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Journal of Analytical Toxicology, Vol. 35, July/August 2011
Determination of L-Ephedrine, Pseudoephedrine,
and Caffeine in Rat Plasma by Liquid Chromatography–
Tandem Mass Spectrometry
Stephen D. Cooper, Brenda L. Fletcher, Melanie A. Rehder Silinski, Sherri S. Brown, Jon W. Lodge,
and Reshan A. Fernando
RTI International, P.O. Box 12194, Research Triangle Park, North Carolina 27709-2194
Bradley J. Collins
NIEHS/National Toxicology Program, P.O. Box 12233, Research Triangle Park, North Carolina 27709-2233
Abstract
A rapid and simple liquid chromatography–tandem mass
spectrometry method was developed and validated for the
simultaneous determination of L-ephedrine, pseudoephedrine, and
caffeine in male Fisher-344 rat plasma at nanogram-per-milliliter
concentrations for use in support of toxicology studies. Only 25 μL
of plasma is required, and extraction is performed using a simple,
single-step protein precipitation. The method was validated over a
range of 2.09 to 5460 ng/mL for L-ephedrine, 2.09 to 5050 ng/mL
for pseudoephedrine and 2.03 to 5340 ng/mL for caffeine. A binary
gradient elution at 0.3 mL/min was used with a Waters XBridge
Phenyl (2.1 × 150 mm, 3.5 μm) column and a Waters XBridge
Phenyl 2.1- × 10-mm guard column at ambient temperature. The
mobile phase consisted of 10 mM ammonium acetate in water
(pH 5.0) and methanol. Caffeine trimethyl-13C3 was used as the
internal standard. The method was evaluated for linearity, recovery,
precision, accuracy, and stability, and it was successfully applied in
toxicokinetic studies of ephedrine, administered alone, in
combination with caffeine, and in the herbal source Ma Huang.
Introduction
Ephedrine alkaloids derived from the plant Ephedra sinica,
known as Ma Huang in traditional Chinese medicine, have
been used in dietary supplements for weight loss and athletic
performance enhancement (1,2). Ephedrine (EPH) and pseu-
doephedrine (PSE), the primary alkaloids found in Ephedra, are
sympathetic agonists which stimulate the brain, increase heart
rate, constrict blood vessels, expand bronchial tubes, and in-
crease metabolism. The combination of EPH and guarana-de-
rived caffeine has been shown to be synergistic for thermoge-
nesis, thus leading to the marketing of various formulations for
the treatment of obesity (3–7). However, a variety of severe ad-
verse side effects have been reported for Ephedra supplements,
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including cardiotoxicity, strokes, heart attacks, seizures, and
death, and the toxicity appears to be greatly enhanced in com-
bination with caffeine (8–11). In 2004, the Food and Drug Ad-
ministration (FDA) banned over-the-counter dietary supple-
ments containing Ephedra due to escalating concerns
regarding their safety (12,13). Other concerns have been raised
regarding product consistency, purity, and quality, because
there are no formal federal regulations for quality control of di-
etary supplements. For example, studies have shown significant
deviations in actual EPH content from labeled amounts, lot-to-
lot inconsistencies, and even a complete lack of content la-
beling (1,2,9,14).
A better understanding of the toxicology of EPH, when ad-
ministered alone, in combination with caffeine, or in the herbal
source Ma Huang, is urgently needed such that appropriate
dosing guidelines or warnings can be issued. The objective of
this work was to develop and validate an analytical method for
the determination of EPH, PSE, and caffeine simultaneously in
a limited volume of plasma at levels low enough to be useful in
toxicokinetic studies.
Experimental
Chemicals and reagents
EPH, sodium salt, PSE hydrochloride, and ammonium ac-
etate were purchased from Sigma-Aldrich (St. Louis, MO). Caf-
feine and caffeine trimethyl-13C3 were from Pfaltz & Bauer
(Waterbury, CT) and Isotec (Miamisburg, OH), respectively.
High-performance liquid chromatography (HPLC)-grade
methanol was obtained from Honeywell Burdick & Jackson
(Muskegon, MI), and ultrapure water was prepared using a Pi-
cosystem UV Plus purification system (Hydro Service and Sup-
plies, Durham, NC). Male Fischer-344 rat plasma was pur-
chased from Hilltop Lab Animals (Scottdale, PA).
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Preparation of standard stock and plasma samples
Isotopically labeled caffeine (caffeine trimethyl-13C3) was
used as the internal standard (IS). A working IS solution was
prepared at 10.0 ng/mL in acetonitrile. For each of the three
analytes, two different standard stock solutions of 500 μg/mL
were prepared in water. The molecular weight for PSE was ad-
justed to correct for the presence of the hydrochloride salt.
Through a series of dilutions, six working standards, used for
calibration standards and quality control samples, were pre-
Figure 1. Structures of L-ephedrine, pseudoephedrine, and caffeine.
Figure 2. Product ion spectra of L-ephedrine (A), pseudoephedrine (B), and caffeine (C).
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Journal of Analytical Toxicology, Vol. 35, July/August 2011
pared from the two stock solutions to cover the range from 5.0
to 250 ng/mL. Calibration standards and quality control sam-
ples were prepared by fortifying 25 μL of blank plasma with 10
μL of the appropriate working standard to obtain plasma con-
centration levels from 2.0 to 100 ng/mL. For actual subject
samples, 25 μL of plasma was spiked with 10 μL of water.
Sample extraction was achieved by the addition of 100 μL of the
IS working solution, vortex mixing, and centrifugation for 10
min. The supernatant was evaporated to dryness and reconsti-
tuted with 50 μL of 10 mM ammonium
acetate in water, pH 5 (mobile phase A).
Preparation of standard stock and
