Journal of Chromatography B, 933 (2013) 15–23

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Journal of Chromatography B
journal homepage: www.elsevier.com/locate/chromb

Development of a simple method for simultaneous determination of

nine subclasses of non-steroidal anti-inflammatory drugs in milk and
dairy products by ultra-performance liquid chromatography with
tandem mass spectrometry
Tao Peng a,1 , Ai-Ling Zhu b,1 , Yue-Ning Zhou c , Ting Hu b , Zhen-Feng Yue d ,
Dong-Dong Chen a , Guo-Min Wang e , Jian Kang a , Chun-lin Fan a ,
Ying Chen a , Hai-Yang Jiang b,∗
a
Chinese Academy of Inspection and Quarantine, Beijing 100123, China
b
College of Veterinary Medicine of China Agricultural University, Beijing 100193, China
c
College of Chemistry of Beijing Institute of Technology, Beijing 100081, China
d
Shenzhen Entry-exit Inspection and Quarantine Bureau, Shenzhen 518045, China
e
Chongqing Entry-exit Inspection and Quarantine Bureau, Chongqing 400020, China

a r t i c l e i n f o a b s t r a c t

Article history: A multi-residue analysis method for simultaneous determination of nine subclasses of non-
Received 22 February 2013 steroidal anti-inflammatory drugs (NSAIDs) in milk and dairy products by ultra-performance liquid
Accepted 14 June 2013 chromatography–tandem mass spectrometry (UPLC–MS/MS) has been established. The sample was ini-
Available online 21 June 2013
tially extracted and deproteinized with ascorbic acid buffer (0.01 M, pH 3) and acetonitrile–ethyl acetate
mixture, followed by centrifugation and evaporation, then reconstituted with acetonitrile-0.1% formic
Keywords:
acid (1 + 1, v/v). After removal of lipid material by n-hexane, the sample was analyzed by UPLC–MS/MS
UPLC–MS/MS
with electro-spray ionization (ESI) interface in Multiple Reaction Monitoring (MRM) mode. The range of
Multi-residue analysis
NSAIDs
limits of detection (LODs) and limits of quantification (LOQs) were 0.03–0.30 ␮g/kg and 0.10–1.00 ␮g/kg,
Milk respectively. The recoveries in milk, milk powder, yogurt, processed cheese and milk beverage ranged
Dairy products from 61.7% to 117%, and the relative standard deviations (RSDs) were less than 17.9% at three spiked
levels (1, 10 and 100 times of the LOQ). Matrix effects were also investigated and it was determined the
signals of the analytes were suppressed from 9.4% to 76.6% in processed cheese. The proposed method
was also applied to incurred sample analysis. The results proved that this method was suitable for the
simultaneous determination of nine subclasses of NSAIDs residues in milk and dairy products.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
NSAIDs are a group of compounds which are extensively used
as anti-inflammatory, analgesic and antipyretic drugs for humans,
companion animals and farm animals [1]. The major mechanism
of action is the inhibition of the synthesis of prostaglandins by
inhibiting cyclooxygenases [2,3]. However, toxicity and adverse
effects can be potential risks for human beings and animals, such as
gastrointestinal ulceration, aplastic anemia, inhibition of platelet
aggregation, renal toxicity, hepatoxicity, cardiovascular risk and
central nervous system depression [4–8]. Moreover, it has been
reported that long-term exposure can induce kidney tumors in
rats and liver tumors in mice [9]. Therefore, to ensure food safety
∗ Corresponding author. Tel.: +86 10 62732802; fax: +86 10 62731032.
E-mail address: haiyang@cau.edu.cn (H.-Y. Jiang).
1
These authors contributed equally to this work.
and human beings’ health, the maximum residue limits (MRLs)
or tolerance for NSAIDs in products of animal origin have been
established in some countries [10–13]. The MRLs range from 0.1 to
1000 ␮g/kg, dependent on the compound and matrix. Especially,
for milk and dairy products, the regulations of NSAIDs are more
strict. The US Food and Drug Administration (FDA) has established
the tolerance for flunixin in milk (2 ␮g/kg) [11]; the MRLs from
European Union have been set at 50, 15, 40, 50 and 0.1 ␮g/kg for
tolfenamic acid, meloxicam, flunixin, metamizole and diclofenac in
bovine milk, respectively, [12]; Japan has also established MRLs for
flunixin, ketoprofen, meloxicam, tolfenamic acid and ibuprofen in
milk (10–50 ␮g/kg) [13]; and in China, paracetamol is prohibited
in dairy cattle [14]. Although MRL values of some NSAIDs such as
firocoxib and vedaprofen have only been set in animal tissues [12],
their residues also are possible to appear in milk and dairy products.
Because of the presence of NSAIDs in products of animal ori-
gin, simple and reliable methods for the determination of NSAIDs
are required to ensure food safety. Some methods including

