Journal of Analytical Toxicology,Vol. 29, March 2005
Multi-Residue Determination of Anti-Inflammatory
Analgesicsin Sera by Liquid ChromatographyMass Spectrometry
Irina RudikMiksa*, Margaret R. Cummings, and Robert H. Poppenga
University of Pennsylvania, School of VeterinaryMedicine, Departmentof Pathobiology, New Bolton Center, Toxicology,
382 WestStreet Road, Kennett Square, Pennsylvania 19348
Abstract
Non-steroidal anti-inflammatories (NSAIDs) are analgesic,
antipyretic, and, as their name implies, anti-inflammatory
drugs, which are widely used for the treatment of a variety
of human and veterinary disease conditions in which control
of pain and inflammation is desired. Acetaminophen (ACE)
is a common over-the-counter analgesic. Detection of a variety
of widely used NSAIDs and ACE in fluid and tissue samples
is an important diagnostic tool. A sensitive and selective
analytical method has been developed for simultaneous
screening of 12 NSAIDs and ACE by liquid chromatographymass spectrometry with an atmospheric pressure chemical
ionization interface set to operate in the negative ion mode
of MS. Following sample preparation, all analytes were separated
on a C18-reversed.phase column with a gradient elution
of acetonitrUe and acetic acid. Full-scan mass spectral
fragmentation profiles were established for each analyte and
individual extracted ion chromatograms were used for
quantitation. Linearity of detection was observed over
the 0.05-25.0 pg/mL range of standard concentrations.
The instrument limits of detection (LOD), based on an individual
analyte quantitation ions, fell between 0.05 and 1.0 pg/mL
for all compounds. The matrix LODs were determined to be
0.05 pg/mL for phenylbutazone (m/z 307); 0.1 pg/mL for
indomethacin (m/z 312), flunixin (m/z 295), and piroxicam
(m/z 330); 0.5 pg/mL for ACE (m/z150), diclofenac (m/z 250),
ketoprofen (m/z 209), and mefenamic acid (m/z240); 1.0 pg/mL
for oxyphenbutazone (m/z 323); 5.0 pg/mL for ibuprofen
(m/z 205), salicylic acid (m/z 137), and tolmetin (m/z 212);
and 10 pg/mL for naproxen (m/z 185).
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs)comprise a
large group of therapeutic agents with anti-inflammatory, anal. Author to whom correspondence should be addressed. E-mail: [email protected].
gesic, and antipyretic actions. The pharmacologic effects of
NSAIDs are primarily due to inhibition of cyclooxygenase
(COX), which is responsible for the biosynthesis of prostaglandins and other related autocoids (1). Acetaminophen
(ACE) also has analgesic and antipyretic actions, but achieves
these effects via a different site of action than COX and is not
classified as an NSAID.ACE is believed to interfere with the endoperoxide intermediates PGG2and PGH2 (2). NSAIDsand ACE
are among the most commonly used over-the-counter preparations for treatment of osteoarthritis and other rheumatic
diseases in human and veterinary medicine. The utilization of
certain NSAIDshas also been correlated with lowering the risk
of Alzheimer's desease in humans (3-5).
Although generally considered safe, acute overdose and
chronic use of NSAIDs are associated with significant adverse
effects on the gastrointestinal, hematopoietic, and renal systems
(1). Chinese patent medicines have been adulterated with
NSAIDs (6) such as indomethacin, phenylbutazone, mefenamic
acid, and diclofenac. In addition, several NSAIDs, such as flunixin and phenylbutazone, have been used unethically in performance horses to mask signs of inflammation and pain. ACE
is hepatotoxic, and acute intoxications are common and can result from overdosage of pediatric formulations or suicide attempts (7). In veterinary medicine, cats are uniquely sensitive
to the toxic effects of ACE because of their inability to glucuronidate the drug (8). ACE intoxication in cats is common because of its administration by well-intentioned owners unaware
of its toxicity. For these reasons, development of an accurate,
sensitive, and selective method for detection of NSAIDs in biological samples has been the goal of a number of clinical and
toxicology laboratories
NSAIDsare a chemically diverse group of drugs that includes
salicylic acid derivatives (e.g., aspirin), para-aminophenol
derivatives (e.g., ACE), indolacetic acids (e.g., indomethacin,
sulindac, etodolac), arylproprionic acids (e.g., ibuprofen,
naproxen, ketoprofen, carprofen), fenamates (e.g., meclofenamic acid), pyrazolones (e.g., phenylbutazone, dipyrone),
enolic acids (e.g., piroxicam, tenoxicam), and heteroaryl
Reproduction(photocopying)of editorialcontentof thisjournalis prohibitedwithoutpublisher'spermission.
