Corticosteroids. Optimizations. Jens Hinge Andersen. National Food Institute Danish Technical University

Transcription

1 Corticosteroids Optimizations Jens Hinge Andersen National Food Institute Danish Technical University

2 Optimizations - corticosteroids : Minimizing ion-suppression from bovine urin Liver: Improving recovery & minimizing work 2 DTU Food

3 Optimizations - corticosteroids : Minimizing ion-suppression from bovine urin Liver: Improving recovery & minimizing work 3 DTU Food

4 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH LC: Superspher 100 RP 18e, 125x4 mm, 4 µm 35% ACN / Water MS/MS: Iontrap APCI+ 4 DTU Food

5 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 5 DTU Food

6 RIVM (ARO/441, 1999) Waters Oasis HLB Hydrophilic-Lipophilic Balanced copolymer DTU Waters Oasis MAX Mixed mode polymeric strong Anion exchange LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 6 DTU Food

7 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water Elute: 2 ml MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 7 DTU Food

8 Se note Recovery - porcine urine Effect of sample concentration in extract (neutral elution) Pig (spike), 6.9 ml urine/ml Pig (spike), 3.5 ml urine/ml Pig (analyte in matrix), 3.5 ml urine/ ml Pig (spike), 1.7 ml urine/ml 100% 0% Triamcinolone-acetonid Prednisolon Flumethason Dexa- + Betamethason 8 DTU Food

9 Se note Recovery - porcine / bovine urine Effect of sample concentration in extract (neutral elution) Pig (spike), 6.9 ml urine/ml Pig (spike), 3.5 ml urine/ml Pig (analyte in matrix), 3.5 ml urine/ ml Pig (spike), 1.7 ml urine/ml 100% Cattle (spike), 6. 9 ml urine/ml Cattle (spike), 3. 5 ml urine/ml Cattle (analyte in matrix), 3.5 ml urine/ml Cattle (spike), 1. 7 ml urine/ml 0% Triamcinolone-acetonid Prednisolon Flumethason Dexa- + Betamethason 9 DTU Food

10 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water Elute: 2 ml MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 10 DTU Food

11 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water Elute: 2 ml 0.1M NH 4 OH in MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 11 DTU Food

12 Se note Recovery - porcine / bovine urine Recovery after neutral or alkaline elution Pig (spike), 6.9 ml urine/ml, neutral elution 100% Cattle (spike), 6.9 ml urine/ml, neutral elution Cattle (spike), 6.9 ml urine/ml, alkaline elution 0% Triamcinolone acetonid Prednisolone Flumethasone Dexa- + Betamethasone 12 DTU Food

13 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water Elute: 2 ml 0.1M NH 4 OH in MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 13 DTU Food

14 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water 6 ml 0.1M NH 4 OH in water Elute: 2 ml 0.1M NH 4 OH in MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 14 DTU Food

15 Se note Recovery - bovine urine Recovery after alkaline wash and elution with alkaline methanol Pig (spike), 6.9 ml urine/ml Pig (spike), 3.5 ml urine/ml Pig (analyte in matrix), 3.5 ml urine/ml Pig (spike), 1.7 ml urine/ml 100% Pig (analyte in matrix), 1.7 ml urine/ml Cattle (spike), 6.9 ml urine/ ml Cattle (spike), 3.5 ml urine/ ml Cattle (analyte in matrix), 3.5 ml urine/ml Cattle (spike), 1.7 ml urine/ml 0% Triamcinolone acetonid Prednisolone Flumethasone Beta/Dexamethasone Cattle (analyte in matrix), 1.7 ml urine/ml 15 DTU Food

16 RIVM (ARO/441, 1999) Waters Oasis HLB 2*3 ml MeOH/0.02M NaOH (40:60) 3 ml Water Elute: 1.5 ml MeOH DTU Waters Oasis MAX 6 ml Water 6 ml 0.1M NH 4 OH in water Elute: 2 ml 0.1M NH 4 OH in MeOH LC: Superspher 100 RP 18e, LC: Zorbax Eclipse XDB-C18 125x4 mm, 4 µm 100x2.1 mm, 1.8 µm 35% ACN / Water 30% ACN / 0.1% HCOOH MS/MS: Iontrap APCI+ MS/MS: Triple quad ESI- 16 DTU Food

17 Ionsuppression - What happened (maybe)? LC: Acidic eluent Neutral and (weakly) acidic components have retention; can elute with analytes SPE (MAX): Mixed mode polymeric strong Anion exchange Eluting with MeOH: Ionised acids are retained; nonpolar components (analytes) elute Eluting with alcaline MeOH: Weak acids are ionised and retained; nonpolar components (analytes) elute 17 DTU Food

18 Ionsuppression - What happened (maybe)? LC: Neutral eluent Neutral and (weakly) acidic components have retention; can elute with analytes SPE (HLB): Hydrophilic-Lipophilic Balanced copolymer Washing with alcaline MeOH/water: Weak acids are ionised and washed out with polar components; nonpolar components (analytes) are retained Eluting with MeOH: Everything (analytes) elute 18 DTU Food

19 Recovery - bovineurine Recovery of spiked standard curve, 4 levels (1-10 µg/l) compared to analytes in eluent (data from validation) 120% 100% 80% 60% 40% 20% 0% Fluocinolone acetonid Triamcinolone acetonid Beclomethasone Flumethasone Dexamethasone Betamethasone MethylPrednisolone Prednisone Prednisolone 19 DTU Food Avg. n=8

20 Zorbax Eclipse XDB-C18 100x2.1 mm, 1.8 µm 30% ACN/0.1% HCOOH, 0.22 ml/min, bar (Agilent 1100) Triple quad ESI- (Micromass Ultima) FLUOC 2 µg L -1 TRIAM 2 µg L -1 BECLO 2 µg L -1 FLUM, 2 µg L -1 BETA, 2 µg L -1 DEXA, 2 µg L -1 MPRED, 2 µg L -1 PRSON, 3 µg L -1 PRLON, 3 µg L -1 Spiked Spiked sample sample Blank k DTU Food min.