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http://dx.doi.org/10.1016/j.jchromb.2013.06.025
16 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23
high performance liquid chromatography (HPLC) [15–20], liq-
uid chromatography mass spectrometry (LC–MS) [21–29], gas
chromatography mass spectrometry (GC–MS) [30–32], capillary
electrophoresis [33–35], and biosensors [36] were applied to
NSAIDs analysis. Compared with other instruments, LC–MS or
LC–MS/MS has obvious advantages such as good separation, high
sensitivity and selectivity, and providing structure information of
analytes. Furthermore, Multiple Reaction Monitoring (MRM) mode
makes it suitable for multi-residue analysis for veterinary drugs,
while maintaining high selectivity and sensitivity at low concentra-
tion levels. Recently, there have been a number of LC–MS methods
developed for NSAIDs analysis in plasma [27,28,37], urine [38–40],
tissues [24–26,41,42]. And research on the determination of NSAIDs
in milk has also been published [22,28,29,43–47]. Daeseleire et al.
[22] developed and validated a method for the detection of flunixin,
its 5-hydroxy metabolite and ketoprofen in raw milk by LC–MS/MS.
Gallo et al. [43] proposed a method for determination of sixteen
NSAIDs in milk by LC–MSn . Dowling et al. [44] established a method
for determination of eight NSAIDs in milk based on rapid resolu-
tion LC–MS/MS. They also published a method for determination of
authorized drugs including 4-formyl amino antipyrine, one of the
metamizole metabolites [46], and performed some improvements
to the method in 2010 [28]. Then, Dubreil-Chéneau et al. [29] and
Gentili et al. [47] reported that twelve and fifteen NSAIDs in milk
can be determined by LC–MS/MS, respectively.
As is well known, NSAIDs can be divided into nine subclasses
according to their inhibitory selectivity and structures: COX non-
selective inhibitors (aniline derivatives, salicylic acid derivatives,
pyrazole derivatives, nicotinic acid derivatives, anthranilic acid
derivatives, propionic acid derivatives, acetic acid derivatives,
enolic acid derivatives) and COX-2 selective inhibitors (coxibs).
However, these reported multi-residue analysis methods were
mainly focused on the COX non-selective inhibitors, other sub-
classes are not covered. As far as we are aware, only one published
method covered both COX non-selective inhibitors and COX-2
selective inhibitors, which was based on LC coupled with QTrap
mass spectrometry [48], but still does not include the aniline
derivatives nor salicylic acid derivatives. Additionally, the entire
matrix in these published methods was only milk, and no dairy
products such as milk powder, cheese, yogurt and milk beverage
were reported. Besides, Campanella [36] described a rapid method
for determination of four NSAIDs in milk and fresh cheese by a
biosensor method based on the inhibition of the COX enzymes.
Based on the aforementioned research, it is necessary to establish a
simple multi-residue analysis method for NSAIDs in milk and dairy
products.
This paper describes the development of a fast, simple and
reliable method based on UPLC–MS/MS to determine and con-
firm twenty NSAIDs, including both groups of COX non-selective
inhibitors and COX-2 selective inhibitors, in milk and dairy prod-
ucts (processed cheese, milk powder, yogurt and milk beverage). All
selected representative analytes in this study covered all nine sub-
classes of NSAIDs which could possibly remain in milk and dairy
products. The objectives of this work were to develop a simple
extraction procedure to overcome the difficulty of extracting mul-
tiple NSAIDs covering all nine subclasses with a wide range of pKa
values, to remove the lipid material, and to apply this method to
incurred sample anlysis to confirm its effectiveness.
2. Experimental
2.1. Chemicals and materials
Paracetamol (PRT), 4-formyl amino antipyrine (4-
FAA), Phenacetin (PNT), Ibuprofen (IPF), Naproxen (NPX),
Ketoprofen (KPF), Flunixin (FLX), Firocoxib (FRC), Meloxicam
(MLX), Diclofenac (DCF), Phenylbutazone (PBZ), Mefenamic acid
(MFN), Vedaprofen (VPF), Tolfenamic acid (TLF), Salicylic acid (SA),
Carprofen (CPF), Piroxicam (PRX), Celebrex (CLC), Meclofenamic
acid (MLF), Rofecoxib (RFC), purity greater than or equal to 99%,
were purchased from Dr. Ehrenstorfer GmbH, Germany. Formic
acid and ammonium acetate of HPLC grade were purchased from
Acros Organics, Belgium. HPLC grade acetonitrile, ethyl acetate,
n-hexane were purchased from J.T. Baker, Germany. Analytical
grade L-ascorbic acid and sodium chloride were from Amresco,
USA and Beihuajingxi Corp., China, respectively. Ultrapure water
(resistivity, 18.2 M) was purified on a Milli-Q Plus apparatus,
Millipore, Billerica, MA, USA.
N-hexane saturated with acetonitrile, was prepared by adding
10 mL acetonitrile into 50 mL n-hexane, shaken by hand for
1–2 min, left to stand and separated by gravity. Aliquots from the
separated upper layer were used for removing lipid material via
liquid–liquid extraction without discarding the acetonitrile.
2.2. Standard solution
Stock solutions containing 100.0 ␮g mL−1 of each individual
chemical were prepared in acetonitrile. A 100 ␮L aliquot of each
stock solution was transferred into a 10.0 mL volumetric flask to
prepare a mixed working standard solution (1.0 ␮g mL−1 ).
2.3. Instrumentation
The analysis was performed using an AcquityTM Ultra per-
formance LC system connected to a Waters Acquity TQ triple
quadrupole mass spectrometer detector with an electrospray ion-
ization (ESI) interface, controlled by Masslynx 4.1 Analyst software.
Chromatographic separation was achieved using an AcquityTM
UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 ␮m). All instruments
mentioned above were purchased from Waters Corp., USA. The
mobile phase was acetonitrile (phase A) and 0.1% formic acid in
water containing 0.5 mmol/L ammonium acetate (phase B). The
gradient conditions were as follows: from 0 to 3 min ramp linearly
10–30% of phase A, hold for 2 min; increased to 50% of phase A at
6 min, hold for 4 min; continuously increased to 90% of phase A at
12 min, hold for 2 min; then reduced to 10% of phase A from 14 to
15 min; and hold for 1 min to re-equilibrate the system with the
initial composition of mobile phase. The flow rate was 0.2 mL/min,
the column temperature was 40 ◦ C, and the injection volume was
10 ␮L.
Acquisition was carried out using ESI in both positive and neg-
ative mode in MRM mode. The MRM parameters (2 transitions)
were summarized in Table 1. Each transition was performed with
a 0.1 s dwell time and a delay time of 0.05 s to achieve more
than 8 points across the peaks. Interface conditions were: capillary
voltage: 3.0 kV in ESI+ mode and 2.5 kV in ESI− mode, source tem-
perature: 120 ◦ C, desolvation temperature: 350 ◦ C, RF lens: 0.1 V,
multiplier: −660 in ESI+ mode and −626 in ESI− mode, cone gas:
nitrogen with flow rate of 100 L/h, desolvation gas was nitrogen
with flow rate of 600 L/h, collision gas: argon with pressure of
2.40 × 10−6 Pa.
2.4. Sample preparation
Milk and milk beverage samples (2.0 mL) were directly mea-
sured. Milk powder, yogurt and processed cheese (2.0 g) were
weighed and then 2 ml of water was added. The samples were vor-
tex vigorously mixed for 30 s. The cheese samples were melted in
an 80 ◦ C water bath and remained there until extraction.
The samples were transferred into a 50 mL centrifuge tube
and 2 mL of L-ascorbic acid buffer (AA buffer, 0.01 M, pH 3) was
 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23 17

