Gas Chromatography/Mass Spectrometry Method for the Quantitation of 3-Chloro-p-toluidine Hydrochloride in Birds Using a Deuterated Surrogate

Article abstract of DOI:10.1021/jf980399z

A method was developed for the quantitation of the avicide 3-chloro-p-toluidine hydrochloride (CPTH) in pigeon breast muscle and gastrointestinal tract. Accuracy and precision were greatly improved by the use of CPTH-d₆ as a surrogate. CPTH-d₆ was synthesized by the nitration of toluene-d₈ followed by chlorination and reduction. The synthetic product provided mass fragments such that CPTH and the surrogate were differentiated by gas chromatography/mass spectrometry (GC/MS). A tissue extraction process used solid-phase extraction to concentrate the free base forms of CPTH and the surrogate from hexane extracts. These compounds were recovered from the SPE columns with n-butyl acetate and quantified by GC/MS. Validation data indicated (1) a linear relation between CPTH/CPTH-d₆ area ratios and CPTH concentration; (2) a method limit of detection of 30 ppb for both tissue matrices; (3) CPTH recoveries from fortified tissues ranging from 56.4 to 92.4%, whereas surrogate corrected recoveries ranged from 95.0 to 101%; and (4) identical extraction efficiencies of both compounds from each matrix.

Full text of DOI:10.1021/jf980399z

4610
J. Agric. Food Chem. 1998, 46, 4610−4615
Ga s Ch r om a togr a p h y/Ma ss Sp ectr om etr y Meth od for th e
Qu a n tita tion of 3-Ch lor o-p-tolu id in e Hyd r och lor id e in Bir d s Usin g a
Deu ter a ted Su r r oga te
Daniel B. Hurlbut,*,† J ohn J . J ohnston,† Stephen R. Daniel,‡ and J eanne Tawara§
USDA APHIS National Wildlife Research Center, 3350 Eastbrook Drive, Fort Collins, Colorado 80525,
Chemistry Department, Colorado School of Mines, Golden, Colorado 80401, and Chemistry Department,
Colorado State University, Fort Collins, Colorado 80523
A method was developed for the quantitation of the avicide 3-chloro-p-toluidine hydrochloride (CPTH)
in pigeon breast muscle and gastrointestinal tract. Accuracy and precision were greatly improved
by the use of CPTH-d6 as a surrogate. CPTH-d6 was synthesized by the nitration of toluene-d8
followed by chlorination and reduction. The synthetic product provided mass fragments such that
CPTH and the surrogate were differentiated by gas chromatography/mass spectrometry (GC/MS).
A tissue extraction process used solid-phase extraction to concentrate the free base forms of CPTH
and the surrogate from hexane extracts. These compounds were recovered from the SPE columns
with n-butyl acetate and quantified by GC/MS. Validation data indicated (1) a linear relation
between CPTH/CPTH-d6 area ratios and CPTH concentration; (2) a method limit of detection of 30
ppb for both tissue matrices; (3) CPTH recoveries from fortified tissues ranging from 56.4 to 92.4%,
whereas surrogate corrected recoveries ranged from 95.0 to 101%; and (4) identical extraction
efficiencies of both compounds from each matrix.
Keyw or d s: 3-Chloro-p-toluidine hydrochloride; gas chromatography; quantitative analysis; tissue
extraction; NMR spectroscopy
INTRODUCTION
As mankind heads into the 21st century, confronta-
tions between man and nature are becoming more
frequent. Airplanes colliding with birds or mammals,
crop predation by pest birds, and telephone cable
damage by subterranean animals are examples of these
ongoing struggles. The mission of the Wildlife Services
(WS) program is to balance “...the needs of humans and
wildlife in many different situations” (USDA, 1997). In
fulfilling this mission, the National Wildlife Research
F igu r e 1. Chemical structures of CPTH and CPTH-d6.
Center (NWRC) “... is the research arm of WS...provid-
ing scientific information on conflicts between humans
consumed first by a scavenger species. The gastrointes-
tinal (GI) tract was analyzed for incurred CPTH resi-
dues because this matrix may contain the greatest
concentration of the avicide shortly after exposure.