plasma samples for extended range
verification
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Standard solutions were prepared as
described, except 12 working standards
were prepared at 10–12,500 ng/mL. Sam-
ples were fortified, extracted with the IS
working solution, dried, and reconsti-
tuted as described, but the reconstituted
extracts were diluted 1:10 with a 20.0
ng/mL solution of IS in mobile phase A.
The extended plasma concentration
ranges were approximately 4–800 and
800–5000 ng/mL for each analyte.
Preparation of standard stock and
plasma samples for caffeine dilution
verification
Stock solutions were prepared as in the
extended range verification, but two
working stock solutions of caffeine, each
prepared directly from one of the 500
μg/mL stocks, were 319,000 and 63,800
ng/mL in water. Two plasma samples were
each prepared in triplicate from these
working stock solutions at 128,000 and
25,500 ng/mL, respectively. These sam-
ples were extracted, dried, and reconsti-
tuted as in the extended range verifica-
tion, but the reconstituted, diluted
extracts were diluted again 1:5 (25,500
ng/mL standard) or 1:25 (128,000 ng/mL
standard) with a 20.0 ng/mL solution of
IS in mobile phase A. These dilution
quality control samples were analyzed
using the 800–5000 ng/mL extended
range calibration curve for caffeine.
Liquid chromatography–tandem mass
spectrometry (LC–MS–MS)
instrumentation and conditions
The LC–MS–MS system consisted of an
Agilent (Palo Alto, CA) 1100 series HPLC
coupled to an Applied Biosystems (Foster
City, CA) API-4000 triple-quadrupole MS
with a TurboIonSpray® (electrospray)
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Journal of Analytical Toxicology, Vol. 35, July/August 2011
source. Chromatographic analysis was performed using a Wa-
ters (Milford, MA) Xbridge phenyl column (150 mm × 2.1-mm
i.d., 3.5-μm particle size) and a Waters Xbridge phenyl guard
column (10 mm × 2.1-mm i.d.). Five microliters of sample
were injected onto the column and elution of the analytes and
internal standard was achieved at ambient temperature using
a binary gradient and a flow rate of 0.3 mL/min. The mobile
phase consisted of 10 mM ammonium acetate at pH 5 (mobile
phase A) and methanol (mobile phase B). MS detection was per-
formed with the electrospray ionization source operated in
positive ion mode, using multiple reaction monitoring (MRM).
The ion transitions were 166 → 133 for EPH and PSE, 195 →
138 for caffeine, and 198 → 140 for caffeine-trimethyl (IS).
The ion spray voltage was 5000 V, the source temperature was
650°C, and the interface heater was operating. The collision ac-
tivated dissociation gas and curtain gas pressures were 10 psi,
the nebulizer gas pressure was 60 psi, and the auxiliary gas
Figure 3. Representative chromatograms for L-ephedrine, pseudoephedrine, and caffeine in blank rat
plasma (A), plasma fortified at the LLOQ for each analyte (B), and a real subject sample collected 30 min created
post-dose of 312.5 mg/kg Ma Huang + 30 mg/kg caffeine (C).
pressure was 50 psi. The optimized compound-dependent pa-
rameters for EPH were declustering potential (DP) = 36 V; en-
trance potential (EP) = 10 V; collision energy (CE) = 27 V; and
collision cell exit potential (CXP) = 6 V. For PSE the parame-
ters were DP = 31 V; EP = 10 V; CE = 29 V; and CXP = 10 V. The
parameters for caffeine trimethyl-13C3 (IS) were the same as
those for caffeine: DP = 46 V; EP = 10 V; CE = 27 V; and CXP =
12 V. Analyst software version 1.4.1 (Applied Biosystems) was
used for data acquisition and integration. Tablecurve 2D ver-
sion 2.01 was used for regression analysis.
Assay validation
The linearity of this method was evaluated by analyzing six
calibration standards for each analyte in duplicate in three an-
alytical runs over the nominal concentration range of 2.0–100
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ng/mL. The regression model for each analyte was a linear,
weighted least-squares algorithm with a weighting factor of
1/concentration-squared (1/x2). Calibra-
tion curves were generated by plotting
the weighted theoretical concentration
(1/x2) against the peak-area ratios of ana-
lyte to IS for each calibration standard.
Recovery was expressed as the percentage
of the response ratio of the analyte in ma-
trix to that in solvent. Intraassay precision
and accuracy were evaluated at six dif-
ferent concentration levels using two in-
dependent calibration curves and one set
of quality control samples prepared on
the same day. Interassay precision and ac-
curacy were evaluated at six different con-
centration levels using two sets of cali-
bration standards and one set of quality
control samples prepared on one day and
one set of calibration standards and one
set of quality control samples prepared
on each of two subsequent days. Stability
experiments were performed at three dif-
ferent concentration levels prepared in
triplicate, and the results were evaluated
by comparing the mean found concen-
tration of a stored sample to that of a
fresh (day 0) sample. Extracted matrix
samples were evaluated following ambient
and refrigerated storage for three days.
Freeze-thaw stability was established for a
set of non-extracted matrix samples
stored frozen at –80°C and subjected to
three freeze-thaw cycles over three days.
Long-term analyte stability in frozen ma-
trix was established for an additional set
of samples prepared in four replicates at
approximately 20 ng/mL and evaluated
after 60 days at –80°C.
Two additional calibration curves were
and validated for each analyte to
demonstrate that real subject samples
with concentrations higher than the
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Journal of Analytical Toxicology, Vol. 35, July/August 2011