Table 1
MRM parameters and retention time of NSAIDs.

No. Analyte Ionization Precursor Product Cone Collision Retention Subclass

mode (ESI) ions (m/z) ions (m/z) voltage (V) energy (eV) time (min)

1 Paracetamol PRT M+H 152.1 93 38 21 1.47 Aniline derivatives

110.1a 38 14
2 Phenacetin PNT M+H 180.2 110.1a 38 20 3.78
138.1 38 15
3 4-formylamino antipyrine 4-FAA M+H 232.2 104.1a 35 23 1.89 Pyrazole derivatives
83 35 24
4 Phenylbutazone PBZ M+H 309.3 160.2a 40 23 9.58
188.2 40 16
5 Salicylic acid SA M−H 137 65 32 26 3.84 Salicylic acid derivatives
93.0a 32 15
6 Piroxicam PRX M+H 332.2 95.0a 32 19 5.58 Enolic acid derivatives
121 32 25
7 Meloxicam MLX M+H 352.2 115.1a 30 20 7.22
141.1 30 20
8 Ibuprofen IPF M+H 207.2 161.1 30 10 6.50 Propionic acid derivatives
M+NH4 224.2 161.2a 21 15
9 Ketoprofen KPF M+H 255.2 105.1a 35 25 7.17
209.2 35 14
10 Naproxen NPX M+H 231.2 170.1 30 25 7.20
185.2a 30 15
11 Carprofen CPF M−H 272.2 228.1a 26 14 8.18
274.2 230.1 30 14
12 Vedaprofen VPF M+H 283.3 155.1a 34 12 13.11
201.1 34 6
13 Flunixin FLX M+H 297.2 264.2 45 34 7.17 Nicotinic acid derivatives
279.2a 45 23
14 Diclofenac DCF M+H 296.1 215.1a 30 20 8.83 Acetic acid derivatives
250.1 30 13
15 Mefenamic acid MFN M+H 242.2 209.2 30 28 10.63 Anthranilic acid derivatives
224.2a 30 15
16 Meclofenamic acid MLF M+H 296.2 243.3a 25 22 10.77
278.2 25 14
17 Tolfenamic acid TLF M+H 261.9 208.8 18 19 11.47
243.9a 18 7
18 Rofecoxib RFC M+H 315.2 269.2a 40 20 6.58 Coxibs
297.2 40 14
19 Firocoxib FRC M+H 337.2 283.2a 39 26 6.95
237.1 39 25
20 Celebrex CLC M+H 382.2 75 60 60 9.88
362.3a 60 28
a
Quantitative ion.