Previous extraction methods using external standards
produced low and variable CPTH recovery data from
fortified tissues. These methods were also laborious and
time-consuming. To permit the correction of variable
recoveries, a residue method was developed that in-
cluded the use of a surrogate material, a deuterated
form of CPTH. This paper discusses the synthesis and
purification of deuterated CPTH (Figure 1) and its use
as a surrogate in a tissue extraction method for quan-
tifying CPTH residues in the breast muscle and GI tract
of pigeons using GC/MS.
and wildlife. It is the only research facility in the world
devoted exclusively to the study of wildlife damage
control” (USDA, 1997).
To satisfy federal and state pesticide registration data
requirements, the NWRC conducts application and
efficacy studies on a number of chemical compounds
including the avicide 3-chloro-p-toluidine hydrochloride
(CPTH). Analysis of test subjects for the presence of
the pesticide assists in confirming the cause of death
and assesses the possibility of secondary hazards to
predator and/or scavenger species that could potentially
consume target animals exposed to the avicide. Gas
chromatography and mass spectrometry (GC/MS) were
used in one such study that evaluated the effectiveness
of CPTH corn baits for reducing pest pigeon populations
(Cummings et al., 1994). Breast muscle was assayed
for CPTH content because this tissue would likely be
P r op er ties. CPTH, also known as DRC-1339 or
Starlicide, is the hydrochloride salt of 3-chloro-p-tolui-
dine (CPT). CPTH is soluble in water and alcohol and
insoluble in hydrocarbon, aromatic, ether, and ketone
solvents. Due to its photosensitivity, CPTH should be
stored in containers that minimize light penetration.
† National Wildlife Research Center.
‡ Colorado School of Mines.
§ Colorado State University.
10.1021/jf980399z CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/28/1998

GC/MS Quantitation of CPTH in Birds
J. Agric. Food Chem., Vol. 46, No. 11, 1998 4611
tetrahydrofuran (THF), J . T. Baker, HPLC grade; hydrochloric
acid (HCl), Fisher, ACS Certified Plus; diethyl ether, Aldrich,
ACS reagent; ethyl acetate, Fisher, HPLC grade; acetonitrile
(ACN), Fisher, Optima; n-butyl acetate, B&J , high purity; H2O,
Milli-Q system.
The pKa of this compound has been reported to be 3.7
(Kimball and Mishalanie, 1994).
Uses. CPTH is used in the United States to control
pest bird populations including starlings, crows, ravens,
pigeons, blackbirds, and grackles (USEPA, 1995). Bait
formulations include water solutions, coatings, and
mixtures in margarine or watermelon pulp. Cattle,
poultry, and swine producers use CPTH baits in and
around animal feedlots to reduce feed consumption and/
or contamination by pest birds. Terrestrial and aquatic
farmers use the avicide to deter bird predation. Animal
refuges use CPTH to control the population of pest birds
that threaten the existence of endangered or indigenous
bird species.
Sym p tom s a n d Mod e of Action . Starlings dosed
with CPTH at levels slightly higher than their acute
LD50 level (3.8 mg/kg) appeared to be normal 20 to 30 h
after dosing (DeCino et al., 1966). However, water
consumption by these birds doubled and then sharply
decreased, while food consumption remained constant.
Four hours prior to death, the dosed birds refused food
and water, became listless, perched themselves with
feathers fluffed, and appeared to doze. Near death, the
birds became comatose and eventually died without
convulsions or spasms.
Internally, Decino found CPTH caused circulatory
impairment in the liver, kidneys, and, to some extent,
brain. Necrosis of the gizzard lining and kidney tubules
caused hemorrhaging and reduction in the excretion of
toxic material. “Death apparently results from uremic
poisoning and congestion of the major organs” (Decino
et al., 1966). A white fatlike material was also observed
within the body cavity, particularly in the pericardial
region. Preliminary tests indicated this material to be
uric acid (Decino et al., 1966). Subsequent testing done
in our laboratory showed that uric acid accounted for
2% of this white material. DeCino also reported that
symptoms exhibited by other CPTH-dosed birds were
similar to those seen for starlings.