upper limit of quantitation (100 ng/mL, ULOQ) could be ana-
lyzed. These curves covered the approximate ranges 4–800
ng/mL and 800–5000 ng/mL for each analyte. For each ex-
tended curve, six calibration standards were analyzed in trip-
licate. A linear, weighted least-squares algorithm with a
weighting factor of 1/x2 was used, and the curves were evalu-
ated for linearity and precision and accuracy of the standards.
Because caffeine levels in real subject samples were found to
exceed 5000 ng/mL, additional dilution verifications were per-
formed for this analyte to demonstrate that extracts could be
diluted into the calibration range. Plasma samples were pre-
pared in triplicate at approximately 125,000 and 25,000 ng/mL,
extracted, and then diluted into the extended 800–5000 ng/mL
curve.
Toxicokinetic study
A toxicokinetic study of EPH administered alone, in com-
bination with caffeine, or in the herbal source Ma Huang
(Jinke Group, Diamond Bar, CA) was performed using the
validated analytical method to determine the plasma levels of
EPH, PSE, and caffeine. Cannulated male Fischer-344 rats
(Taconic, Germantown, NY) were administered EPH intra-
venously at 6.25 mg/kg or by oral gavage at 6.25, 12.5, or
25 mg/kg, in combination with caffeine (25 mg/kg EPH + 30
mg/kg caffeine), or in Ma Huang (312.5 mg/kg alone or in
combination with 30 mg/kg caffeine). Blood samples were
collected at 5, 10, 15, and 30 min and 1, 2, 4, 8, 12, and 16 h,
processed to plasma, and stored at –80°C until LC–MS–MS
analysis.