95
Journal of Analytical Toxicology, Vol. 29, March 2005
acetic acids (e.g., diclofenac) (1). Because NSAIDs and ACE
comprise a heterogeneous group of compounds, most available
detection methods are optimized to detect a single drug
and one or more of its metabolites. Available reports suggest
that liquid chromatography-mass spectrometry (LC-MS)
and LC with UV detection in conjunction with solid-phase
extraction (SPE) are the most commonly used methods for
detection and quantitation of single NSAIDs in biological
samples (9-23). Other single-drug methods include gas chromatography-mass spectrometry (GC-MS) (24,25), spectrofluorimetric (26) and spectrophotometric methods (27), capillary
electrophoresis (23,28-31), and thin-layer chromatography
(32). Several multi-drug screening procedures have been
developed for groups of NSAIDs based on their chemical
properties. These include detection of salicylates (33-35),
fenamates (36), naproxen and other naphthalene derivatives
(37), anthranilic (38-40), and lecithin-based derivatives (41).
Available multi-residue screening methods for NSAIDsinclude
high-performance liquid chromatography (HPLC) with electrothermal or IN detection for a group of 3-16 different
analytes present in a single sample with reported limits of
detection (LODs) ranging from 0.01 to 3.6 lag/mL (42--45).
Successful screening of up to 21 NSAIDsand their metabolites
has been reported using a variety of GC-MS procedures
(46-48). Difficulties in optimization of an HPLC method for
phenylbutazone and mefenamic acid and an inability to detect
oxyphenbutazone have been reported (42). An involved gradient profile, a combination of organic solvents, or a buffering
system are usually required in HPLC procedures to achieve
the best peak separation and analyte identification. GC-MS
methods, although useful for screening purposes in clinical
and forensic toxicology (LODsrange 0.005-0.05 tag/mL),prove
laborious because of the need for sample derivatization
with methyl iodide to improve chromatographic properties
(46,48). A multi-analyte method for separation of diclofenac,
ketoprofen, indomethacin, and flunixin present in the same
sample has recently been reported for an LC-MS system with
an atmospheric pressure chemical ionization (APCI) interface
set to operate in the negative ion mode (49). The use of APCI
is advantageous in that it can transform fragile thermolabile
species into ions without decomposition, although sometimes
fragment ions are absent. The method developed for the
four NSAIDsshowed that LC-MS affords high analyte specificity
and short analysis time. The use of a non-routine CN-column
and an organic/buffer mobile phase for elution and deprotonation of compounds might imply difficulties with such an
approach.
This article describes a simple, rapid, and reproducible multiresidue screening method for simultaneous detection of
12 NSAIDs and ACE in serum. Sample preparation omits
any SPE columns, and drug elution is achieved on an C18reversed-phase LC column without the need for derivatization.
Under the method conditions, full-scan mass spectral flagmentation patterns, obtained in the negative ion mode of APCI,
are reported for each compound. Accurate identification and
quantitation of all analytes are achieved over a wide range of
concentrations without the need for complete chromatographic
resolution.
96
Material and Methods
Materials
All pure analyte standards/solutions, except for flunixin, and
bovine adult serum (sterile-filtered) were purchased from
Sigma (St. Louis, MO). Flunixin was a gift from ScheringPlough Corp (Kenilworth, NJ). Acetonitrile (ACN), methanol
(MET), sodium chloride (NaCl), and glacial acetic acid were obtained from Fisher Scientific (Pittsburgh, PA). All reagents
were HPLC grade. An ultra pure water system from Millipore
(Billerica, MA) was used to generate water with resistivity of 18
M~.cm (Milli-Q RG and Milli-RO10). LC autosampler vials (2
mL, wide opening) designed for a robotic arm tray with 250-1JL,
high-recovery,fiat-bottom inserts were purchased from Agilent
Technologies (Wilmington, DE).
Preparation of standards
Individual stock standards of 1000 lag/mL for flunixin, indomethacin, ketoprofen, mefenamic acid, oxyphenbutazone,
phenylbutazone, and piroxicam were prepared by dissolving
10 mg of each compound in 10 mL ofACN. Naproxen and tolmetin 1000 lJg/mL standards were made with 50% ACN/water,
and diclofenac standard was placed in MET.Standards for ACE,
ibuprofen, and salicylic acid were purchased as solutions at
1000 l~g/mLin MET.Two 100 l~g/mLstandard mixes (A and B)
were prepared. Mix A was made by combining 100 lJL of individual 1000 lJg/mL standards of ACE, salicylic acid, piroxicam,
tolmetin, ketoprofen, naproxen, and diclofenac with 300 pL of
ACN, and mix B was prepared by addition of 100 IJL of 1000
IJg/mL of oxyphenbutazone, phenylbutazone, flunixin, indomethacin, ibuprofen, and mefenamic acid to 400 IJL of ACN.