Selectivity a n d Toxicity. Although other avicides
are available (Schafer, 1984), CPTH uniquely possesses
a high degree of selectivity between birds and mammals
(Savarie and Schafer, 1987). This selectivity was also
observed within bird species; starlings, red-winged
blackbirds, and crows were susceptible to CPTH, whereas
other bird species including ducks, sparrows, and hawks
were more resistant to the avicide (DeCino et al., 1966).
Toxicological data indicated that CPTH is highly toxic
to most pest bird species (oral LD50 of 1.0-10 mg/kg)
and less toxic to most mammals and predatory birds
(oral LD50 of 250-1000 mg/kg) (Schafer, 1984). CPTH
has a moderately acute toxicity to cold- and warm-water
fish and is acutely toxic to aquatic invertebrates (USEPA,
1995). CPTH was also found to be a chronic rather than
an acute toxicant to sensitive birds (Schafer et al., 1977).
This chronic toxicity appears to be related to irreversible
kidney damage (Schafer, 1991).
Chemicals used include the following: Na2CO3, Aldrich;
SbCl3, Fisher, ACS Certified; 10% palladium-on-carbon, Ald-
rich; NaBH4, Aldrich, 99%; p-toluidine, Aldrich, 99%; NaCl,
Fisher, USP/FCC granular; CPTH, Purina Mills, 97%; 2-chloro-
p-nitrotoluene, Chem Services; p-nitrotoluene, Chem Services;
Cl2(g), Aldrich, 99.5%+; NaOH, Fisher, 50% w/w in H2O.
Ap p a r a t u s. High-Performance Liquid Chromatography
(HPLC). To avoid byproducts in the final synthesis material,
intermediate products were isolated after each reaction using
HPLC. These separations were done using a Hewlett-Packard
(HP) 1090 liquid chromatograph equipped with an ultraviolet
(UV) diode array detector (DAD) and a Phenomenex prepara-
tive column (25 cm × 21.2 mm i.d., 5 µm particle size).
Instrument parameters included mobile phase flow rate of 5.0
mL/min, ambient column temperature, and DAD wavelength
settings of 272 and 300 nm. Each purification step discussed
under Synthesis and Purification used a distinctive mobile
phase composition and injection volume during the isolation
of intermediate products. Liquid fractions were collected using
an Eldex Universal fraction collector (Eldex Laboratories, Inc.).
GC/ MS. The GC/MS system consisted of an HP 5890 gas
chromatograph and an HP 5970 mass selective detector.
A
DB-1 capillary column (J &W Scientific), 30 m × 0.25 mm i.d.
with a 0.25 µm film thickness, was used with a 4 mm
Cyclosplitter (Restek Corp.) glass injection port liner. The
temperatures of the injection port and transfer line were 280
and 300 °C, respectively. The flow rates of the helium carrier
gas through the split and purge vents were 60 and 1 mL/min,
respectively, and the column head pressure was 15 psi.
Qualitative evaluations of reaction products were performed
using the following GC/MS parameters: oven program, 70 °C,
held for 1 min, then increased to 190 °C at 15 °C/min, then
increased to 265 °C at 30 °C/min; solvent delay, 4.5 min; purge
time, 1.0 min; MS acquisition mode, SCAN, m/z 30-200; run
time, 16.50 min.
Aliquots of the n-butyl acetate solutions, including stan-
dards and tissue extracts, were analyzed using the following
GC/MS parameters: oven program, 90 °C for 0.25 min, then
increased to 300 °C at 35 °C/min; purge time, 1.0 min; solvent
delay, 2.25 min; MS acquisition mode, single ion monitoring
(SIM), m/z 106, 140, 141 for CPTH and 112, 147, 149 for
CPTH-d6; run time, 14.25 min. A macro was developed to sum
the abundance data from each set of SIM ions into one
chromatogram. Therefore, each GC/MS analysis generated
two chromatograms, one each for CPTH (Figure 2A) and
CPTH-d6 (Figure 2B).