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Table I. Summary of Calibration Curves and Back-Extracted Concentrations from L-Ephedrine, Pseudoephedrine,
and Caffeine

Calculation No. Regression Parameters

Compound Run No. Day 1 2 3 4 5 6 Slope Intercept r

L-Ephedrine 2.09 8.72 20.9 43.6 66.8 109

1 1 2.02 9.18 19.7 44.9 64.4 117 0.01845 0.001724 0.9983
2 2.14 9.00 19.3 42.9 63.5 115
3 2 2.13 7.97 20.9 44.1 68.9 110 0.01783 0.01480 0.9984
4 2.13 7.69 21.4 44.6 69.2 112
5 3 2.13 8.09 19.4 41.9 70.8 111 0.01811 –0.003821 0.9983
6 2.10 8.66 20.0 43.7 72.9 113
n 6 6 6 6 6 6
Mean 2.11 8.43 20.1 43.7 68.3 113
% RE 0.9 –3.3 –3.7 0.2 2.2 3.7
% CV 2.2 7.1 4.2 2.6 5.4 2.3

Pseudoephedrine 2.09 8.08 20.9 40.4 66.8 101

1 1 2.24 8.79 19.2 41.6 64.6 110 0.03111 0.00972 0.9969
2 1.92 8.18 19.0 38.7 64.8 108
3 2 2.01 7.02 21.0 41.0 69.4 100 0.03091 0.01899 0.9969
4 2.28 7.12 21.2 42.4 71.4 102
5 3 2.03 7.31 18.4 38.2 71.4 102 0.03194 –0.007106 0.9965
6 2.23 7.99 20.4 40.7 75.6 105
n 6 6 6 6 6 6
Mean 2.12 7.74 19.9 40.4 69.5 105
% RE 1.4 –4.3 –4.9 0.1 4.1 3.5
% CV 7.1 9.0 5.8 4.1 6.1 3.7

Caffeine 2.03 8.56 20.3 42.8 64.8 107

1 1 1.95 9.54 20.8 44.8 64.8 113 0.02298 0.01151 0.9999
2 2.04 8.63 20.5 42.0 63.9 107
3 2 2.06 8.53 20.4 43.2 65.7 105 0.02297 0.01539 0.9998
4 2.01 8.32 20.8 43.4 65.2 104
5 3 2.08 8.97 19.3 42.8 67.2 107 0.02515 0.005427 0.9994
6 1.97 8.54 19.9 42.6 65.8 105
n 6 6 6 6 6 6
Mean 2.02 8.76 20.3 43.1 65.4 107
% RE –0.6 2.3 –0.1 0.8 1.0 –0.2
% CV 2.5 5.0 2.9 2.2 1.7 3.0

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Results and Discussion Selectivity, sensitivity, and recovery