added. The mixture was vigorously vortex mixed for 30 s. Then,
10 mL acetonitrile–ethyl acetate (1 + 1, v/v) and 1 mL sodium chlo-
ride solution (1%, w/w) were added. The sample was vigorously
vortex mixed for 1 min, followed by ultrasonic treatment in a
water bath for 10 min and centrifuged at 10,000 rpm for 5 min
at 4 ◦ C. The supernatant was transferred into a 50-mL tube and
the process above was repeated. The supernatants were combined
and evaporated to dryness under a gentle stream of nitrogen at
40 ◦ C. The residue was reconstituted with 1 mL acetonitrile-0.1%
formic acid (1 + 1, v/v), then 3 mL n-hexane saturated with ace-
tonitrile was added to remove the lipid material. After mixing for
30 s, the sample was allowed to stand for 2 min, the supernatant
was discarded and the lower layer was centrifuged at 10,000 rpm
for 3 min. The final extract was filtered through a 0.2 ␮m nylon
membrane filter and transferred into a 2 mL-vial, and analyzed
by UPLC–MS/MS.
2.5. Method validation
The method validation was performed in accordance with Com-
mission Decision 2002/657/EC [49] in terms of specificity, linearity,
accuracy, precision and analytical limits. To investigate the speci-
ficity of the method, two MRM transitions were monitored for each
of the twenty analytes. Ion ratios, retention times, and interfering
peaks obtained from matrix-matched standard solutions, different
negative samples, and spiked samples were examined.
Matrix-matched calibration curves were constructed for quan-
tification on each validation day. Standard solutions diluted with
negative blank sample extract, as prepared in Section 2.4, were pre-
pared to create five concentration levels in the range of 0–100 ␮g/kg
of each compound. The calibration curves were created using 1/x
weighted linear regression (origin excluded).
For estimation of accuracy, negative samples were spiked at
three concentration levels (1, 10, and 100 times of the LOQ of each
compound). Seven replicates of each matrix were analyzed at each
spiked level, the relative standard deviation (RSD) was used to eval-
uated the precision of method.
3. Results and discussion
3.1. LC–MS/MS determination
According to our previous study [24,25] and the methods pub-
lished [28,44], almost all NSAIDs can be detected by LC–ESI-MS
in the positive mode or negative mode. To confirm every ana-
lyte, one precursor ion and two daughter ions were selected to
monitor, which met or exceeded the requirement of four iden-
tification points as specified in 2002/657/EC [49]. The optimum
parameters of ionization mode, capillary voltage, cone voltage and
collision energy were determined for each analyte and displayed
in Table 1. And MRM was divided into ten scan functions to ensure
more than eight points across the chromatographic peak in both
18 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23
Fig. 1. Daughter scan of carprofen.
ionization modes at the same scanning speed. Unlike what has been
reported by other researchers [28,44], in our study, good responses
for most of the NSAIDs belonging to the group of COX non-selective
inhibitors were obtained in positive mode. Only SA and CPF ionized
well enough in the negative mode to achieve acceptable sensitivity.
However, the fragmentation of CPF only generated one daugh-
ter ion (m/z 272.2 > 228.1). The presence of chlorine isotopes in
CPF made it possible for another precursor ion with the same
fragmentation pattern [M−H−COO]− to qualify as a transition to
achieve the number of requisite identification points (Fig. 1). Fur-
thermore, two protonated species [M+H]+ and [M+NH4 ]+ for IPF
were observed in positive mode. Both of the two precursor ions of
IPF had high responses, but only one daughter ion was obtained for
each, presumably because IPF could easily fragment into smaller or
unstable ions while the collision energy was above the optimized
voltage when fragment [M+H−COO]+ or [M+NH4 −COO]+ produced.
Therefore, the two suitable transitions of m/z 207.2 > 161.1 and m/z
224.2 > 161.2 had been selected for IPF to satisfy the purposes of
confirmation (Fig. 2). The latter transition was used for quantitative
purpose for its stronger and more stable response.
Fig. 2. MS scan and daughter scan of ibuprofen.
Chromatographic separation was performed on an AcquityTM
UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 ␮m). The different pKa
values ranging from 3 to 9.5 presented the main difficulty in separa-
tion of the selected NSAIDs. Acetonitrile (phase A) and 0.1% formic
acid in water containing 0.5 mmol/L ammonium acetate (phase B)
described in our previous studies [24–26] was finally chosen as the
mobile phase. The gradients and scan time were optimized, and
then the symmetric and sharp peaks were obtained. Typical MRM
chromatograms of spiked samples are shown in Fig. 3.
3.2. Sample preparation
Some variables in extraction procedure needed to be optimized
to achieve the best condition of sample preparation, including
extraction solutions, clean up step, and the composition of recon-
stitution solutions.
3.2.1. Optimization of the extraction procedure
Dairy products (milk powder, yogurt, processed cheese, milk
beverage) were complex matrices containing a high proportion of
fat and protein. According to references, acetonitrile [28,42,44,47]
or methanol [29,43] was always used to extract NSAIDs from milk.
And in our previous study [24,25], higher recoveries were obtained
by using acetonitrile–phosphoric acid (80 + 1, v/v) because of the
good solubility of NSAIDs and deproteinization. Acidic acetonitrile
was effective for stronger polar drugs, but not good for weaker polar
or the non-polar drugs in milk. Ethyl acetate is partially immiscible
with water, so non-polar drugs could more readily transfer into
the ethyl acetate phase. An acetonitrile/ethyl acetate mixture was
then compared to the acidic extraction. Eventually, the acetoni-
trile/ethyl acetate solution improved the recoveries and sensitivity
of some NSAIDs. Although some methods reported that adding
antioxidant agents such as ascorbic acid (AA) can avoid oxida-
tion of PBZ during analysis [28,43,44], some researchers indicated
that adding AA (0.1 M) prior to the extraction led to a loss of
 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23 19

