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Innovation • Urement • Innovation • Science • Expertise Multiresidue analysis of non-steroidal anti-inflammatory drugs in kidney using LCMSMS. ment • innovatio re n 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 Validation data has been generated for Carprofen, Diclofenac, Flunixin, Ibuprofen, hChromatograms 1: Lowest QC - Pig kidney spiked with half Meloxicam, Naproxen, Niflumic acid, NSAIDs in 3 species (horse, cattle and pigc MRLCHROMATOGRAMS levels for each1: Lowest compound.Qc – Pig kidney spiked with half MRMRLL levels of each compounds. • 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 and 2002/657/EC. 0 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 n e e i c a s s u • r e n m o i t e a n v t o • n i n No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or any retrieval system, without the written permission of the copyright holder. © LGC Limited, 2009. All rights reserved. 2160/TK/0609.
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