Method development
The molecular structures of EPH, PSE, and caffeine are
shown in Figure 1. The MS parameters were optimized in pos-
itive ESI mode by infusing approximately 1 μg/mL of each an-
alyte. The full scan Q1 mass spectra showed an [M+H]+ of m/z
166.1 for both EPH and PSE and m/z 195.1 for caffeine.
Product ion spectra for each analyte were optimized by sys-
tematically ramping the voltages, gases, or source temperature
during infusion or flow-injection analysis. APCI was also tested,
but with no improvement in sensitivity over ESI. The final
product ion spectra are shown in Figure 2. For EPH and PSE,
the product ion m/z 133 was selected over the more abundant
No endogenous peaks were observed in the chromatogram of
blank plasma (Figure 3A). The minor alkaloid constituents of
Ma Huang, namely methylephedrine, norephedrine,
methylpseudoephedrine, and norpseudoephedrine, were also
analyzed to verify that they did not interfere with detection of
the analytes of interest.
The lower limit of quantitation (LLOQ) was the lowest stan-
dard that could be accurately quantitated within 20% of the
theoretical value and at which six replicates could be repro-
duced within 20% CV. The LLOQ for EPH, PSE, and caffeine
were 2.09, 2.09, and 2.03 ng/mL, respectively. The chro-
matogram for the LLOQ standards is shown in Figure 3B. A
real subject sample chromatogram is presented for plasma
collected 30 min following an oral dose of 312.5 mg/kg Ma

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m/z 148, because the latter was likely generated by a non-spe-
cific loss of water. The selected MRM transition for both of
these analytes was m/z 166 → 133. For caffeine, m/z 195 → 138
was used.
Extraction was initially performed using a Waters Oasis HLB
SPE cartridge, which was able to retain both caffeine and the
ephedrines, for an HPLC–UV analysis. However, with MS de-
tection, a simple protein precipitation technique with ace-
tonitrile was found to be sufficient. Several HPLC columns
were tested, including Waters Nova-Pak Phenyl, Waters At-
lantis, and Zorbax SB-Phenyl. A Waters XBridge Phenyl was fi-
nally selected as the most suitable, and was able to sufficiently
separate the EPH isomers.
Huang + 30 mg/kg caffeine (Figure 3C).
Six replicates of the LLOQ plasma standards were analyzed
to determine the standard deviations in the responses for each
analyte. The limit of detection (LOD) was defined as three
times the standard deviation of the response expressed as con-
centration. The LODs for EPH, PSE, and caffeine were 0.98,
0.82, and 0.58 ng/mL, respectively.
Recovery was expressed as the percentage of the response
ratio of the analyte in matrix to that in solvent. The average re-
coveries for two sets of six matrix standards over a concentra-
tion range of 2.0 to 100 ng/mL were 100% (3.7% CV), 105%
(10% CV), and 109% (11% CV) for EPH, PSE, and caffeine, re-
spectively.

Table II. Intra- and Interassay Precision and Accuracy for L-Ephedrine, Pseudoephedrine, and Caffeine

Intraassay Interassay
Nominal Mean calc. Mean calc.
Concentration conc. conc.
Compound (ng/mL) n (ng/mL) % RE % CV n (ng/mL) % RE % CV

L-Ephedrine
QC 1 2.09 3 2.16 4 7.2 7 2.08 –0.7 7.9
QC 2 8.72 3 8.70 –0.2 7.8 7 8.32 –4.5 6.9
QC 3 20.9 3 19.0 –9.3 5.0 7 19.6 –6.4 6.4
QC 4 43.6 3 43.9 0.6 2.3 7 43.6 0.1 5.1
QC 5 66.8 3 62.4 –6.5 4.3 7 65.6 –1.8 6.0
QC 6 109 3 114 4 3.7 7 111 2 3.6