21 Optimizations - corticosteroids : Minimizing ion-suppression from bovine urin Liver: Improving recovery & minimizing work 21 DTU Food

22 RIVM (ARO/441, 2003) 5 g Liver, 10 ml Ac-buffer Add MeOH for extraction ( ml) to 2 ml Add 6 ml of water 22 DTU Food Centrifuge Alltech SPE C18, 1000 mg 5 ml Acetone/10% Na 2 CO 3 (20:80); 5 ml Water; 5 ml Heptane. Dry Elute: 5 ml EtAc, redisolve LCMSMS: C18 / Iontrap APCI+

23 RIVM (ARO/441, 2003) DTU 5 g Liver 1 g Liver, 10 ml Ac-buffer Add MeOH for extraction ( ml) to 2 ml Add 6 ml of water, 2 ml Ac-buffer Add MeOH for extraction (2 + 2 ml) to 1 ml Add 3 ml of water Centrifuge Alltech SPE C18, 1000 mg 5 ml Acetone/10% Na 2 CO 3 (20:80); 5 ml Water; 5 ml Heptane. Dry Elute: 5 ml EtAc, redisolve LCMSMS: C18 / Iontrap APCI+ 23 DTU Food Centrifuge Lichrolut RP18, 500 mg 3 ml Acetone/10% Na 2 CO 3 (20:80); 3 ml Water; 3 ml Heptane. Dry Elute: 2.5 ml EtAc, redisolve LCMSMS: C18 / Triple quad ESI-

24 Recovery - porcine and bovine liver Recovery: Evaporation before SPE 100% 80% 60% 40% 20% 0% 24 DTU Food Se note Porcine liver Bovine liver Fluocinolone acetonid Triamcinolone acetonid Beclomethasone Flumethasone Betamethasone Dexamethasone MethylPrednisolone Prednisone Pr ednisolone

25 RIVM (ARO/441, 2003) DTU 5 g Liver 1 g Liver, 10 ml Ac-buffer Add MeOH for extraction ( ml), 2 ml Ac-buffer Add MeOH for extraction (2 + 2 ml) Centrifuge Alltech SPE C18, 1000 mg 5 ml Acetone/10% Na 2 CO 3 (20:80); 5 ml Water; 5 ml Heptane. Dry Elute: 5 ml EtAc, redisolve LCMSMS: C18 / Iontrap APCI+ 25 DTU Food Centrifuge Lichrolut RP18, 500 mg 3 ml Acetone/10% Na 2 CO 3 (20:80); 3 ml Water; 3 ml Heptane. Dry Elute: 2.5 ml EtAc, redisolve LCMSMS: C18 / Triple quad ESI-

26 RIVM (ARO/441, 2003) DTU 5 g Liver 1 g Liver, 10 ml Ac-buffer Add MeOH for extraction ( ml) to 2 ml Add 6 ml of water, 2 ml Ac-buffer Add MeOH for extraction (2 + 2 ml) Centrifuge Add 13.5 ml of water (to ( 20% MeOH/water) Add HCOOH to ph < 4 Centrifuge Alltech SPE C18, 1000 mg 5 ml Acetone/10% Na 2 CO 3 (20:80); 5 ml Water; 5 ml Heptane. Dry Elute: 5 ml EtAc, redisolve LCMSMS: C18 / Iontrap APCI+ 26 DTU Food Centrifuge Lichrolut RP18, 500 mg 3 ml Acetone/10% Na 2 CO 3 (20:80); 3 ml Water; 3 ml Heptane. Dry Elute: 2.5 ml EtAc, redisolve LCMSMS: C18 / Triple quad ESI-

27 Recovery - porcine and bovine liver Recovery of spiked standard curve, 5 levels (1-15 µg/kg) compared to analytes in eluent (data from validation) 120% 100% 80% 60% 40% 20% 0% Fluocinolone acetonid Triamcinolone acetonid Beclomethasone Flumethasone Dexamethasone Betamethasone MethylPrednisolone Prednisone Prednisolone 27 DTU Food Avg. n=9

28 Addendum method: RIVM-method did not work well in our laboratory because of interfering peaks for several of the corticosteroids in the blind samples. No systematic search for the reason for these peaks were done. No evaluation of ion-suppression in the RIVM-method was done. The two clean-up methods could have the same aim: to remove weak acids. The RIVM-method by ionising them and washing them from the SPE column before elution of analytes ours by ionising them and retaining them on the anionic exchange SPE while eluting the analytes. Liver method: Ion suppression was found acceptable. No significant differences between species (porcine/bovine) were found. 28 DTU Food