Syn th esis a n d P u r ifica tion . The synthesis of CPTH-d6
(Figure 3) used three chemical reactions: (A) nitration of
toluene-d8 to produce p-NO2-toluene-d7, (B) chlorination of the
product from reaction A to produce 2-chloro-p-NO2-toluene-
d6, and (C) reduction of the product from reaction B to produce
CPT-d6. A 0.05 N HCl in ethyl acetate solution was used to
form the HCl salt, CPTH-d6. All synthesis reactions were done
in air hoods using appropriate personal protective equipment
including gloves, lab coat, and safety glasses.
Nitration Reaction. The first step in the synthesis was the
nitration of toluene-d8, based on the method by Dannley and
Crum (1968). A 0.10 mol sample of toluene-d8 was placed in
a 50-mL round-bottom flask, which was submerged in an ice
bath. A mixture of D2SO4(con) (8 mL) and DNO3(con) (7 mL)
cooled to 5 °C was carefully added and vigorously mixed. The
two-phase solution was warmed to ambient and mixing
continued for another 115 min. The top organic layer was
removed and washed with three 20-mL aliquots of D2O, 10
mL of a 5% Na2CO3 in D2O solution, and 10 mL of D2O. The
organic layer, yellow in color, was evaporated at room tem-
perature to a constant volume to remove any residual toluene-
d8 material. The products from this reaction, determined by
GC/MS, included o-, m-, and p- isomers of NO2-toluene-d7,
along with some (NO2)2-toluene-d6 compounds.
MATERIALS AND METHODS
Rea gen ts. Deuterated Materials. The starting material for
the synthesis, toluene-d8 (99+ atom % D), was purchased from
the Aldrich Chemical Co. (Milwaukee, WI). Other deuterated
chemicals used included D2SO4, 99.5+ atom % D; DNO3, 99+
atom % D; D2O, 99+ atom % D; DCl, 99.5+ atom % D.
Solvents used included the following: hexane, J . T. Baker,
HPLC grade; CH2Cl2, Fisher, Optima; methanol (MeOH),
Fisher, HPLC grade; isopropyl alcohol (IPA), Mallinckrodt,
analytical reagent; petroleum ether, Fisher, pesticide grade;

4612 J. Agric. Food Chem., Vol. 46, No. 11, 1998
Hurlbut et al.
F igu r e 2. CPTH and CPTH-d6 chromatograms from fortified breast tissue extract.
F igu r e 3. CPTH-d6 synthetic reactions.
The target product, p-NO2-toluene-d7, was isolated using a
two-step HPLC separation process. To separate the mono- and
dinitro compounds, 75-µL aliquots from the nitration product
solution were chromatographed with a hexane/CH2Cl2 (55:45)
mobile phase. Fractions containing the mononitro compounds
were collected during the 24-35-min retention time window.
These fractions were combined and evaporated at room tem-
perature to a constant volume. An equal volume of MeOH
was added and mixed, and 100-µL aliquots were separated
using a hexane/CH2Cl2/IPA (75.0:24.5:0.5) mobile phase. Frac-
tions containing p-NO2-toluene-d7 were collected during the
45-60-min retention time window and analyzed by GC/MS.
Fractions that were 99+% in the target product, based on
relative area response, were combined and saved. Fractions
having relative area responses <99% p-NO2-toluene-d7 were
combined, evaporated, dissolved in MeOH, and repurified by
HPLC. All LC fractions of 99+% purity were combined and
evaporated, producing a pale yellow crystalline material.
Chlorination Reaction. A molar ratio of 1:38, SbCl3(s)/p-
NO2-toluene-d7 was placed in a 50-mL graduated glass cen-
trifuge tube. This reaction vessel was placed in a 55 °C water
bath, liquefying both components (Davies, 1921). A small stir
bar was placed in the tube to mix the reaction solution, and
Cl2(g) was carefully bubbled into the mixture for 6 h. The
reaction vessel was removed from the warm water bath, and
0.300 mL of a 0.5 N DCl in D2O was added, forming a white
precipitate. The reaction solution was extracted with petro-
leum ether, and the combined extracts were removed and
evaporated to dryness. The residue was dissolved in a minimal
amount of CH2Cl2, and 100-µL aliquots were purified by HPLC
using a hexane/CH2Cl2 (85:15) mobile phase. Fractions con-
taining the 2-chloro-p-NO2-toluene-d6 target product were
collected during the 50-62-min retention time window and
confirmed by GC/MS. These fractions were combined, and the
mobile phase was evaporated, leaving a white precipitate.