Pseudoephedrine
QC 1 2.09 3 2.07 –1 7.8 7 2.04 –3 4.9
QC 2 8.08 3 8.05 –0.4 10 7 7.54 –6.7 9.0
QC 3 20.9 3 18.8 –10 2.8 7 19.3 –7.5 6.7
QC 4 40.4 3 40.2 –0.5 3.6 7 40.2 –0.5 6.8
QC 5 66.8 3 63.1 –5.5 4.3 7 66.5 –0.5 5.6
QC 6 101 3 107 6 2.8 7 104 3 3.9

Caffeine
QC 1 2.03 3 2.03 –0.2 3.5 7 2.06 1 4.2
QC 2 8.56 3 8.95 4.5 5.7 7 8.69 1.5 5.9
QC 3 20.3 3 20.3 0.2 2.8 7 19.8 –2 5.2
QC 4 42.8 3 43.4 1 3.2 7 43.0 0.4 3.6
QC 5 64.8 3 64.1 –1 0.97 7 64.3 –0.7 4.6
QC 6 107 3 110 2 2.8 7 108 0.8 2.9

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Linearity, precision, and accuracy
The calibration curves for EPH, PSE, and caffeine were linear
in plasma from 2.0 to 100 ng/mL with correlation coefficients
r > 0.99. Average back-calculated values with accuracy (percent
relative error, % RE) and reproducibility (percent coefficient
of variation, % CV) for each matrix standard are shown in
Table I.
The intraassay precision and accuracy for each analyte were
evaluated at six different concentration levels using two sets of
independent calibration standards and one set of quality con-
trol samples prepared on the same day. The interassay precision
and accuracy for each analyte were evaluated at six different
concentration levels using two sets of calibration standards
and one set of quality control samples prepared on one day, and
one set of calibration standards and one set of quality control
samples prepared on each of two subsequent days. The intra-
and interassay precision was less than 10% CV for each of the
three analytes, and accuracy was within ± 10% RE (Table II).
Stability
Stability studies were performed to evaluate both matrix
and extract stability of the analytes. Post-preparative stability
was demonstrated for extracted samples of EPH, PSE, and caf-
feine at both room temperature and at ~5°C for three days. The

Table III. Stability Results for L-Ephedrine, Pseudoephedrine, and Caffeine

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Nominal Mean Mean Day 0
Stability Storage Conc. Found Conc. Found Conc. Mean %
Compound Type Conditions (ng/mL) (% CV) (% CV) Difference

L-Ephedrine Refrigerator stability 5°C, 3 days 8.72 8.18 (3.0)* 7.49 (2.6)† 9.2
of extracts 20.9 19.9 (12)* 19.6 (4.9)† 2
66.8 55.9 (6.8)* 62.7 (4.3)† –11
Ambient stability Room temperature, 8.72 8.73 (5.9)* 7.49 (2.6)† 16.5
of extracts 3 days 20.9 19.9 (3.7)* 19.6 (4.9)† 2
66.8 62.3 (3.5)* 62.7 (4.3)† –0.6
Freeze-thaw stability –80°C, 8.72 8.21 (4.2)* 7.49 (2.6)† 9.7
3 cycles over 3 days 20.9 19.3 (4.3)* 19.6 (4.9)† –1
66.8 62.8 (4.4)* 62.7 (4.3)† 0.2
Long-term frozen stability –80°C, 60 days 20.0 23.0 (4.7)‡ 19.5 (5.8)‡ 18

Pseudoephedrine Refrigerator stability 5°C, 3 days 8.08 7.08 (2.9)* 6.89 (16)† 2.7
of extracts 20.9 19.7 (15)* 20.1 (4.8)† –2
66.8 55.6 (5.4)* 64.6 (4.8)† –14
Ambient stability Room temperature, 8.08 7.48 (7.2)* 6.89 (16)† 8.6
of extracts 3 days 20.9 19.7 (2.3)* 20.1 (4.8)† –2
66.8 61.5 (2.3)* 64.6 (4.8)† –4.8
Freeze-thaw stability –80°C, 8.08 7.57 (4.5)* 6.89 (16)† 9.9
3 cycles over 3 days 20.9 19.4 (2.6)* 20.1 (4.8)† –4
66.8 62.8 (3.5)* 64.6 (4.8)† –2.8
Long-term frozen stability –80°C, 60 days 20.0 22.5 (4.9)§ 20.2 (2.5)§ 11

