Multiresidue analysis of non-steroidal anti-inflammatory drugs in kidney using LCMSMS. ent • innova rem tion John Points, Ratharamaran Ganeshasunderam, Nicoletta Castiglione, Liam Gormley,su Iain Patterson • s LGC, Teddington, UK; E-mail: [email protected] ea cie m n Introduction Method Validation Chromatograms • ce Carprofen, Diclofenac, Flunixin, Ibuprofen, Validation data has been generated for hChromatograms 1: Lowest QC - Pig kidney spiked with half CHROMATOGRAMS 1: Lowest Qc – Pig kidney spiked with half MRMRLL levels of each compounds. • Meloxicam, Naproxen, Niflumic acid, NSAIDs in 3 species (horse, cattle and pigc MRL levels for each compound. r Qc 0.5 Pig 2 e Phenylbutazone, Tolfenamic acid and 09 SNSS 061009-011 2: MRM of 14 Channels ES- kidney) according to the requirements of 10.50 100 317.27 > 289.35 a 8.44e4 % x Phenylbutazone-D10 Vedaprofen are anti-inflammatory drugs used 2002/657/EC. 0 09 SNSS 061009-011 2: MRM of 14 Channels ES- e 10.61 307.24 > 279.42 p 100 for cattle, pigs (flunixin) and horses. They are 3.30e4 Three batches containing 7 individual blanks % Phenylbutazon7.95eT1 s 0 e 09 SNSS 061009-0112: MRM of 14 Channels ES- given for pain relief and to reduce swelling for and 7 fortified replicates of kidney from three 9.62 294.07 > 250.11 100 1.15e5 e % Diclofenac T1 8.73 r muscular and skeletal injuries and disorders. different species at ½, 1 & 2 MRL levels of each 0 r 09 SNSS 061009-011 1: MRM of 10 Channels ES- 353.15 > 289.31 t 100 1.71e6 There are Maximum Residue Limits (MRLs) compounds were analysed. The calculated % i Meloxicam-D3 • 0 s 09 SNSS 061009-011 1: MRM of 10 Channels ES- prescribed for their residues in edible tissues 350.12 > 286.33 concentrations and linear regressions of some 100 1.59e6 % e MeloxicamT1 when they are used in the approved manner; compounds are shown in Figures 1 to 4. 0 s 09 SNSS 061009-011 1: MRM of 10 Channels ES- 100 297.55 > 254.13 i 6.86e5 % administering them to any other species Flunixin-D3 0 • Selectivity s 09 SNSS 061009-011 1: MRM of 10 Channels ES- constitutes illegal use. 100 295.17 > 251.13 2.93e6 % Flunixin T1 Two precursor-produy ct transitions are 0 q 09 SNSS 061009-011 1: MRM of 10 Channels ES- 5.51 l 100 229.07 > 169.85 5.98e4 measured for each NSAIDs, giving 4 % Naproxen T2 u Extraction procedure 0 Time a 4.00 6.00 8.00 10.00 12.00 14.0016.00 identification points. No interference peaks a Sub-sample kidney 2g were found. n Fortify matrix calibration QCs. Add Internal Standards Chromatograms 2: Lowest QC - Pig kidney spiked with half MRL levels for each compound. l Performancea limits CHROMATOGRAMS 2: Lowest Qc - Pig kidney spiked with half MRL levels of eaceachh compounds. i Qc 0.5 Pig 2 t 09 SNSS 061009-011 3: MRM of 7 Channels ES- 13.64 100 327.19 > 237.37 y Add 4ml Elga water, Add 0.5ml 10M KOH Calculate using the procedure in ISO 11843 9.28e4 • % Vedaprofen T3 Vortex & Homogenise 0 and 2002/657/EC. 09 SNSS 