Reduction Reaction. A 1.38 mmol sample of 2-chloro-p-NO2-
toluene-d6 was combined with 5.5 mL of THF and 55.1 mg of
a 10% palladium-on-carbon catalyst in a 50-mL round-bottom
flask (Petrini et al., 1987). The reaction solution was placed
in an ice bath and mixed for 5 min. NaBH4(s), 3.47 mmol,
was added to the flask in three portions over 10 min. The ice
bath was removed and mixture warmed to ambient; mixing
continued for another 15 min. The flask was returned to the
ice bath, and 2.3 mL of a 2 N HCl(aq) solution was slowly
added to the mixture to decompose any residual NaBH4
present. Diethyl ether, 9.7 mL, was added and mixing
continued for another 5 min. The two liquid layers were
separated, and the pH of the aqueous layer was adjusted to 8
with 2 N NaOH(aq) and extracted with hexane. The diethyl
ether and hexane layers were combined and evaporated to
constant volume at room temperature. The residue was
dissolved in and chromatographed with a hexane/CH2Cl2/IPA
(8.5:90.0:1.5) mobile phase. Fractions collected during the 34-
60-min retention time window and analyzed by GC/MS
contained only the free base product, CPT-d6.
HCl Salt Formation. All of the CPT-d6-containing LC
fractions were combined, and the mobile phase was evaporated
to a volume of 35 mL. The concentrated solution was
transferred to a 50-mL tube and evaporated at room temper-
ature to a constant volume. The residual material was
dissolved in 5 mL of hexane, and three 7-mL aliquots of a 0.05
N HCl in ethyl acetate solution were added, forming the white
precipitate, CPTH-d6. After each addition, the solution was
vortex mixed and centrifuged for 2 min. The final organic

GC/MS Quantitation of CPTH in Birds
J. Agric. Food Chem., Vol. 46, No. 11, 1998 4613
F igu r e 4. Mass spectra for CPTH-d6 and CPTH.
layer was removed, and the white precipitate was further
washed with two 5-mL volumes of hexane. The CPTH-d6
precipitate was analyzed by GC/MS and nuclear magnetic
resonance (NMR) to confirm the identity and isotopic purity
of the final product. A 13.3-mg sample of the CPTH-d6
material was dissolved in 10 mL of MeOH. A 100-µL aliquot
was subsequently diluted to 10 mL with H2O. A CPTH
solution with a known concentration was prepared in the same
manner. Aliquots of 10 µL from both solutions were chro-
matographed with an acetonitrile/H2O (80:20) mobile phase
flowing through a C-8 column (Keystone, 25 cm × 4.6 mm i.d.,
5 µm particle size) at a rate of 1.0 mL/min. UV absorbance
was monitored at 241 nm. On the basis of the CPTH area
responses and the molecular weight difference between the
two compounds, the purity of the CPTH-d6 material was found
to be 97.1%.
Tissu e Extr a ction s. To quantify CPTH residues in ground
breast muscle and GI tract from pigeon, an extraction proce-
dure was developed using this deuterated material as a
surrogate. Appropriate protective equipment was used includ-
ing gloves, lab coat, and safety glasses.
Glassware and SPE Conditioning. To minimize any binding
of CPTH to glassware, glass centrifuge tubes were rinsed with
a 7 µg/mL p-toluidine in n-butyl acetate solution prior to use.