Working standards were made by adding either 250 or 100 pL
of each A and B mix to 500 IJL or 800 t~Lof ACN/water (60:40)
solution for 25 and 10 IJg/mL standard mixes, respectively.All
other standards were made by serial dilution of the 25 and 10
tJg/mL working standard mixes with ACN/water. All samples
were stored at 4~ when not in use.
Sample handling
Negative control bovine serum, not containing any detectable
amounts of NSAIDsor ACE, was analyzed prior to analyte fortification. Aliquots of negative control serum (0.4 mL) were
spiked with standard mix solutions at 0.05, 0.10, 0.50, 1.0, 2.0,
5.0, 10.0, and 20.0 IJg/mL levels. Each fortified sample was
combined with 0.05 g of NaC1and 0.8 mL of ACN.All samples
were vortex mixed for 15 s and centrifuged for 10 rain at 14,000
rpm. Clear supernatants (0.5 mL) were placed into an autosampler vial and analyzed by LC-MS.
LC-MS conditions
A series 1100 LC-MS Hewlett-Packard system (Wilmington,
DE) was equipped with a dual LC pump, a diode-array detector,
a degasser, a column thermostat, and an autosampler. A Betasil
C18-reversed-phase column (150 x 4.6 ram, 5-pro particle size)
from Keystone Scientific (Bellefonte, PA)was equilibrated with
the mobile phase (50% ACN and 50% of 0.25% acetic acid in
water) at 0.5 mL/min prior to analysis. The LC profiles were developed at 40~ with a mobile phase and flow rate gradients.
Journal of Analytical Toxicology, Vol. 29, March 2005
The following 17-rain program was used: 50% ACN was
pumped at 0.5 mL/min for 5 rain, increased to 80% ACN over
5 rain at 0.7 mL/min, held for 5 rain, then reduced to 50% ACN
over the next minute, and the column re-equilibrated for 1.0
rain. Each assay was based on the 20-1JL injections. MS was set
to operate in a negative mode of APCI, detection was set with
the 2-min time delay, gain at 10, fragmentor at 70, threshold of
50, and a step-size of 0.1. A full-scan MS mode was used (m/z
115-200 and rn/z 150-400) in order to obtain a complete representation of the total ion chromatogram for each compound
analyzed. To optimize for ACE and salicylic acid during NSAID
standard mix analyses, mass-to-charge ratio ion range of
115-200 was followed between 2 and 5.2 rain, with the rest of
the compounds monitored (m/z 150--400) between 5.2 and
14.5 rain. MS was optimized for the following settings: dry gas
flow rate of 3 mL/min, corona at 20 IJA,nebulizer pressure of 50
psi nitrogen, dry gas and vapor temperatures of 300~ and
capillary voltage of 2500V.
Linearity, precision, and instrument
limit of detection (ILOD)
Linearity of instrument standard response was determined for
all analgesics over the range of concentrations: 25.0, 10.0, 5.0,
1.0, 0.50, 0.25, 0.10, and 0.05 I~g/mL. Individual calibration
curves were constructed for every analyte
based on peak areas under the corresponding
Table I. Chromatographic and Fragmentation Behavior of NSAIDs and ACE
individual extracted current-ion profiles. A plot
Iont
(%)
RT*
TRw
of concentration versus peak area resulted in a
Name
MW*
(m/z)
Abundance (min)
(pg/mL)
linear curve for individual drugs based on triplicate analysis (n = 3) at corresponding exACE
151.2
150#
100
3.20
28 (canine)
tracted ion profiles. This resulted in average
correlation coefficients (R2) between 0.9930
Diclofenac
318.1'*
250#
100
12.1
2.5 (humans,150 rag)*t
294
55
and 0.9998 for each standard curve repeated in
triplicate for all mass-to-charge ratios listed in
Flunixin
296.3
295#
100
11.2
1.1-2.2 (bovine)
Table I (quantitation and confirmation ions).
303
35
0.25-1 (canine)
The percent standard deviation (%SD =
251
15
[square root of {nSx2 - (Sx)2/n(n - 1)}]*100%,
Ibuprofen
206.3
161
100
12.7
12-15 (humans)
where n is the number of measured values for
10 (canine)
205#
85
particular measurements, x) for all the curves
Indomethacin
357.8
312#
100
12.0
was between 0.005 and 0.514%, indicating that
0.5-3.0 (humans)
356
30
detection was linear from 0.05 to 25.0 l~g/mL.
