NON-STEROIDAL ANTI-INFLAMMATORY DRUGS ANALYSIS IN MILK BY QUECHERS AND LC-MS: LOW AND HIGH
RESOLUTION DETECTION AND CONFIRMATION APPROACHES A. Rúbies1, L. Guo2, I. Beguiristain1, F. Centrich1, M. Granados2
1. Laboratori Agència de Salut Pública de Barcelona, 2. Departament de Química Analítica - Universitat de Barcelona.
*
Non-steroidal anti-inflammatory drugs (NSAIDs) are used as anti-inflammatory, analgesic and
antipyretic drugs in medicine and veterinary. Their action mechanism is based on the blocking of
the biosynthesis of prostaglandins. NSAIDs are highly effective and extensively used, but they have
some adverse side effects, such as hepatotoxicity, renal disorders or allergic reactions.
In the European Union, to assure food safety and protect consumers, maximum residue limits have
been established for some authorised NSAIDs in food products. Therefore, high throughput and
reliable analytical methodology is required for the effective control of NSAIDs in food from animal
origin. Liquid chromatography (LC) coupled to mass spectrometry (MS) is currently the technique
of choice in confirmatory analysis of NSAIDs residues. We present a new method for the
determination of representative NSAIDs in milk based on QuEChERS methodology, LC-MS/MS and
UHPLC-HRMS.
HPLC – MS/MS (QqQ)
NON-STEROIDAL ANTI-INFLAMATORY DRUGS (NSAIDs)
NSAIDs IN MILK: EXTRACTION AND CLEAN-UP
MRM chromatogram of a spiked sample at 1.5 µg kg-1 Validation results (HPLC-QqQ)
INTRODUCTION
NSAID MRL
(µg kg-1)
5-hydroxyflunixin 40
Flunixin 40
Diclofenac 0.1
Tolfenamic acid 50
Meloxicam 15
EU Maximum Residue Limits (MRLs)
for NSAIDs in milk.
NSAID Concentration
(µg kg-1)
Phenylbutazone
Oxyphenbutazone
Diclofenac
5
Ibuprofen
Naproxen
Mefenamic acid
10
Recommended NSAIDs concentrations
to be monitored according to
Community Reference Laboratory
PROPIONIC ACID
DERIVATIVES
ACETIC ACID
DERIVATIVES
OXICAMS ANTHRANILIC ACID DERIVATIVES
Ketoprofen
Flufenamic acid
Meclofenamic acid
Niflumic acid
NICOTINIC ACID DERIVATIVES
5-Hydroxyflunixin
Flunixin
Diclofenac Meloxicam
PYRAZOLONES
Phenylbutazone
Oxyphenylbutazone
The method is straightforward, reliable,
and well suited for high throughput
confirmatory analysis of NSAIDS in milk
samples
Addition of ascorbic acid
Milk + Na2SO4 + ammonium acetate
Clean-up by d-SPE C18
Addition of Na2SO4
Extraction with 5% acetic acid in ACN
Evaporation and filtration
QuEChERS PROPOSED METHOD
CONCLUSIONS
NSAIDs have quite different chemical structures and different functional groups.
Optimisation of sample treatment for a NSAIDs multiresidue method is a complex issue.
UHPLC conditions:
Column: Kinetex, Phenomenex (2.1x100)mm, 1.7 µm
Column temperature: 40ºC
Mobile phases: A: water containing 0.1% formic acid
B: ACN
Gradient: 70/30 (0’), 45/55 (10’). Flow rate: 300 µL min-1
Injection volume: 5 µL
Quantification: Matrix matched surrogate standards method
Internal standards: Meloxicam-D3, Phenylbutazone-13C12,
Niflumic acid-13C6, Flufenamic-13C6.
Working mode: PRM with intermediate Collision Energy
Ionisation: HESI+ and HESI-
Milk samples were spiked in decreasing concentracions
and extracted to check performance of the instrument:
1 µg·kg-1, 0.7 µg·kg-1, 0.5 µg·kg-1, 0.3 µg·kg-1, 0.1 µg·kg-1
Precursor ions were detected at all concentrations.