Caffeine Refrigerator stability 5°C, 3 days 8.56 7.91 (0.48)* 8.41 (6.7)† –5.9
of extracts 20.3 19.6 (8.8)* 19.9 (2.8)† –1
64.8 56.7 (6.5)* 65.5 (0.75)† –13
Ambient stability Room temperature, 8.56 8.61 (5.1)* 8.41 (6.7)† 2.3
of extracts 3 days 20.3 18.9 (0.38)*,# 19.9 (2.8)† –5.3
64.8 59.8 (0.51)* 65.5 (0.75)† –8.8
Freeze-thaw stability –80°C, 8.56 8.62 (5.0)* 8.41 (6.7)† 2.5
3 cycles over 3 days 20.3 18.8 (1.6)* 19.9 (2.8)† –5.7
64.8 59.6 (0.35)* 65.5 (0.75)† –9.0
Long-term frozen stability –80°C, 60 days 20.0 21.9 (5.9)" 19.8 (2.5)" 10

* Calculated using the linear regression equation from “Day 3” (Table I).
† Calculated using the linear regression equation from “Day 2” (Table I).
‡ Linear regression equation: y = 0.01838x + 0.02736 (r = 0.9943).
§ Linear regression equation: y = 0.03124x + 0.04098 (r = 0.9944).
# Only two replicates were used; the third was deemed an outlier (Grubb’s test).

" Linear regression equation: y = 0.02544x + 0.01726 (r = 0.9993).

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analytes were also stable in frozen plasma (–80°C) for 60 days
and could withstand three freeze-thaw cycles in three days. The
found concentrations of the stability samples were compared to
those of freshly prepared samples, and the percent difference
was calculated. The results obtained were within acceptable
limits (% difference ≤ ±20%, % CV ≤ 20%; Table III).
Extended range and dilution verification
Extended calibration curves for EPH, PSE, and caffeine were
linear in plasma from approximately 4 to 800 ng/mL and 800
to 5000 ng/mL with correlation coefficients r > 0.99. Average
back-calculated values with accuracy and reproducibility for
each matrix standard are shown in Table IV. For the diluted caf-
feine extracts with nominal plasma concentrations of 128,000
and 25,500 ng/mL, the average back-calculated values were
toxicology study. Rats were administered EPH intravenously at
6.25 mg/kg; by oral gavage at 6.25, 12.5, or 25 mg/kg; in com-
bination with caffeine (25 mg/kg EPH + 30 mg/kg caffeine); or
in Ma Huang (312.5 mg/kg alone or in combination with 30
mg/kg caffeine). The concentration of EPH ranged from below
the limit of quantitation (BLOQ, < 2.09 ng/mL) to 2290 ng/mL;
PSE ranged from BLOQ (< 2.09 ng/mL) to 115 ng/mL; and caf-
feine concentrations ranged from BLOQ (< 2.03 ng/mL) to
22,800 ng/mL. The toxicokinetic analysis is in preparation to be
published elsewhere (15).
Conclusions

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129,000 ng/mL (0.8% RE, 0.78% CV) and 24,700 ng/mL (–3%
RE, 4.2% CV), respectively.
Analysis of dosed animal specimens
The validated method was successfully used to analyze
plasma samples from male Fischer-344 rats dosed during a
EPH, PSE, and caffeine can be simultaneously quantitated in
a small volume of rat plasma using this rapid and simple LC–
MS–MS method. Each of the three analytes can be precisely
and accurately quantitated over the approximate range of 2 to
5000 ng/mL, and samples with caffeine concentrations as high
as 123,000 ng/mL can be diluted and analyzed. The method re-