Precision of standard mixes and fortified
Ketoprofen
254.3
209~
100
9.30
1.1 (equine)
serum
was determined by three consecutive
253
10
injections (n = 3) of 20-1JL aliquots of the 1.0
197
10
~g/mL standard mix and 20 ~g/mL serum
Mefenamic
241.3
240#
100
13.4
10-20 (humans, lg)*t
spikes. Peak area of each extracted ion was esacid
196
10
tablished by a Hewlett-Packard integration alNaproxen
230.3
185#
I00
9.60
2-5 (canine)
gorithm and quantitation of repeatable
229
10
10 (equine)
injections was evaluated for % recovery based
170
50
on calibration curves constructed between
0.05 and 25 lJg/mL. Data were observed to be
Oxyphen324.4
323#
100
9.60
Not available
reproducible and %SD values fell below 10%
butazone
280
15
(n = 3) for ACE, naproxen, phenylbutazone,
204
25
salicylic
acid, ketoprofen, oxyphenbutazone,
Phenyl308.4
307#
100
12.9
5-10 (canine)
and
piroxicam
in a standard mix for all quanbutazone
1-4.4 (equine)
titation
ions
(Table
I). The %SD values for di10-20 (bovine)
clofenac, flunixin, ibuprofen, indomethacin,
Piroxicam
331.4
330#
100
8.10
0.3-1.0 (canine)
mefenamic acid, and tolmetin in a standard
266
25
mix fell between 10.2 and 14.4%. For more
210
5
precise quantitation of a 1.0 IJg/mL standard,
Salicylic acid
138.1
137#
100
4.95
< 20% (humans, topical)
calibration curves based on lower standards
(0.05-5 lJg/mL) alone were constructed for
every
analyte at its ion of quantitation (Table
Tolmetin
257.3
212#
100
8.30
40 (humans, 400 rag)t*
I). A range of percent recoveries observed was
* Molecular weight.
between 79 and 93% with %SD range of
~"All available mass fragment ions observed under method's conditions.
1.0-14.7 for all analytes. Quantitation of all
* Retention time.
Therapeutic range (2,7).
analytes in a 201Jg/mL fortified serum repeat# Ion of quantitation.
able injections was evaluated for all ions listed
** DIC sodium complex.
tf Therapeutic range has not been established. Reported are the peak plasma concentrations at highest oral
with mass-to-charge ratios in Table I. The
dosage.
%SD values fell between 2.6 and 19.3% based
97
Journal of Analytical Toxicology, Vol. 29, March 2005
on all quantitation ions and 5.8 and 16.3% for all confirmation
ions listed with mass-to-charge ratios.
Between-day variability was evaluated for a 10 pg/mL fortified
serum spike based on a three-day minimum design. The %SD
of a three-day analysis for all ions listed with mass-to-charge
ratios, except for indomethacin (Table I), was 1.2-10.0%. Detection of indomethacin showed slightly higher between-day
variation with %SD of 13.2 and 13.8% at m/z 312 and 356,
respectively.
ILOD was defined as the lowest concentration of each analyte
present in a standard mix that could be detected and expressed
as a concentration at signal-to-noise ratio (S/N) of 3:1 based on
the individual extracted-ion chromatograms.
Analyte recovery, LOD, and limit of
quantitation (LOQ)
Extraction efficiency for every NSAID and ACE was evaluated at 8 fortified serum concentrations in replicates of 4 (n =
4). The amount of drug extracted during the purification procedure as compared to the concentration used to fortify the
sample was expressed in terms of percent drug recovered evaluated at each ion listed with mass-to-charge ratio. Individual extracted ion (re~z) peak areas were plotted onto individual
A
Results
Chromatography and MS fragmentation
A complete chromatographic and fragmentation pattern was
collected for all 12 individual 10 pg/mL NSAID and ACE standards under given conditions in order to establish the correct
peak assignments and mass spectral behavior of each analyte
when present in a mix. A different degree of fragmentation was
seen for the 13 drugs analyzed. The full-mass spectral fragmentation profile for ACE, phenylbutazone, and salicylic acid
MEFA
B
IBU
161.2
100
standard calibration curves, and the corresponding concentration of each drug in fortified samples was determined. The ratio
of two concentrations (recovered vs. fortified) times 100% was
used to report % drug recovery.
Sample LODs were defined as the lowest concentration of
each analyte that can be detected in a fortified serum sample at
3:1 S/N. The lower sample LOQ was defined as the lowest measured concentration (based on LOD) of each analyte that can be
recovered from the fortified serum sample within 20% of true
value based on an individual ion of quantitation (Table I).