Fig. 3. UPLC–MS/MS chromatograms of processed cheese samples spiked with twenty NSAIDs at LOQ levels.

sensitivity and recovery for some analytes (KPF, VPF, FLX and NPX)
and did not significantly improve the recoveries [29,47]. This lim-
itation was overcome by using low concentration of AA solution
(0.01 M, pH 3) during extraction procedure. Finally, a comparison
between acidic acetonitrile, ethyl acetate–acetonitrile (1 + 1, v/v),
and the two extraction solutions followed by adding AA buffer
(0.01 M, pH 3) was performed. Acetonitrile–ethyl acetate (1 + 1,
v/v) with the addition of AA buffer (0.01 M, pH 3) was found to
be better than the other three extractions. Results for the extrac-
tion of processed cheese are given in Fig. 4, and similar results
were obtained in other matrices. Meanwhile, the best pH value for
the extraction was optimized by comparing the recoveries at the
various pH values, and pH 3 was found to be suitable for the extrac-
tion of most analytes. The acidic extraction solvent improved the
20 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23
Fig. 4. Comparison of extraction recoveries with different extraction solution in processed cheese.
protein precipitation and protonation of NSAIDs with increasing
pKa value.
3.2.2. Selection of the cleanup step and reconstitution solutions
Liquid-liquid partition and solid phase extraction were the
common methods for cleanup, which can remove lipid mate-
rial co-extracted by organic solvents and reduce matrix effect. In
the published multi-residue analysis methods of NSAIDs in milk,
some different SPE cartridges, such as C18 [43], HLB [47], Evo-
lute ABN [28,44], were widely used to purify the extract. But
Dubreil-Chéneau et al. [29] observed high variation of results with
purification by SPE cartridges, and they used methanol for single
extraction. However, it was necessary to remove the lipid mate-
rial to ensure the sensitivity, recovery and protect the LC column.
N-hexane was a non-polar solvent forming distinct partitioning
phases with acetonitrile and ethyl acetate, and could be a con-
venient cleanup step to remove co-extracted lipid material [43].
Since HLB and MCX cartridges have already been successfully used
to enrich NSAIDs from tissues [24,25], a comparison of HLB, MCX
cartridges and n-hexane was performed in the study, and similar
results were obtained in all cases. Considering efficiency, consum-
ing time, and cost, liquid-liquid partition was selected.
Acetonitrile and 0.1% formic acid described in our previous study
[25,26] were selected as reconstitution solution components for
NSAIDs. The efficiency of different reconstitution solutions that
contained 10, 50 and 90% organic, respectively, was investigated.
The reconstitution solution containing 10% organic resulted in low
intensity of some analytes such as FLX, PBZ, VPF, MLF, and MFN,
possibly because of low solubility of these compounds. And for the
90% organic solution, leading peaks for some analytes were found.
However, the solution containing 50% organic yielded the high-
est intensity and characteristic shaper, symmetric peaks without
retention time shift. Hence, acetonitrile-0.1% formic acid solution
(1 + 1, v/v) was selected for reconstitution. Comparison of recover-
ies in processed cheese with the different reconstitution solutions
are presented in Fig. 5, and the results of other matrices are similar
to the cheese.
3.3. Method validation
3.3.1. Specificity
In the specificity study, matrix-matched standard solutions,
spiked samples and negative samples were analyzed. Specificity
was found to be satisfactory, with no chromatographic interfer-
ences observed at the retention time of the target compounds.
Typical MRM chromatograms of spiked cheese samples are shown
in Fig. 3. The criteria for retention times and ion ratios in matrix-
matched standard solutions and spiked samples were examined.
The retention time of the peaks in samples compared with matrix-
matched standard solution was within the tolerance of 2.5%
(Table 1). Furthermore, two transition ions were monitored for each
of the twenty analytes. The more intense ion was used for quantifi-
cation. Compared with matrix-matched standard solution, all ion
ratios of samples spiked at three levels of 1, 10 and 100 times of the
LOQ of each analyte met the required tolerances outlined in Com-
mission Decision 2002/657/EC. The results of ion ratios in processed
cheese and matrix-matched standard solutions at three levels are
shown in Table 2.
3.3.2. Matrix effect
In order to investigate matrix effect, twenty negative samples
for each matrix were prepared as Section 2.4 described, then spiked
with standard solution and analyzed. The results obtained revealed
reduction of the peak areas of some analytes compared with the
peak areas in the standard solution, due to the signal suppression
from matrix. Compared with other dairy products, the signal sup-
pression in cheese was the most severe, and is shown in Table 3.
The signal suppression of TLF and SA were 76.6% and 52.8%, with the
RSDs of 8.5% and 10.2%, respectively. Eventually, matrix-matched
standard curves were used to compensate for the matrix effect and
allow for reliable quantitative analysis.
3.3.3. Linearity
The matrix-matched calibration curves were created by diluting
standard solutions with negative blank sample extract, as pre-
pared in Section 2.4, containing five concentration levels of each
compound in linearity range shown in Table 2. The calibration
curves were created using 1/x weighted linear regression (origin
excluded). Correlation coefficients (r) were all better than 0.99.
3.3.4. Accuracy, precision and repeatability
Acceptable recovery and repeatability, expressed as RSD, were
obtained using negative samples spiked at 1, 10 and 100 times
of the LOQ level. Within the range of linearity, the mean recov-
eries at three spiked levels varied from 69.6% to 108% with RSDs
of 3.2–15.8% in bovine milk, from 67.3% to 117% with RSDs of
4.3–15.6% in milk powder, from 62.1% to 109% with RSDs 2.2–17.9%
in yogurt, from 61.7% to 109% with RSDs 3.9–17.4% in milk beverage,
and from 64.8% to 109% with RSDs 3.3–14.2% in cheese, respec-
tively. The detail results are shown in Table 2 and within required
tolerance [50].
Table 2
Mean recoveries (M.R, n = 7), repeatability (RSD), mean ion ratios of analytes in matrix matched standard solution and processed cheese, linearity range, LOD and LOQ of NSAIDs in dairy products.
Analyte Spiked (␮g/kg) Milk Milk powder Yogurt Milk beverage Processed cheese Linearity LOD (␮g/kg) LOQ (␮g/kg)
range (␮g/kg)

