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Extraction of Phenylbutazone from Beef Using Strata-X-A on Kinetex 2.6u XB-C18 50x2.1 by LC/MS/MS

Sample Preparation Details

Part No:8B-S123-EBJ
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 100 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Sample Pretreatment:
1. In an Erleneyer flask (or similar), combine 2 ±0.1 g sample (well-homogenized ground beef or pork sausage) with 2 mL 0.1N NaOH and 50 uL of 4 ug/mL Int Std (phenylbutazone-D10) solution
2. Cap the flasks and mix vigorously for at least 10 sec
3. Add 8 mL 100% MeOH to the flask and cap the vessel
4. Place the flasks securely on a lab shaker and shake the vessels for 55-60 min at medium-high to high setting
5. Transfer the contents of the flask into a 15 mL conical tube and centrifuge them for 10 min @ 4000-4500 rpm
6. Remove 2 mL of supernatant and combine with 2 mL DI water into a glass tube and mix
7. Proceed to SPE method
Dry:5 mins at 10" of Hg
Final Prep and Analysis: Extraction Procedure:
1. Condition a Strata-X-A, 100 mg/6 mL with 3 mL 100% MeOH followed by 3 mL DI water
2. Load sample from the final step of pretreatment section
3. Wash with 2 mL DI H2O
4. Wash with 2 mL 100% ACN
5. Wash with 2 mL 100% Ethylacetate
6. Dry the SPE bed for 4-5 min @ max vacuum
7. Elute with 2x1.5 mL 1% formic acid in MeOH
8. Evaporate the eluate to dryness under gentle nitrogen stream at 45-50 C
9. Re-suspend the residue with 500 uL 50:50 MeOH:0.1% formic acid in DI H2O Inject: 5 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Phenylbutazone 25  101

Application Detail (App ID: 21872)

Part No:00B-4496-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% Formic Acid in DI Water
Elution B:0.1% Formic Acid in Methanol
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 50 50
2 0.5 50 50
3 2 5 95
4 3.5 5 95
5 3.51 50 50
6 5 50 50

Flow Rate:0.45 mL/min
Col. Temp: 45 °C
Detection: Mass Spectrometer (MS) @ amu (0°C)
Detector Info: AB Sciex API 4000
Note:Mass spec conditions:
API 4000, ESI TurboIon Spray, + Ionization;
CAD: 6.00
CUR: 20.00
GS1: 50.00
GS2: 50.00
IS: 5500.00
TEM: 600.00
ihe: ON
DP 50.00
EP 10.00


Analyte Details

Analyte Detail not available

1
Phenylbutazone
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