100
240.1 [M-H]
205.0 [M-H]
80
80NH
~ 6o
"1=
= 60
<
,.o
,<
40
OH
40
m/c 161
20
OH
2o-~
d
2~ ,,., ,1!,I,I!~.,,,,,h ,,i,!,1h!H I,h,. ......=.......,m,~,.~...,.=,~,,,,I.............~, ,:.,,;,
0 t ...........
,l
175
200
m/z 250
C
100
m/z
225
200
DIC
H 2C~--I~ O H
D 100
=
60
irdZ 312
311.6
o
H20/LIOHI--U
[ND
80
8O
r
250
m/z
-
O
II
249.8
I! 96.2
250
252,1
293.7[M-H]"
.~: 60
<~'=:40
I
c=o
313.9
[~
<~
40
I
96.1
20
J
I
I ~,
..............................
260
m/~
IIJ,
280
I
20
3SS.8[M-H]I
cl
0
r ....
320
m/Z
[
' ,
r
I
,I
340
Figure I. Full-scan mass spectrum profiles generated by ibuprofen, mefenamic acid, didofenac, and indomethacin (5 pg/mL) in MS with negative mode of
APCI, Fragmentation of ibuprofen (A), diclofenac (C), and indomethacin (D) generated molecular species (all at 100% abundance) corresponding to the loss
of one CO2 molecule from each parent ion resulting m/z 161,2.50, and 312, respectively. An ion at m/z 196 (10% abundance) was seen for the loss of CO2
from the parental structure of mefenamic acid (B), Deprotonated molecular ion was seen for every compound ([M-H]-), Complete chemical structure, fragment generation, and the resulting mass-to-charge ratio values are shown for every drug in their corresponding panels,
98
Journal of Analytical Toxicology, Vol. 29, March 2005
under negative APCI resulted in generation of molecular ions
only ([M-H]-]) at m/z 150, 307, and 137, respectively (data not
shown). Fragmentation spectra for tolmetin resulted in a single
ion at m/z 212 corresponding to the loss of CO2from the molecular structure (not shown), ([M-H]-) ion for tolmetin at m/z of
256 was not detected.
Fragmentation of ibuprofen, mefenamic acid, diclofenac, and
indomethacin present in a 5 IJg/mL standard mix resulted in
two mass-to-charge ratio ions for each molecule (Figure 1). In
addition to the [M-H]- ions for each of these drugs at m/z 205
(85% abundance, ibuprofen), 240 (100% abundance, mefenamic acid), 294 (55% abundance, diclofenac), and 356 (30%
abundance, indomethacin), further fragmentation resulted in
the loss of one CO2 unit from each of ibuprofen (m/z 161),
mefenamic acid (m/z 196), diclofenac (m/z 250), and indomethacin (m/z 312) as shown in Figures 1A-1D, respectively.
The five remaining NSAIDsproduced three possible detectable
ions upon fragmentation (Figure 2). Each compound resulted
in [M-H]- ion at m/z 330, 323, 253, 229, and 295 for piroxicam, oxyphenbutazone, ketoprofen, naproxen, and flunixin,
respectively. Loss of one CO2 unit from ketoprofen, naproxen,
and flunixin structures generated ions at m/z 209, 185, and
251, respectively. Further fragmentation of ketoprofen (Figure
2C) and naproxen (Figure 2D) resulted in the additional loss of
a methyl unit ([[M-H]-(CO2)-(CH3]]-) and produced ions at m/z
197 (10% abundance; ketoprofen) and 170 (50% abundance;
A
naproxen). The third detectable ion of flunixin at m/z 303
(Figure 2E) could be attributed to the formation of a mixed anhydride between the parental structure and the acetic acid present in the mobile phase as proposed in Figure 3A. The loss of
two fluoride units is not surprising because fluoride is considered to be a 'good' leaving group. As in the case of flunixin, pattern of fragmentation of piroxicam generated three ions,
formation of one of which could be attributed to the presence
of the acetic acid in the mobile phase. The observed ion at m/z
210 in the fragmentation pattern of piroxicam can be explained
by the loss of (O=C-NH-CsH4N)unit from the parental structure
as shown in Figure 2A. Further loss of H20, one CH3 unit, and
a reaction between the newly formed piroxicam fragment and
the acetic acid would lead to a new and stable mixed anhydride
with m/z 266 as suggested in Figure 3B. The two additional ions
observed for oxyphenbutazone were possibly due to the initial
loss of a 3-carbon unit (CH3-CH2-CH2)from the parental ion resuiting with m/z 280 followed by the loss of the phenyl group
(C6H6)with the final m/z 204 (25% abundance) as proposed in
Figure 2B. A complete summary of elution times for each individual drug present in a standard mix, their corresponding
mass-to-charge ratio values and mass spectral abundances are
reported in Table I.
