see below).
The complete report, entitled "Performance Evaluation of Particle Beam Liquid Chroma-
tography/Mass Spectometry for the Measurement of Acid Herbicides, "(Order No.
PB90-270 547/AS; Cost: $17.00, cost subject to change) will be available only from:
National Technical Information Ser/ice
5285 Port Royal Road
Springfield, v'A 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted a .':
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, NV 89193-3478
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Penalty for Private Use $300
EPA/600/S4-90/022
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