UHPLC-HRMS/MS (Q-Orbitrap)
RT: 0.00 - 12.01
0 1 2 3 4 5 6 7 8 9 10 11 12
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0
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RT: 4.37AA: 261978BP: 311.0650
RT: 5.12AA: 485442BP: 255.1013
RT: 6.33AA: 18071BP: 255.1015
RT: 5.37AA: 394403BP: 323.1403
RT: 5.55AA: 2643895BP: 295.0696
RT: 5.54AA: 2871771BP: 352.0417
NL: 5.27E4
m/z= 311.0634-311.0666 F: FTMS - p ESI Full ms2 [email protected] [50.00-650.00] MS ICIS 15102038
NL: 8.62E4
m/z= 255.0999-255.1025 F: FTMS + p ESI Full msx ms2 [email protected] [email protected] [50.00-740.00] MS ICIS 15102038
NL: 8.18E4
m/z= 323.1386-323.1418 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-740.00] MS ICIS 15102038
NL: 3.79E5
m/z= 295.0685-295.0715 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-740.00] MS ICIS 15102038
NL: 4.47E5
m/z= 352.0402-352.0438 F: FTMS + p ESI Full msx ms2 [email protected] [email protected] [50.00-740.00] MS ICIS 15102038
5-OH-flunixin
Ketoprofen
Oxyfenbutazone
Flunixin
Meloxicam
RT: 0.00 - 12.01
0 1 2 3 4 5 6 7 8 9 10 11 12
Time (min)
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RT: 8.36AA: 2843522BP: 281.0543
RT: 6.66AA: 8255BP: 281.0544
RT: 8.81AA: 5534BP: 281.0545
RT: 8.54AA: 22981BP: 294.0085
RT: 9.44AA: 560494BP: 307.1453
RT: 9.82AA: 47178BP: 307.1453
RT: 10.64AA: 598493BP: 280.0590
RT: 10.79AA: 143350BP: 294.0095
NL: 3.51E5
m/z= 281.0529-281.0557 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-620.00] MS ICIS 15102038
NL: 8.83E3
m/z= 294.0081-294.0111 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-620.00] MS ICIS 15102038
NL: 5.66E4
m/z= 307.1437-307.1467 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102038
NL: 7.75E4
m/z= 280.0577-280.0605 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102038
NL: 2.09E4
m/z= 294.0081-294.0111 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102038
Niflumic acid
Diclofenac
Phenylbutazone
Flufenamic acid
Meclofenamic acid
Analyte Level
(µg·kg-1)
Extraction
Recovery (%)
Precision
(RSD%) Trueness (%)
CCα (µg·kg-1)
CCβ (µg·kg-1)
Flunixin
2.5
5.0
10.0
25.0
92.9
90.2
95.2
89.3
10.3
13.5
7.9
10.8
97.6
94.6
105.1
102.3
1.5
1.9
5-OH-Flunixin
2.5
5.0
10.0
25.0
40.0
89.7
87.1
88.2
88.2
88.3
14.3
9.7
11.5
10.0
1.7
91.2
103.8
85.7
101.1
101.9
No MRL
1.1
1.5
MRL= 40 µg·kg-1
41.2
42.3
Phenylbu-
tazone
2.5
5.0
10.0
25.0
78.1
89.1
95.3
86.1
16.9
12.6
11.8
14.9
98.8
101.8
99.1
88.9
0.4
0.8
Oxyphen-
butazone
2.5
5.0
10.0
25.0
94.7
89.3
91.1
85.6
11.7
13.1
12.2
13.7
103.2
94.6
98.8
100.7
0.9
1.3
Niflumic acid
2.5
5.0
10.0
25.0
93.4
90.6
93.4
93.9
9.2
7.8
10.1
10.1
108.4
107.4
94
100.8
1.0
1.4
Flufenamic
acid
2.5
5.0
10.0
25.0
96.0
90.9
96.5
91.9
10.3
7.9
8.3
9.4
108.8
108
95.5
101.2
0.8
1.2
Diclofenac
2.5
5.0
10.0
25.0
92.3
91.0
97.1
90.9
9.2
8.8
12.8
12.2
92.4
100.0
102.0
90.8
1.5
1.9
Ketoprofen
2.5
5.0
10.0
25.0
81.0
95.6
92.2
87.5
10.4
12.0
10.7
8.6
87.6
97.8
98.8
103.1
0.4
0.8
Meloxicam
2.5
5.0
10.0
15.0
25.0
94.4
92.5
92.0
91.2
90.5
15.2
9.0
7.8
12.2
98.0
102.8
102.0
104.3
No MRL
1.0
1.4
MRL= 15 µg·kg-1
15.8
16.5
Meclofenamic
acid
2.5
5.0
10.0
25.0
91.2
90.4
93.3
95.3
10.8
10.4
10.4
12.2
105.6
102.8
84.1
94.1
0.5
0.9
Chromatogram of an extract from a milk sample (0.5 µg·kg-1 ):
Precursor ions
QuEChERS is a high throughput technique that can be used in the field of veterinary drugs residues analysis for complex matrices such as milk.
Biphenyl-RP is a stationary phase that provides good performance in the chromatographic separation of NSAIDs due to its interaction with the aromatic
rings.
HRMS/MS is a powerful tool which provides excellent selectivity and enables lower limits of quantification and better signal/noise ratios when compared
to MS/MS. Coelutions and subsequent cross-talk problems can be resolved using HRMS/MS.
Successful participation in Fapas 02274 inter-laboratory exercise has been achieved: phenylbutazone was confirmed at 7 µg·kg-1 (Z-score=0)
UHPLC-MS/HRMS instrumentation: Thermo
Accela coupled to a Q-Exactive mass
spectrometer with heated electrospray
ionization source (HESI).