061009-011 3: MRM of 7 Channels ES- 13.64 100 281.28 > 237.29 Add a further 0.5ml 10M KOH, Vortex thoroughly 2.94e5 % Vedaprofen T1 • 0 In-housee reproducibility examples 09 SNSS 061009-011 3: MRM of 7 Channels ES- 11.81 100 265.98 > 222.33 4.89e5 13 % Tolfenamicacid C6 s c 0 Hydrolysis for 15 mins @ 60°C in water bath 09 SNSS 061009-011 3: MRM of 7 Channels ES- 11.81 i 100 260.11 > 216.17 Vortex thoroughly and allow to cool 2.32e5 % Tolfenamic acid T1 a Adjust pH to 2.0 ± 0.1 with H3PO4 0 09 SNSS 061009-011 2: MRM of 14 Channels ES- v 7.07 100 281.11 > 237.37 9.63e5 % Niflumic acid T1 f r 8.53 9.78 0 09 SNSS 061009-011 2: MRM of 14 Channels ES- e 8.89 100 275.06 > 231.31 Add 4ml water and vortex 1.18e7 % CarprofenD3 e 7.12 Centrifuge @ 4000rpm for 25 mins 0 09 SNSS 061009-011 2: MRM of 14 Channels ES- t 8.94 100 272.12 > 228.23 6.42e6 s % CarprofenT1 y 0 09 SNSS 061009-011 2: MRM of 14 Channels ES- 9.31 100 205.13 > 161.17 9.43e4
% Ibuprofen T1 SPE cleanup (Oasis MAX 6cc, 150 mg cartridge) 6.55 • 0 Time 1. Condition with 3ml TBME, 3mlMeOH & 3 ml H2O Figure 1: Precision of 3 batches (n=7 per species, per concentration) 4.00 6.00 8.00 10.00 12.00 14.0016.00 •
2. Load samples for Phenylbutazone T1 (307.38>279.42).
3. Wash with 5 ml 50mM NaOAc buffer pH=7.0
Results s y
4. Wash with MeOH:H2O (60:40) and dry cartridges Table 1: Performance limits from three validation batches.
t under vacuum e MRL 5. Elute with 5 ml Formic acid:TBME:MeOH (5:75:20) Compound Species CCα CCβ
e (µg/ml) Collect in 15 ml graduated falcon tubes r
f Carprofen Horse 1000 1064 1102
Cattle 1000 1064 1102 v a
Spike Post extraction matrix standards Pig Not set 42 83 i
c
Add 3ml 50mM Amm formate in H2O:TBME:MeOH (5:20:75) s Diclofenac Horse Not set 1.5 2.9
Add 500µl Mobile phase, Dry down to 500µl, under N2(g) @ 40°C
Cattle 10 11 12 e
• Pig 10 11 12
Figure 2: Precision of 3 batches (n=7 per species, per concentration) for Flunixin Horse 200 202 205
Adjust final volume to 1 ml with mobile phase Carprofen T1 (272.12>228.23). •
y
Filter into LC MS/MS vial, LC MS/MS analysis Cattle 100 102 105
-- t
a
i Pig 30 32 35
l Ibuprofen Horse Not set 2 3
Cattle Not set 2 3 n Instrument conditions a
Pig Not set 2 3 a
u Meloxicam Horse 65 67 68
l
y
q Cattle 65 67 68
Pig 65 67 68 s
• Naproxen Horse Not set 2 5
i
Cattle Not set 2 5 s
Figure 3: Precision of 3 batches (n=7 per species, per
e Pig Not set 2 5 concentration) for Tolfenamic acid T1 (259.63>216.06).
•
s Niflumic acid Horse Not set 1.2 2.4
i
Cattle Not set 1.2 2.4 t
r
r Pig Not set 1.2 2.4
Phenylbutazone Horse Not set 0.58 1.15 e e
Cattle Not set 0.58 1.15 s
p
Pig Not set 0.58 1.15 e
x
Tolfenamic acid Horse Not set 4 8 a
e
r Cattle 100 104 110
c
• Pig 100 104 110
h
Vedaprofene Horse 1000 1106 1211
Figure 4: Precision of 3 batches (n=7 per species, per •
c Cattle Not set 300 598 concentration) for Meloxicam T1 (350.12>286.33). m
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