Table IV. Summary of Extended Range Calibration Curves and Back-Extracted Concentrations from L-Ephedrine,
Pseudoephedrine, and Caffeine

Concentration (ng/mL)
Compound Run No. 1 2 3 4 5 6 7 8 9 10 11 12

L-Ephedrine* 4.16 8.72 20.9 43.6 104 874 835 1090 2090 3280 4180 5460
1 3.53 7.72 20.2 43.1 108 887 776 1170 2090 3170 4240 5590
2 4.54 9.00 20.2 40.9 108 889 828 1080 2210 3280 4180 5190
3 4.33 10.2 20.6 42.1 102 892 840 1100 2170 3121 4150 5430
n 3 3 3 3 3 3 3 3 3 3 3 3
Mean 4.13 8.97 20.3 42.0 106 889 815 1120 2160 3190 4190 5410
% RE –0.6 2.9 –2.7 –3.6 1.9 1.8 –2.4 2.4 3.3 –2.7 0.3 –1.0
% CV 13 14 1.1 2.6 3.3 0.28 4.2 4.5 2.8 2.5 1.1 3.7

Pseudoephedrine† 4.16 8.08 20.9 40.4 104 808 835 1010 2090 3030 4180 5050
1 3.95 7.38 20.4 38.3 110 827 782 1120 2020 2860 4240 5200
2 4.61 7.96 19.7 37.3 109 866 848 1010 2270 2990 4130 4920
3 4.29 8.01 20.3 39.8 106 850 810 986 2140 2970 4220 4980
n 3 3 3 3 3 3 3 3 3 3 3 3
Mean 4.28 7.78 20.1 38.5 108 848 813 1040 2140 294 4200 5030
% RE 3.0 –3.7 –3.7 –4.8 4.2 4.9 –2.6 2.9 2.5 –3.0 0.5 –0.3
% CV 7.7 4.5 1.9 3.2 1.9 2.3 4.1 6.6 5.8 2.4 1.4 3.0

Caffeine‡ 4.04 8.56 20.3 42.8 102 854 811 1070 2030 3200 4060 5340
1 4.12 8.28 20.7 41.8 107 827 783 1140 2000 3110 4050 5640
2 4.09 8.55 20.3 42.2 104 839 828 1070 2120 3220 3970 5290
3 3.94 8.69 19.8 43.8 104 845 800 1040 2030 3030 4020 5460
n 3 3 3 3 3 3 3 3 3 3 3 3
Mean 4.05 8.51 20.3 42.6 105 837 804 1080 2050 3120 4010 5460
% RE 0.2 –0.6 –0.2 –0.5 2.9 –2.0 –0.9 1.5 0.9 –2.6 –1.1 2.3
% CV 2.4 2.4 2.2 2.5 1.6 1.1 2.8 4.7 3.0 3.0 1.1 3.3

* Standards 1–6 used linear regression equation y = 0.02102x + 0.02426 (r = 0.9957); standards 7–12 used linear regression equation y = 0.002033x – 0.09799 (r = 0.9980).
† Standards 1–6 used linear regression equation y = 0.03600x + 0.01223 (r = 0.9974); standards 7–12 used linear regression equation y = 0.003613x – 0.1537 (r = 0.9971).
‡ Standards 1–6 used linear regression equation y = 0.02361x + 0.02216 (r = 0.9996); standards 7–12 used linear regression equation y = 0.002160x – 0.02877 (r = 0.9985).

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quires only a single-step protein precipitation, and its applica-
tion was successfully demonstrated for a toxicokinetic study of
EPH administered alone, in combination with caffeine, and in
the herbal source Ma Huang.
Acknowledgments
This project has been funded in whole or in part with federal
funds from the National Institute of Environmental Health
Sciences under Contract No. N01-ES-65554. The authors
thank Dr. Brian F. Thomas of RTI International and Dr. Cynthia
S. Smith of NIEHS/NTP for reviewing this work.
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