Multi-residue detection and ILOD
Mass spectral data for a mixture of 12 NSAIDsand ACE was
OH
329,7
0
/H2
H2
--C --CH 3
~176
9 80
21o
60
CHz;C
100-
8O
r-
m/z 280
B
[M-H]-
100
322.7
[M-H]"
u
'~ 60 2
.D
< 40
.....2,!0
'
250m/Z
m/z 1 9 7 - ~
=) 80
200
184.8
100-
'~60
<
< 40
170.1
~, m/z 185
3
c
~
~H
H
40
20
253.2
"
0
229~-H]"
.
200
225 m/z
250
150
J. . . .
200
7
_300
o!
[oo
294.7
H~C
CF3 [M-HI"
~ 40 j m/z251
303.2
'o'
............II........ m& 1
20
m/z
El00 1
OCH3
i_ ~
,_%7-0
,~ 6o
I
360
D
209.0
204.1
20"
....................................................................................
200
ClO o
4O
266.1
20
I
20 i
251
L
m/z
250
275 m/z
300
Figure 2. Complete MS fragmentation data obtained for piroxicam, oxyphenbutazone, ketoprofen, naproxen, and flunixin in a full-scan negative monitoring
mode of APCI. All spectra were collected for 5 tJg/mL individual standards under identical conditions. Structures of parental compounds and MS generated
daughter ions corresponding to the detected mass-to-chargeratio ion fragments are shown by arrows in corresponding panels. [M-H]- ion was found to be most
abundant (100%) for piroxicam (A), oxyphenbutazone (B), and flunixin (E). Lossof one CO2 unit generateddaughter ions at 100% abundance for ketoprofen
(m/z 209) (C) and naproxen (m/z185) (D).
99
Journal of Analytical Toxicology, Vol. 29, March 200g
collected for a range of standard concentrations (0.05 to 25.0
pg/mL) in a full-scan mode of MS with negative APCI (m/z
115-400). The total ion chromatogram for a 5 pg/mL standard
mix resulted in 11 well resolved MS peaks as shown in Figure 4.
Nine out of 12 NSAIDs compounds were well separated and
showed good chromatographic behavior. All available peaks
were assigned to the individual analyte with observed co-elution
of oxyphenbutazone and naproxen at 9.5 min and co-elution of
indomethacin and diclofenac at 12 rain. For unambiguous identification and quantitation of each compound present in a mixture, extracted current-ion profiles for all analytes were obtained
for every available mass-to-charge ratio mass fragment. Chromatographic co-elution of diclofenac and indomethacin could
be resolved based on the two non-shared individual extracted
ions. Indomethacin fragmentation resulted in ions at m/z 356
and 312, not shared by diclofenac with mass-to-charge ratio
fragments of 294 and 250 (data not shown). The same reasoning was applied to oxyphenbutazone and naproxen where
three different ions can be used for identification and confirmation of each analyte (Table I). In the case of diclofenac, the
two characteristic ions at m/z 253 and 251 (protonation products ofm/z 250) appear in the extracted current ion profiles of
ketoprofen and flunixin (data not shown). However, it is clear
that elution of ketoprofen and flunixin mass spectral peaks
occur 1-2 min earlier as compared to diclofenac. Thus, clean
peak separation allows for correct mass-to-charge ratio assignments at the two ions m/z 253 and 251, rendering them useful
for confirmation of ketoprofen and flunixin in a sample. Extracted-ion chromatograms for m/z 210 (piroxicam) and m/z
303 (flunixin) was problematic at or below 5 pg/mL, suggesting
that their presence cannot be used in confirmation of piroxicam
and flunixin.
ILODs were determined for all compounds present in a standard mix based on individual quantiation ions (Table I) with
values of 0.05 pg/mL for indomethacin, phenylbutazone, and
piroxicam; 0.1 pg/mL for diclofenac, flunixin, ketoprofen, mefenamic acid, and oxyphenbutazone; 0.5 pg/mL for ACE,
ibuprofen, salicylic acid, and tolmetin; and 1.0 pg/mL for
naproxen. This range of ILODs values (0.05-1.0 pg/mL) falls
well below the established therapeutic range for all the analytes
as summarized in Table I. Although higher ILODs were obtained for the available confirmation ions with the range of
0.5-5.0 pg/mL, the presence of most analytes could still be
confirmed at or below the individual therapeutic ranges. The list
of all the available therapeutic ranges for 13 different analytes,
their mass-to-charge ratio quantitation ions, and all available
confirmation ions are listed in Table I.
