Improved SPE for LC-MS Determination of Ractopamine and Zilpaterol in Bovine Liver: The Oasis PRiME MCX Method

Significance of the Topic

Ractopamine and zilpaterol are beta agonist veterinary drugs regulated with low maximum residue limits in bovine liver. Analysis of these compounds is complicated by high levels of co-extracted phospholipids in liver tissue, which cause matrix effects, instrument contamination, and reduced analytical performance.

Study Objectives and Overview

This study presents an improved solid phase extraction cleanup using the Oasis PRiME MCX cartridge to efficiently remove phospholipids from methanolic bovine liver extracts before UPLC MS/MS determination of ractopamine and zilpaterol. The performance of this method is compared with the AOAC Official Method 2011.23.

Methodology and Instrumentation

Sample Preparation: Homogenized bovine liver (5 g) was extracted three times with methanol, combining supernatants to a final volume of 50 mL.
SPE Cleanup: The Oasis PRiME MCX Vac cartridge (60 mg) was used without conditioning. Two mL of extract was loaded, followed by a 2 mL aqueous ammonium formate/ formic acid wash and a 2 mL methanol wash. Analytes were eluted with 2 mL of 5% ammonia in methanol, evaporated under nitrogen at 40 °C, and reconstituted in 1 mL of 20:80 methanol/water.

• Liquid Chromatography: ACQUITY UPLC H-Class system with BEH C18 1.7 µm, 2.1×100 mm column; mobile phase A: 0.02% formic acid in water; phase B: 50:50 acetonitrile/methanol; gradient from 95% A to 2% A over 9.3 min; column at 40 °C; injection volume 4 µL.
• Mass Spectrometry: Xevo TQ-S micro in positive electrospray ionization MRM mode; source 120 °C; desolvation 300 °C at 1000 L/hr; cone gas 30 L/hr; collision gas 0.15 mL/min; transitions: zilpaterol 262.2>185.1 and 262.2>201.1; ractopamine 302.2>164.1 and 302.2>284.2; data acquired with MassLynx v4.2.

Key Results and Discussion

Recoveries for ractopamine ranged from 91% to 105% (5–100 ng/g) and for zilpaterol from 95% to 98% (1–20 ng/g), with RSDs below 8%. The Oasis PRiME MCX cartridge removed over 90% more phospholipids compared to the traditional MCX SPE protocol, resulting in cleaner baselines, reduced matrix interference, and improved signal stability.

Benefits and Practical Applications

The streamlined SPE workflow requires no cartridge conditioning, accelerates sample processing, and reduces solvent consumption. Enhanced cleanup prolongs column life and decreases maintenance, making the method well suited for routine food safety and residue monitoring laboratories.

Future Trends and Opportunities

Potential advancements include integration with online SPE automation, extension to other polar veterinary drugs and complex matrices, miniaturization of sample preparation, and adoption of greener solvents to meet regulatory and sustainability goals.

Conclusion

The Oasis PRiME MCX method offers a rapid, high-recovery cleanup for bovine liver analysis of beta agonists, surpassing existing SPE protocols in phospholipid removal, sensitivity, and instrument robustness.

References

1. AOAC Official Method 2011.23 Determination and confirmation of parent and total ractopamine in bovine, swine, and turkey tissues