Misadventures in Mass Spectrometry and Drug Residue Analysis

Tom Thompson
Alberta Agriculture, Food & Rural Development
Food Safety Division
Agri-Food Laboratories Branch

Abstract

There are currently numerous laboratories attempting to switch over from HPLC-based methods to LC-MS procedures. While MS and MS/MS techniques offer high sensitivity and specificity, analysts determining residues of veterinary drugs in animal tissues, dairy products, cereal crops, honey, fruits, vegetables and foods in general are still faced with inherent challenges. Many non-volatile mobile phase additives and ion-pair reagents are not suitable for MS methods. Also, matrix effects observed in atmospheric pressure ionization techniques often result in difficulties in performing accurate quantitation. Furthermore, there is a lack of established methods that have been validated by interlaboratory comparison.

Despite some of the drawbacks of LC-MS techniques, it is nonetheless an extremely powerful analytical technique. This is illustrated by the preliminary investigation towards the development of a rapid method for the analysis of drug residues in honey. Oxytetracycline (OTC) is the only drug registered in Canada and the United States for the control of American foulbrood (AFB) disease in honey bees. Tylosin and lincomycin are currently being evaluated for use in the control of OTC-resistant strains of AFB-causing bacteria. During initial field studies, a method employing solid phase extraction and LC-APcI-MS was developed and subsequently implemented. While the method was found to be applicable to honey samples containing a wide range of drug concentrations (10 to 10,000 ppb), it was found to be a labour-intensive procedure when large numbers of samples were processed. An alternative procedure employing LC-MS/MS was proposed.

In the "dilute and shoot" method, honey was spiked with analytes and surrogate standards (clindamycin and roxithromycin for lincomycin and tylosin, respectively) and diluted with a mixture of acetonitrile and water. The diluted honey was injected directly into the LC-MS/MS without further cleanup. Using a gradient elution program with 0.04% aqueous heptafluorobutyric acid and acetonitrile, the drugs were sufficiently isolated from the bulk of the honey matrix. A six-port rotary valve was utilized to divert the LC effluent away from the MS/MS immediately before and after the elution of the analyte and surrogate compounds to minimize the contamination of the MS source region. The use of multiple reaction monitoring of two product ions per target analyte increases the confidence of compound identification. No potential interferences were observed in any of the MRM traces. The MS/MS procedure was found to be linear over a range from 5 to 1,000 ppb. Excellent accuracy and precision was observed for tylosin in honey samples spiked with 5 to 800 ppb of each drug. The precision for lincomycin was not as good and elevated recoveries (> 130%) were consistently observed despite the use of matrix-matched standards. While the method appears to have considerable potential, further work is needed to refine it.