QuEChERS Cleanup and Analysis of Veterinary Drugs in Milk by LC/MS/MS

By: Olga I. Shimelis, and Jennifer Claus, Reporter US Volume 34.1


Sample Preparation

Milk sample (2 mL) was placed into a 50 mL centrifuge tube (Product No. 55248-U). The analytes were spiked and the sample was left to sit for 30 minutes. Acetonitrile (8 mL) was added, mixed for 1 min and centrifuged. The supernatant was separated into a 15 mL centrifuge tube. Concentrated formic acid (0.1 mL) and 500 mg Z-Sep+ (Product No. 55296-U) were added or, alternatively, 500 mg of DSC-18 sorbent (Product No. 52600-U). Samples were shaken for 1 min and centrifuged. The supernatant was evaporated at 50 °C to 0.75 mL. Acetonitrile (0.15 mL) was added along with water to total volume of 1.0 mL. The samples were filtered prior to LC/MS analysis using 0.45 μm filter.

HPLC Analysis

An Ascentis® Express column with Fused-Core® particle architecture was used to achieve faster separations while using a standard Agilent 1200 HPLC system. Two different column stationary phases, C18 and Reversed Phase Amide (RPA), were tested for this separation. The RPA HPLC column was chosen because it provided better retention for more polar analytes, such as salbutamol and sulfanilamide (Figure 1). Matrix effects were evaluated by spiking the blank extracts with analytes and comparing the obtained recovery values to the calibration curves in solvent. Solvent standards were used for all quantitation.

Veterinary Drugs Using Ascentis Express RP-Amide HPLC Column

Figure 1. Veterinary Drugs Using Ascentis Express RP-Amide HPLC Column in + ESI Mode

Results and Discussion

Development of the sample extraction procedure was based on available published literature for veterinary drug analysis.1-8 First, the “true” QuEChERS extraction into 100% acetonitrile and the salt-out protocol was tested. It was quickly determined that polar drugs, such as salbutamol and levamisol, did not extract well into pure acetonitrile. Therefore, as a compromise, 20% water was included in the final extraction solvent, and no extraction salts were used for the final procedure.

Addition of 1% formic acid to the extract prior to Z-Sep+ cleanup was necessary to improve the recoveries for compounds that had chelating properties. This additive was not necessary when C18 cleanup was used.

The removal of phospholipids, in particular, phosphatidylcholine, (PC) from milk samples was evaluated and was found to be more complete using Z-Sep+ in comparison to that using C18. The PC chromatogram in cleaned milk samples is shown in Figure 2.

Evaluation of ionization effects (Table 2) indicated that ion suppression was higher in the case of the more polar and less retained analytes: salbutamol, sulfanilamide, and lyncomycin. This ion suppression effect was more pronounced for Z-Sep+ cleaned-samples. The ion suppression effect was higher for later eluting abamectin when C18 cleanup was used. In fact, the abamectin signal was extremely low and almost non-detectable in C18-cleaned samples.


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