M.R (%) RSD (%) M.R (%) RSD (%) M.R (%) RSD (%) M.R (%) RSD (%) M.R (%) RSD (%) Ion ratio a Ion ratio b
PRT 0.1 92.4 8.9 82.1 7.1 65.6 2.2 84.2 9.9 84.2 5.6 0.05 0.04 0.1–20.0 0.03 0.1
1.0 85.9 6.7 88.7 8.1 77.1 3.4 89.9 7.1 89.9 7.2 0.05 0.06
10.0 86.3 5.3 90.3 6.8 83.1 9.8 90.8 6.7 82.8 8.8 0.04 0.05
4-FAA 0.1 104 5.9 78.3 10.3 72.4 8.3 80.8 9.6 72.9 3.3 0.83 0.86 0.1–20.0 0.03 0.1
1.0 76.9 10.3 82.1 11.7 86.8 5.3 83.6 10.2 66.7 10.4 0.90 0.80
10.0 85.2 7.3 80.8 6.6 88.7 6.5 82.1 9.5 96.1 5.5 0.80 0.82
PNT 0.1 69.6 7.9 97.8 5.4 109 5.9 61.7 8.7 97.5 6.7 0.05 0.05 0.1–20.0 0.03 0.1
1.0 78.9 11.2 87.7 6.5 89.4 4.8 76.3 8.6 79.3 8.6 0.06 0.06
10.0 92.4 9.2 83.9 6.9 104 10.1 84.3 7.6 84.2 11.2 0.05 0.05
SA 0.2 76.1 3.2 95.6 9.5 84.2 8.7 87.9 9.6 90.2 13.7 0.03 0.03 0.2–50.0 0.06 0.2
2.0 95.3 6.8 92.9 7.2 84.3 9.1 72.9 8.6 79.8 6.8 0.04 0.05
20.0 98.4 7.5 90.8 15.6 97.5 11.2 83.3 14.1 88.6 8.7 0.03 0.04
PRX 0.1 73.3 8.2 109 9.8 84.1 7.6 96.3 9.3 85.3 8.6 0.30 0.31 0.1–20.0 0.03 0.1
1.0 96.9 6.7 97.6 5.7 87.5 7.5 88.5 8.8 84.5 7.5 0.26 0.31
10.0 84.5 6.6 92.3 5.3 88.6 9.2 91.7 7.8 96.2 7.3 0.34 0.32
IPF 0.3 96.2 5.6 79.6 7.5 69.3 6.3 95.2 5.2 79.4 5.6 0.43 0.42 0.3–50.0 0.10 0.3
3.0 98.3 7.2 88.9 8.6 78.7 4.3 101 7.5 79.3 7.2 0.50 0.54
30.0 88.9 5.8 82.2 9.4 105 14.2 99.5 8.3 93.2 8.1 0.39 0.39
RFC 0.3 79.8 10.3 83.7 5.2 85.2 3.9 90.8 11.2 83.4 5.8 0.67 0.65 0.3–50.0 0.10 0.3
3.0 93.3 7.9 97.7 8.5 74.3 7.4 96.7 6.7 78.6 7.6 0.73 0.74