Silica (1 g) solid-phase extraction (SPE) columns (International
Sorbent Technology) were preconditioned by eluting 2 mL of
n-butyl acetate followed 2 mL of the p-toluidine solution and
2 mL of n-butyl acetate through the sorbent bed. The columns
were vacuum-dried and rinsed with three 5-mL volumes of
hexane. The columns were stored in 2 mL of hexane prior to
use.
equipped with a screw cap. The CPTH-d6 surrogate material,
5 µg, was added to the tissue and was vortex mixed. Both
analytes were allowed to absorb into the tissue for 30 min. A
5-mL aliquot of NaCl(s)-saturated H2O was added, and the
sample was vortex mixed and then sonicated for 5 min. A 10-
mL volume of a NaCl(s)-saturated 2 N NaOH(aq) solution was
mixed with the sample. After 5 min, 15 mL of hexane was
added and the sample was mixed and mechanically shaken
(horizontally) for 10 min. After shaking, 200 µL of IPA was
added and the sample shaken by hand. The tube was
centrifuged for 2 min at 835g. The hexane layer was removed
and eluted through a preconditioned SPE column by gravity
flow. The sample extraction and centrifugation were repeated
two additional times using 10 mL of hexane and 100 µL of
IPA. After the elution of the last hexane extract, the SPE
column was vacuum-dried. Analytes were recovered from the
column by eluting 2 mL of n-butyl acetate through the SPE
cartridge into a prerinsed centrifuge tube. Vacuum was
applied to the column to recover any residual n-butyl acetate
present. The resulting extract in the centrifuge tube was
diluted to a final volume of 5.00 mL with n-butyl acetate, which
was then analyzed for both the surrogate, CPTH-d6, and CPTH
by GC/MS.
GI Tract Extraction. The same extraction process used for
ground breast muscle was used for ground GI tract with the
following modifications: (1) 2 g of ground tissue was extracted;
(2) 2 µg of CPTH-d6 was combined with the tissue sample; (3)
5 mL of a NaCl(s)-saturated 2 N NaOH(aq) solution was used;
and (4) the final volume of the n-butyl acetate solution was
2.00 mL.
Standard Preparation. To optimize reproducibility of the
GC/MS responses, the analytes were injected into the GC/MS
in their free base forms of CPT and CPT-d6. Intermediate
solutions of each compound were prepared by combining an
aliquot from a concentrated aqueous solution with an equal
volume of 2 N NaOH(aq). The free base analyte was parti-
tioned into and diluted with n-butyl acetate. The final CPTH
and CPTH-d6 concentrations of these intermediate solutions
were 100 and 50 µg/mL, respectively. Aliquots from these
n-butyl acetate solutions were used in preparing calibration
standards.
RESULTS AND DISCUSSION
Sp ectr a l Da ta . Figure 4 contains the mass spectra
of CPTH and CPTH-d6. The analyte concentration for
each solution was 100 µg/mL. The spectral patterns
indicate the two analytes can be distinguished from each
other without the concern of mass fragment overlap
between the two compounds. Extracted ion chromato-
grams for CPTH were generated by summing abun-
dance data for the three most abundant mass fragments
(m/z 106, 140, and 141). CPTH-d6 chromatograms were
Breast Tissue Extraction. A 5-g sample of CPTH-fortified
ground breast tissue was placed in a 35-mL Teflon tube

4614 J. Agric. Food Chem., Vol. 46, No. 11, 1998
Hurlbut et al.
Ta ble 1. Recover y a n d Rep ea ta bility Resu lts for th e
Deter m in a tion of CP TH Resid u es in F or tified Br ea st
Mu scle a n d GI Tr a ct
produced by summing abundance data for mass frag-
ments (m/z) 112, 147, and 149.
The structures of both compounds (Figure 1) were
determined by combining proton, carbon-13, and deu-
terium NMR spectroscopy using a Bruker spectrometer.