Detection of analytes in fortified sera and matrix
LOD and LOQ
Negative control bovine serum was fortified at 8 different
concentrations of a standard mix of 12 NSAIDs and ACE: 0.05,
0.10, 0.50, 1.0, 2.0, 5.0, 10.0, and 20.0 pg/mL. Samples were
prepared and analyzed as described in Methods. Total mass
spectral profile was obtained for a negative serum sample
and for a 20 pg/mL serum spike (data not shown). No characteristic ions of possible analytes were observed in the total
MS profile of the drug-free sample at the corresponding reten-
A
o
mc\
__N
m/z
__N
Cn3On
O
o
,
H~C
I~C
o//\\o
0
!1
296
/cH~
H
CH2F
re~Z238
O O
/
CFa
H
N~ "
G--O--C--CH
m/z 252
II
II
o
o
m/z 94
a
o.
m/z
o
O
"~
302
0
0
m/z 266
Figure 3. Proposedchemical reactionsfor generation of observed mass-to-chargeratio daughter ions of flunixin (A) and piroxicam (B) under chromatographic
conditions with negative ionization mode of APCI. Parental structure of flunixin can undergo two different modification pathways, generating ion m/z 252
for the loss of COx or reacting with the acetic acid molecule prior to the lossof CO2 forming a mixed anhydride at m/z 302 (A). Piroxicam (B) can undergo
one proposed type of the chemical modification, loosing a CH~OH unit and reacting with the acetic acid molecule leading to the loss of a phenyl amine
structure (m/z 94).
100
Journal of Analytical Toxicology, Vol. 29, March 2005
tion times. The total MS ion chromatogram of a fortified sample
contained 7 out of 11 analyte peaks that were clearly assigned
to the specific analgesics based on a response of a standard
mix (Figure 4). This demonstrates that the peak behavior
and the elution patterns of most compounds are similar to a
standard mix. Peak assignment was not possible for salicylic acid
and ACE in the total MS of the fortified sample. The loss of clear
resolution between piroxicam and tolmetin, and ketoprofen,
oxyphenbutazone and naproxen was observed and showed the
effect of matrix interference. However, mass spectral comparison of a fortified sample to a control serum demonstrated the
specificity of the method. Difficulties in peak assignment for
fortified samples were overcome by the data obtained in the
extracted current-ion profiles of all analytes. Figure 5 shows
the absence of matrix interferences and the clean peak resolution observed in the extracted single current-ion profiles for
13 analytes at the corresponding quantitation ions (Table I).
'~
x 10es
,Nos.~c
35
30
PBU't
~
:
:
PIR
OPSUTR4Ap FLU
KET
Extracted ion current profiles for the confirmation ions were
the same (data not shown) and could be used for verification of
drug identity.
Sample analyte recoveries and the corresponding %SD values
were determined for n = 4 replicates over a range of concentrations, 0.05-25.0 lag/mL (data not shown). All analgesics in
fortified samples were quantitated based on extracted single
current-ion profiles at corresponding mass-to-charge ratio
values (Table I). Matrix LODs were determined for all available
mass-to-charge ratio ions, expressed in units of concentration
and defined as the lowest ion signal seen for a spiked serum at
S/N 3:1. LOQs were based on all data as described in Methods.
Values of LODs and LOQs are reported for all available mass-tocharge ratio ions and summarized in Table II. As seen in Tables
I and II, most of the analgesics analyzed could be detected,
quantitated, and confirmed when present in a fortified serum at
or below their corresponding therapeutic ranges. Confirmation above therapeutic levels could be obtained for ibuprofen,
ketoprofen, and naproxen. Average recoveries at individual
LODs for all analytes (n = 4 replicate analysis) were expressed
in % recovery (• %SD and summarized in Table II.
MEFA
Discussion
15
10
The development of an analytical method using LC-MS with
negative APCI for simultaneous separation and identification of
0
13 analgesics is reported. All compounds were separated using
4
6
8
10
12
14
Time (min)
a traditional C18 column and an organic/acetic acid/water moFigure 4. Total ion full-scan mass spectral profile for a 5 pg/mL mix of 12
bile phase. APCIwas chosen for this multi-residue screening beNSAIDs and ACE standards analyzed with negative APCl. All peaks are
cause of its ability for sensitive determination of analytes with
labeledwitha 3-lettercodeas designatedforeachdrugdescribedin this
moderate polarity and molecular mass. In the case of the 12
study.Oxyphenbutazone/naproxenand indomethacin/diclofenacshows
NSAIDs and ACE included in this method, the mass-to-charge
the co-elution of thesetwo pairs of compounds at 9.6 and 12 rain, reratios for [M-HI- ions ranged between 138 and 356 (Table I).
spectively.