T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23

30.0 101 8.2 106 6.8 95.7 5.8 102 8.9 91.3 11.3 0.67 0.64
FRC 1.0 86.3 7.5 67.3 4.7 83.8 6.1 69.4 9.8 97.2 8.7 0.65 0.69 1.0–100.0 0.30 1.0
10.0 86.8 4.6 80.9 10.3 83.4 12.9 83.2 9.7 102 9.4 0.60 0.61
100.0 96.3 6.8 81.7 7.4 86.3 8.3 82.6 7.4 94.9 6.6 0.68 0.68
KPF 0.3 99.4 9.9 99.1 9.9 82.7 7.2 109 7.5 86.1 4.2 0.89 0.91 0.3–50.0 0.10 0.3
3.0 101 5.8 107 9.3 98.9 10.4 88.5 6.8 88.4 11.5 0.84 0.84
30.0 94.7 6.1 91.8 7.2 107.6 9.6 89.3 7.8 96.6 7.1 0.85 0.88
FLX 0.1 75.1 8.8 113 11.1 83.4 17.3 93.3 10.2 87.5 5.9 0.26 0.26 0.1–20.0 0.03 0.1
1.0 97.4 7.1 98.7 6.3 85.7 4.8 91.7 8.3 79.9 4.5 0.27 0.30
10.0 98.3 6.6 88.5 13.9 97.1 11.4 90.8 7.8 88.5 6.3 0.28 0.25
MLX 0.5 88.7 6.7 88.1 4.3 89.8 12.3 82.6 6.1 97.1 8.4 0.24 0.24 0.5–100.0 0.15 0.5
5.0 104 11.4 99.3 9.8 106 8.7 109 9.7 78.0 9.8 0.23 0.20
50.0 95.6 6.5 82.3 7.9 109 10.2 83.7 13.6 85.2 5.5 0.20 0.27
NPX 0.2 72.2 7.4 106 8.3 103 6.7 101 8.6 96.7 5.1 0.14 0.12 0.2–50.0 0.06 0.2
2.0 87.0 5.3 85.9 4.8 87.4 6.7 87.7 5.1 86.3 7.4 0.15 0.16
20.0 102 6.2 99.2 4.8 95.2 7.4 91.2 9.9 96.6 10.2 0.14 0.14
CPF 0.5 77.1 7.2 109 5.6 62.1 9.7 104 10.1 82.4 6.4 0.33 0.31 0.5–100.0 0.15 0.5
5.0 87.7 7.9 76.5 6.4 82.6 9.5 95.3 3.9 98.8 8.2 0.35 0.29
50.0 84.4 8.2 87.4 7.5 99.5 7.6 92.1 13.6 95.3 9.5 0.30 0.33
DCF 1.0 81.9 4.6 87.9 6.2 80.2 8.9 90.2 11.2 95.5 6.8 0.91 0.93 1.0–100.0 0.30 1.0
10.0 81.1 5.7 85.8 7.8 95.2 9.3 85.2 8.3 97.6 5.3 0.87 0.85
100.0 108 7.9 83.3 6.1 85.7 10.6 84.9 17.4 108 9.5 0.92 0.89
PBZ 1.0 99.6 3.7 104 4.9 99.1 12.3 103 8.0 64.8 4.7 0.71 0.67 1.0–100.0 0.30 1.0
10.0 99.7 6.7 95.1 7.1 81.9 8.6 96.8 11.2 85.5 12.1 0.66 0.72
100.0 106 4.2 96.8 9.8 84.4 8.6 94.3 7.8 97.1 8.7 0.73 0.73
CLC 1.0 86.6 4.6 99.6 6.7 79.1 8.2 77.4 8.1 96.2 14.2 0.63 0.59 1.0–100.0 0.30 1.0
10.0 98.7 7.3 103 9.6 89.7 7.4 104 7.7 106 8.8 0.65 0.63
100.0 97.9 13.7 97.4 14.9 103 6.7 81.7 15.6 109 6.4 0.65 0.66
MFN 1.0 70.4 9.8 98.9 10.9 96.1 5.6 99.4 9.4 93.5 9.2 0.49 0.43 1.0–100.0 0.30 1.0
10.0 108 6.5 106 5.1 99.2 8.9 102 7.8 94.2 4.9 0.46 0.50
100.0 97.1 8.8 92.3 6.4 83.8 7.7 100 8.2 85.5 7.6 0.53 0.52
MLF 0.3 94.5 7.8 117 8.8 99.5 10.3 63.2 11.2 98.1 5.8 0.87 0.88 0.3–50.0 0.10 0.3
3.0 99.5 5.6 99.5 5.6 98.2 8.2 96.5 10.3 86.9 6.1 0.93 0.84
30.0 97.1 7.8 90.7 4.3 88.5 6.2 98.4 6.6 89.8 7.8 0.91 0.91
TLF 1.0 98.3 5.9 91.5 6.4 102 7.8 99.1 8.8 95.6 7.4 0.50 0.46 1.0–100.0 0.30 1.0
10.0 99.1 6.9 98.9 6.9 96.4 11.2 90.8 5.4 97.4 6.3 0.50 0.49
100.0 91.2 15.8 94.5 5.6 100 9.9 109 9.6 99.3 8.7 0.52 0.54
VPF 1.0 89.6 7.3 97.4 6.3 92.2 9.2 95.8 7.4 93.7 9.7 0.60 0.53 1.0–100.0 0.30 1.0
10.0 84.3 8.2 87.7 12.7 84.3 17.9 89.7 12.9 84.3 9.2 0.56 0.62
100.0 103 12.9 86.7 9.4 89.7 10.1 91.2 6.7 94.5 11.2 0.60 0.59
n, the number of samples per matrix; M.R.: mean recoveries.
a
Ion ratios of analytes in matrix-matched standard solution.
b
ion ratios of analytes in spiked processed cheese samples.