CPTH: 1H (300 MHz, D2O, 4.70 ppm) δ 2.24 (s, 3,
CH3), 7.11 (dd, 1, J ) 2.3, 8.2 Hz, H-6), 7.30 (d, 1, J )
8.2 Hz, H-5), 7.33 (d, 1, J ) 2.3 Hz, H-2); 13C (75 MHz,
D2O) δ 21.6 (CH3), 124.1 (C-6), 126.2 (C-2), 134.9 (C-5),
131.3, 137.6, 140.4 (C-1, C-3, C-4). Absolute 13C assign-
ments were made by HETCOR.
breast
GI tract
RSD mean RSD
CPTH level
(µg/g)
compd
mean
s
s
(A) Recovery Data (%) Using Surrogate
2.00
0.500
0.100
101
98.3
95.0
1.6
3.6
5.6
1.7
3.7
5.9
99.3 2.0 2.0
99.4 3.7 3.7
97.8 5.2 5.3
(B) Absolute Recovery Data (%)
2.00
CPTH
CPTH-d6
CPTH
CPTH-d6
CPTH
CPTH-d6
79.9
79.8
64.8
65.0
3.8
4.6
4.2
3.2
4.7
5.8
6.5
4.9
65.1 2.6 3.9
66.3 2.6 4.0
56.4 1.9 3.3
56.0 2.6 4.6
92.4 9.1 9.9
90.9 8.2 9.0
CPTH-d6: 2H (76.75 MHz, H2O, 4.80 ppm) δ 2.28 (s,
3, CD3), 7.21 (s, 1, D-6), 7.41 (bs, 2, D-2, D-5).
0.500
0.100
The synthetic scheme produced CPTH-d6 with suf-
ficient chemical and isotopic purity to be used as a
surrogate for the quantification of incurred CPTH
residues in pigeon GI tract and breast muscle.
68.2 10
68.7 7.9 12
15
to 3 times the peak to peak baseline noise at the analyte
retention time in the control chromatograms. Five
ground tissue samples from each matrix were extracted
to evaluate potential chromatographic interferences. An
additional five samples from each matrix were fortified
with 0.050 µg of CPTH/g and 1.00 µg of CPTH-d6/g and
extracted.
The extraction of nonfortified tissue samples indicated
no chromatographic response with the same retention
time as CPTH. Chromatograms of nonfortified tissues
and the low-level fortified tissues were used to deter-
mine MLODs. For each tissue matrix, the MLOD was
calculated to be 0.030 µg/g.
Va lid a tion of Extr a ction Meth od . The extraction
method was validated by evaluating (1) area ratios
(CPTH/CPTH-d6) from the MS detector response for
CPTH concentrations from 0.025 to 2.00 µg/mL and
CPTH-d6 at 1.00 µg/mL versus CPTH concentration, (2)
matrix interferences, (3) method limit of detection
(MLOD) using tissue samples fortified with 0.050 µg/g
CPTH and 1.00 µg/g CPTH-d6 and baseline responses
from the extraction of nonfortified tissues, (4) recovery
and repeatability at three different CPTH fortification
levels of 2.00, 0.500, 0.100 µg/g. The CPTH-d6 concen-
tration in these tissue samples was 1.00 µg/g.
Composite samples of each tissue matrix were pre-
pared by blending dissected tissues from 10 pigeons not
exposed to CPTH. All statistical analyses, including
linear regressions and t tests, were done using SAS
(version 6.11, Cary, NC).
Recovery and Repeatability. Five ground samples
from each matrix were fortified at each of three CPTH
levels; 2.00, 0.500, and 0.100 µg/g. The CPTH-d6
content in all of these samples was 1.00 µg/g. After
fortification, the samples were vortex mixed. After 30
min, the samples were extracted and analyzed as
previously described.
Using calibration data that included CPTH-d6 re-
sponses, the surrogate-corrected CPTH recoveries ranged
from 95.0 to 101% (Table 1A). Using calibration data
that excluded the use of the surrogate data, the mean
CPTH recovery values ranged from 56.4 to 92.4% (Table
1B). In addition, the recovery variability using the
surrogate-corrected values ranged from 1.7 to 5.9%,
whereas the nonsurrogate data exhibited variabilities
ranging from 3.3 to 15%.
Further evaluation of the absolute recovery data in
Table 1B showed no statistical difference between the
mean recovery values for both CPTH and CPTH-d6 from
fortified breast and GI samples. For example, a t test
performed on the recovery data from tissues samples
fortified at 0.500 µg of CPTH/g of tissue and 1.00 µg of
CPTH-d6/g of tissue levels resulted in p values of 0.9152
and 0.7834 for breast tissue and GI tract, respectively.