Analyte HESI Precursor
ion (m/z)
Product ion
(m/z)
Neutralized
Collision
Energy (V)
5-OH- Flunixin - 311.0650 267.0750 20
Ketoprofen + 255.1012 209.0948 20
Oxyphenbutazone - 323.1402 295.1448 32
Flunixin - 295.0700 251.0798 25
Meloxicam + 352.0420 141.0110 15
Niflumic acid - 281.0543 237.0642 20
Diclofenac - 294.0096 250.0191 10
Phenylbutazone - 307.1452 280.1524 15
Flufenamic acid - 280.0591 236.0683 28
Meclofenamic acid - 294.0096 258.0324 10
Meloxicam D3 + 355.0587 --- 10
Flufenamic acid C13 - 286.0793 --- 10
Phenylbutazone C13 - 319.1862 --- 10
Niflumic acid C13 - 287.0752 --- 10
Euroresidues, May 2016. Egmond aan Zee
Analyte ESI Fragmentor
Energy (V)
Precursor
ion (m/z)
Collision
Energy (V)
Product
ions* (m/z)
5-OH-flunixin + 110 313 25
35
295
280
Diclofenac - 90 294 10 250
Phenyl-
butazone - 150 307
10
15
279
131
Flunixin - 70 295 15
35
251
209
Flufenamic
acid - 130 281
15
25
237
216
Ketoprofen + 130 255 15
50
105
77
Meclofenamic
acid + 110 297
12
15
279
264
Niflumic acid - 130 281 15
35
237
177
Oxyphen-
butazone - 130 323
15
25
295
134
Meloxicam + 130 352 25
15
141
115
Flufenamic
acid 13C6 + 110 288 15 270
Meloxicam
D3 + 130 355 15 115
Niflumic acid 13C6
+ 130 288 35 250
Phenyl-
butazone 13C12
+ 130 321 25 166
In bold quantification transition.
Conditions for NSAIDs MS/MS (QqQ)
Column: Biphenyl, Phenomenex (2.1x100)mm, 2.6 µm
Column temperature: 40ºC
Mobile phases: A: water (0.1% formic acid)
B: ACN (0.1% formic acid)
Flow rate: 0.4 mL min-1
Injection volume: 20 µL
Quantification: Matrix matched surrogate standards method
Internal standards: Meloxicam-D3, Phenylbutazone-13C, Niflumic
acid-13C, Flufenamic-13C.
LOQ = 2,5 µg/kg
Working range: 2,5-40 µg/Kg. Ionisation: ESI+ and ESI-
LC-MS equipment: Agilent 1290 UHPLC coupled to Agilent 6460
triple quadrupole (QqQ) mass spectrometer with electrospray
ionization source.
LC Conditions for NSAIDs
NSAIDs HRMS parameters
Chromatogram of an extract from a milk sample (0.1 µg·kg-1 )
(precursor ions, left, product ions right) C:\TraceFinderData\...\Data\15102816 10/28/15 19:50:29 ME5C:5 Inj Vol 10.000000 Mass Tolerance 5.0 ppmC:\TraceFinderData\32\Projects\Aines\151028_2\Methods\Aines_FULLMS\AINES_FULL MS.meth
RT: 0.00 - 12.02
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RT: 8.32AA: 3584190BP: 281.0553
RT: 8.74AA: 9007BP: 281.0555
RT: 8.59AA: 2205BP: 294.0090
RT: 9.49AA: 21916BP: 307.1454
RT: 10.71AA: 48390BP: 280.0587
NL: 3.98E5
m/z= 281.0529-281.0557 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [50.00-620.00] MS ICIS 15102816
NL: 1.94E3
m/z= 294.0081-294.0111 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-620.00] MS ICIS 15102816
NL: 5.48E3
m/z= 307.1437-307.1467 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102816
NL: 4.87E4
m/z= 280.0577-280.0605 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102816
NL: 1.81E3
m/z= 294.0081-294.0111 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS 15102816
Niflumic acid
Diclofenac
Phenylbutazone
Flufenamic acid
Meclofenamic acid
C:\TraceFinderData\...\Data\15102816 10/28/15 19:50:29 ME5C:5 Inj Vol 10.000000 Mass Tolerance 10.0 ppmC:\TraceFinderData\32\Projects\Aines\151028_2\Methods\Aines_FULLMS\AINES_FULL MS.meth
RT: 0.00 - 12.02
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RT: 5.56AA: 5253126BP: 115.0326
RT: 6.27AA: 11883BP: 115.0327
RT: 8.30AA: 4138652BP: 237.0654
RT: 8.59AA: 329838BP: 250.0193
RT: 10.70AA: 701330BP: 236.0691
NL: 8.01E5
m/z= 115.0309-115.0333 F: FTMS + p ESI Full msx ms2 [email protected] [email protected] [50.00-740.00] MS ICIS 15102816
NL: 4.61E5
m/z= 237.0618-237.0666 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-620.00] MS ICIS 15102816
NL: 6.93E4
m/z= 250.0166-250.0216 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-620.00] MS ICIS 15102816
NL: 0
m/z= 280.1496-280.1552 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [email protected] [email protected] [50.00-675.00] MS 15102816
NL: 1.17E5
m/z= 236.0659-236.0707 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-675.00] MS ICIS 15102816
NL: 0
m/z= 258.0298-258.0350 F: FTMS - p ESI Full msx ms2 [email protected] [email protected] [email protected] [50.00-675.00] MS 15102816