Full-scan mass spectra examined by means of negative APCI
under the conditions of the method produced
deprotonated molecular ions ([M-H]-) for all
el0 ~
el0 ~
the
compounds except for tolmetin. Fragmen,n/z 150
m/z 240
[
tational data was specific for each drug without
50
6
8
10
12
14
16
recourse to HPLC separation allowing clear
4
6
$
10
12
14
16
ndz 185
.'
identification of individual components of the
m/z 250
I',
200
i;,
J
4
6
8
10
12
14
16
mixture. Detection and quantitation of 12
4
6
$
10
12
14
16
i
m/z 323
/l
NSAIDs and ACE was based on individual ex500 J m/z 295
':
tracted ion chromatograms and identification
4
6
g
I0
12
14
16
o i
i- i
,
o
by their fragment-ion spectra. The most com,'
~
L
''Vz
330
4
6
8
10
12
14
16
monly generated fragments for NSAIDs were
m/z 205
20
"g
ndz 137
those resulting from the loss of one CO2 unit as
50 [
,'
4
6
g
10
12
14
16
observed for ibuprofen, mefenamic acid, dim/z 312
;,
clofenac, indomethacin, flunixin, naproxen, ke]
m/z 212
toprofen,
and tolmetin. The loss of a methyl
4
6
g
10
12
14
16
4
6
8
10
12
14
16
group
generated
a third fragment ion for
i
m/: 209
"=r
20000 L ~ z307 . . . .
I00 i
naproxen and ketoprofen (Figure 2). The pres4
6
g
10
12
14
16
4
6
8
10
12
14
16
ence of the acetic acid in the mobile phase for
Time (min)
Time (rain)
LC elution did not pose any problems in ion
Figure 5. Extracted single current-ion profiles obtained in a negative mode of APCI for a fortified
generation or interpretation. Addition of acetic
serum sample at 10 pg/mL. Chromatograms of 13 possible quantitation ions are shown at their
acid across parental structure was seen only
corresponding mass-to-charge ratios. No matrix interferences were observed. For analyte idenfor flunixin and piroxicam with suggested
tification, refer to Table I.
mechanisms shown in Figure 3. In compar5
101
Journal of Analytical Toxicology, Vol. 29, March 2005
ison with the previously published data on multi-residue detection of diclofenac,flunixin, indomethacin, and ketoprofen by
negative APCI (49), the present paper shows that the highest
abundance can be achieved for ions other than [M-H]-, especially in the case of diclofenac, indomethacin, and ketoprofen
(see Table I), and an [M-H]- ion can be observed for flunixin
under present conditions.
[t is often suggested that the MS detector should be set to operate in the selected ion monitoring mode (SIM)in order to improve sensitivityand specificityof the method. Running analysis
in a full-scan monitoring mode is known to produce less sensitive LODs as compared to SIM. However, detection of 12
NSAIDs and ACE by the present method was shown to be reproducible and specific for each analyte. The chromatography
allowed optimization of the low molecular weight compounds
(ACEand salicylicacid)with detection of two ranges of mass-tocharge ratio ions at different time ranges, m/z 115-200 from 2
to 5.2 rain and rn/z 150--400 from 5.2 to 14.5 rain. The re-
Table II. Matrix Recoveries,tODs, and tOQs
Name
(m/z)
LOD*
(pg/mL)
ACE
150
0.5
1.0
81.8
12.9
Diclofenac
250
294
0.5
1.0
0.5
1.0
92.2
93.8
13.5
3.22
Flunixin
295
251
0.1
2.0
0.1
2.0
95.1
85.3
14.2
10.1
Ibuprofen
205
161
5.0
10
82.1
52.9
23.6
9.49
Indomethacin 312
356
0.1
0.5
Ion
LOQ* %Recoveryt
(pg/mL)
at LOD
10
20
0.1
0.5
Ketoprofen
209
253
[97
0.5
10
10
20
20
20
Mefenamic
acid
240
196
0.5
5.0
Naproxen
185
170
10
10
Oxyphenbutazone
323
280
204
1.0
10
5.0
Phenylbutazone
307
0.05
0.05
Piroxicam
330
266
0.1
0.5
0.5
1.0
Salicylic
acid
137
5.0
Tolmetin
212
105
116
%SD
3.20
18.2
43.8
58.5
50.6
6.54
6.66
12.4
107
79.2
9.19
5.94
20
20
30.4
38.2
4,20
7.81
1.0
10
5
75.4
70.4
64.8
0.5
5.0
20
103
73.2
71.9
52.9
19.6
14.3
24.4
4.79
13.9
14.3
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62.2
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* LODs and LOQs were determined as described in Methods.
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102
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Manuscript received November 20, 2003;
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