This provides additional evidence that the behavior of
the surrogate mimics that of the analyte in both
matrices.
The synthetic reactions produced a CPTH-d6 material
that was well suited as a surrogate in the development
of an extraction method for the analysis of CPTH
residues in pigeon tissues. Regression analysis indi-
cated a linear and directly proportional relation between
instrument response (CPTH area/CPTH-d6 area) and
CPTH concentration. The MLOD of 30 ppb for both
tissue matrices offered acceptable sensitivity for residue
determination. Observed recoveries based on external
standard solution responses indicated similar extraction
efficiencies for CPTH and CPTH-d6 from both the breast
Response Linearity. Prior to assessment of analyte
and surrogate-containing calibration solutions, a set of
CPTH-d6 solutions ranging from 0.100 to 2.00 µg/mL
were injected and evaluated. Regression analysis in-
dicated a linear relation (r2 ) 0.995, p < 0.0001)
between analyte response and CPTH-d6 concentration.
Furthermore, the y-intercept was not found to be
significantly different from zero (p[intercept)0] ) 0.844),
indicating a directly proportional relation between ana-
lyte response and CPTH-d6 concentration. Evaluation
of a 2.00 µg/mL CPTH-d6 solution did not produce any
chromatographic response in the corresponding CPTH
chromatogram. These data indicate that the GC/MS
response of the CPTH-d6 did not contribute to the
response of CPTH, and surrogate responses were found
to be linear for recoveries as low as 10%. Therefore,
using a 1 µg/mL CPTH-d6 concentration in standard and
extract solutions was acceptable.
After the CPTH-d6 evaluation, calibration solutions
containing 1.00 µg/mL of the surrogate along with
CPTH concentrations ranging from 0.025 to 2.00 µg/mL
were prepared and analyzed. Regression analysis in-
dicated a linear relation (r2 ) 0.999, p < 0.0001)
between area ratio (CPTH/CPTH-d6) and CPTH con-
centration. In addition, the y-intercept was not found
to be significantly different from zero (p[intercept)0]
)
0.234), indicating a directly proportional relation be-
tween area ratio and CPTH concentration. Injection of
a 2.00 µg/mL CPTH solution did not produce any
response in the corresponding CPTH-d6 chromatogram.
Matrix Interference and MLOD Determination. The
MLOD calculations were based on the mass of CPTH
that would generate a chromatographic response equal

GC/MS Quantitation of CPTH in Birds
J. Agric. Food Chem., Vol. 46, No. 11, 1998 4615
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muscle and GI tract samples. As surrogate-corrected
recoveries accurately reflected CPTH fortification levels,
this approach would generate highly precise and ac-
curate quantification data for incurred CPTH residues
in pigeons. Furthermore, this method permitted the
analysis of 24 tissue samples in an 8-h day. The
methodology is well suited for the support of registration
studies or monitoring wildlife damage management
control efforts. This analytical method should be ap-
plicable to the quantitation of CPTH in other relevant
matrices such as soil, plants, and other animal tissues.
CPTH-d6 may also be a suitable surrogate for analysis
of CPTH residues by HPLC/MS.
Schafer, E. W., J r.; Brunton, R. B.; Cunningham, D. J .;
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ACKNOWLEDGMENT
We thank Dr. Robert Krull for providing the NMR
spectral data for CPTH and the surrogate material and
Dr. J ohn Beck for providing us with the Petrini citation,
which was essential in producing CPTH-d6.
U.S. Department of Agriculture (USDA). Animal Plant Health
Inspection Service. What’s In a Name? Inside APHIS; U.S.
Government Printing Office: Washington, DC, 1997; Vol.
17 (4), p 1.
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Received for review April 20, 1998. Revised manuscript
received September 10, 1998. Accepted September 15, 1998.
We thank the USDA and the NWRC for funding this work.
Mention of commercial products is for identification only and
does not constitute endorsement by the U.S. Department of
Agriculture.
J F980399Z