non-steroidal anti-inflammatory drugs analysis in … 034.pdf · non-steroidal anti-inflammatory...

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

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

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


NL: 4.47E5


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)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

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


NL: 8.83E3


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


NL: 2.09E4


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

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

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


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

0 1 2 3 4 5 6 7 8 9 10 11 12

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

0

50

100

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


NL: 4.61E5


NL: 6.93E4


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


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

non-steroidal anti-inflammatory drugs analysis in … 034.pdf · non-steroidal anti-inflammatory...

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