21
22 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23

Fig. 5. Comparison of recoveries with diffident reconstitution solutions in processed cheese.

Table 3
Matrix effect (as % signal suppression) of NSAIDs in processed cheese (n = 20).

Compound Matrix effect (%) RSD (%) Compound Matrix effect (%) RSD (%)

PRT 28.8 6.4 KPF 40.1 11.9

4-MAA 14.2 11.6 PRX 49.1 7.3
PNT 37.6 8.2 FLX 21.0 12.6
IPF 9.4 12.8 FRC 29.9 11.7
RFC 9.9 9.3 MLX 37.9 7.5
NPX 24.6 12.8 DCF 23.7 7.7
MFN 13.9 8.1 PBZ 21.8 11.7
VPF 5.4 12.9 CLC 31.0 5.6
TLF 76.6 8.5 CPF 37.0 11.1
SA 52.8 10.2 MLF 45.2 10.3

3.3.5. Limit of detection (LOD) and limit of quantitation (LOQ)
To test LODs and LOQs of the method, twenty replicated negative
samples per matrix were spiked, prepared according to Section 2.4,
and analyzed. The signal to noise ratio (S/N) at the time window the
analyte appeared was calculated. The LODs were the concentrations
while responses of analytes were better than three times of S/N, and
the LOQs were ten times of S/N. LODs and LOQs of this method were
0.03–0.30 ␮g/kg, and 0.10–1.00 ␮g/kg, respectively. The results are
shown in Table 2 and the UPLC–MS/MS chromatograms are shown
in Fig. 3.
3.4. Method application
The effectiveness of the method in measuring trace levels of
NSAIDs was verified by analyzing thirty-nine dairy products pro-
vided by local Inspection and Quarantine Bureaus (ten pieces of
processed cheese, milk powder and yogurt, respectively, and nine
pieces of milk beverage). Three replicates of each sample were ana-
lyzed. Two samples of cheese were found to contain trace residues
of FLX, NPX, PRX and RFC. Typical MRM chromatograms of posi-
tive cheese samples are shown in Fig. 6. The results indicated that
the method was suitable for NSAIDs monitoring in milk and dairy
products.

4. Conclusions

A fast, simple and reliable liquid–liquid partition UPLC–MS/MS

method for the determination of nine subclasses of NSAIDs residues
in milk and dairy products has been developed. The sample treat-
ment is simple and the extraction efficiency is good. The LODs and
LOQs ranged from 0.03 ␮g/kg to 0.30 ␮g/kg and from 0.10 ␮g/kg
to 1.00 ␮g/kg, respectively. To the best of our knowledge, there is
no published method suitable for the simultaneous determination
and confirmation of nine subclasses of NSAIDs residue in milk and
dairy products (cheese, milk powder, yogurt and milk beverage).
Foremost, the proposed method is satisfactory to the requirement
of residue analysis of NSAIDs and provides an analytical tool for the
determination and confirmation of all nine subclasses of NSAIDs in
milk and dairy products.

Acknowledgements

This study was supported financially by grants from the

Ministry of Science and Technology of China (2012BAD33B02,
Fig. 6. UPLC–MS/MS chromatograms of positive processed cheese samples. 2012BAK08B01, 2011BAK10B01), and the General Administration
 T. Peng et al. / J. Chromatogr. B 933 (2013) 15–23
of Quality Supervision, Inspection and Quarantine of the People’s
Republic of China (201210016, 201210086, 2009IK314).
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