The use of antibiotics in food animal production has resulted in benefits throughout the food industry; however, their use has led to animal and human health safety concerns. This article presents an overview of current approaches and new developments in the application of liquid chromatography-mass spectrometry for the analysis of animal-derived foods for residues of one important class of antibiotics: macrolides.
by Dr Leslie C. Dickson and Dr Jian Wang
Macrolides are a group of antibiotics that have been widely used to treat many respiratory and enteric bacterial infections in humans and animals. The pharmacological activity of these basic and lipophilic drugs arises from the presence of a macrocyclic lactone ring with 14, 15 or 16 atoms to which one or more deoxy sugars are attached [Figure 1]. Some of the more commonly used macrolides are erythromycin, josamycin, oleandomycin, roxithromycin, spiramycin, tilmicosin, tulathromycin and tylosin. Tilmicosin, tulathromycin and tylosin are not used in human medicine. Some macrolides are also administered to food animals at sub-therapeutic concentrations as feed additives to prevent disease and to promote growth. The world market for all antibiotics for use as feed additives is estimated to be $US 4 billion.
An important issue in the use of antibiotics in food animal production is the development of bacterial strains that are resistant to some antibiotics, and the potential for the transfer of this resistance to pathogens that can cause disease in humans. Another concern is that residues of antibiotics and their metabolites in foods may have adverse effects on allergic or sensitive consumers. National agencies and international organisations have set regulatory limits on the concentrations of antibiotic residues in foods of animal origin. Residue limits vary from zero (no assigned limit) to 15 mg/kg, depending on the commodity and jurisdiction. These limits are enforced in part through chemical analyses of appropriately collected samples of animal-derived foods such as meat, milk and eggs. This article reviews current approaches and new developments in the analysis of animal-derived foods for residues of macrolide antibiotics using liquid chromatography-mass spectrometry.
Current approaches
One of the most difficult challenges in chemical analysis is the unequivocal identification and reliable quantitation of trace concentrations of drug residues in complex matrices such as food. Drug residues are present at much lower concentrations — usually mg/kg to ng/kg — than the proteins, carbohydrates, fats, vitamins, minerals and water that make up the bulk of the sample and potentially could interfere with the analysis. Methods suitable for identification and quantitation of drug residues at these concentrations require efficient and selective sample preparation and analytical separation techniques, coupled with highly selective and sensitive detection systems [1-3].
Sample preparation
Sample preparation involves several sequential procedures for the extraction of the macrolide residues from the bulk of the sample, removal of co-extracted proteins, fats and other interfering compounds, and concentration of the macrolide residues. The technique most commonly used to clean-up and concentrate macrolide residues from samples is solid-phase extraction (SPE). The types of SPE stationary phases that have been used to clean-up extracts of food samples include mixed retention mode (nonpolar and ion exchange) resin-based materials and nonpolar bonded silica. SPE is usually a manual procedure, but SPE separations can be automated to eliminate the tedious manual steps and minimise the residue losses that often occur with manual procedures. Thus, automated SPE provides a solution for high-sample throughput. The technique of liquid-liquid partitioning is often used in conjunction with SPE to further clean-up sample extracts, for example to remove residual fats. There is no single sample procedure that can be applied to clean-up samples for the determination of macrolide residues from all diverse matrices. The development of the appropriate methodology for sample preparation depends on the performance requirements of the analysis and the nature of the matrices [4].
Chromatographic separation and detection
Instrumental analytical approaches for the determination of macrolide residues in food include liquid chromatography (LC) with ultraviolet (UV) or fluorometric detection, and liquid chromatography with mass spectrometry (LC-MS). Mass spectrometry, serving as a sensitive and universal detection technique, has largely replaced UV and other selective detectors for multi-residue analyses of trace concentrations of drug residues in complex samples [4]. The essential stages in an LC-MS analysis are chromatographic separation and isolation of the analytes in the sample, the ionisation of the analyte molecules, the measurement of the mass-to charge ratio (m/z) of the analyte ions and their subsequent detection.
Liquid chromatography is a fundamental separation technique used in food analysis and other fields of chemical analysis. Conventional high performance liquid chromatography (HPLC) is often used in conjunction with MS to separate analytes from each other and from potentially interfering matrix components that could compromise the reliability of the determination. Chromatographic separations of macrolides rely on the use of reversed-phase (RP) LC columns. In most studies, the non-polar stationary phase is a modified silica using particles 3-5 μm in diameter with surface-bonded octadecylsilyl chains, commonly referred to as ODS-silica or C18-silica. The mobile phase for RP LC is a mixture of water, organic solvents such as acetonitrile and methanol, and volatile modifiers such as formic acid, ammonium acetate or ammonium formate.
The most common ionisation technique used for analyses of macrolide residues is electrospray ionisation (ESI), which is a process in which ions that are in solution are brought into the gas phase by evaporation and ion desorption. The technique is applicable to polar and medium polarity analytes that cover a broad mass range, and is the ionisation source of choice for macrolides in all different types of matrices. One disadvantage of ESI is that it is susceptible to matrix effects, which are the effects of components of a sample other than the analyte and can change the ionisation process and reduce the reliability of the results. Other ionisation techniques that are less susceptible to matrix effects include atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photoionisation (APPI), but these techniques have had limited applicability to macrolide analysis [4].
Mass spectrometry is a technique for measuring the m/z and intensities of ions produced in the ion source from sample components eluting from the LC column. This detection technique can be used to confirm the presence and quantitate the amount of a target compound in a sample. Mass spectrometers with various designs, performance and functions that are commonly used for macrolide analysis include the triple-quadrupole (QqQ) and the quadrupole ion trap (QIT).
The QqQ MS, sometimes referred to as tandem MS or MS/MS, is the most commonly used mass analyser for the determination of macrolide residues in food matrices. A QqQ MS consists of a quadrupole mass filter (Q1), a collision cell (q2) and a second quadrupole (Q3). The coupling of multiple mass filters provides greater selectivity, sensitivity, dynamic range and more structurally-dependent information than using a single mass filter alone. QqQ MS is commonly operated in selected reaction monitoring (SRM) mode for quantitation and confirmation. In this mode, an ion with a given m/z is selected by Q1, fragmented in q2, and the fragment ions selected by Q3. The QqQ’s other functions, such as product-ion, precursor-ion and neutral-loss scans, can facilitate macrolide confirmation [Figure 2]. The QIT MS traps, fragments and detects ions within a small three-dimensional volume. It is unique in its ability to conduct two or more stages of mass fragmentation and detection, referred to as MSn. A QIT MS has a higher duty cycle and greater scanning sensitivity than QqQ MS and can be more versatile and affordable, but can exhibit poor repeatability and be less reliable for quantitative analyses of residues in difficult and complex matrices [4].
Recent developments and future trends
Although conventional HPLC using 3-5 μm particles currently plays a dominant role for the determination of macrolides, ultra-high-performance liquid chromatography (UHPLC) using sub-2 μm particle sizes and other fast chromatographic methods have recently gained in popularity. When coupled with MS instruments capable of high-speed data acquisition, these methods offer significant advantages in resolution, speed and sensitivity. Although there are a limited number of reported applications of fast chromatographic methods in the literature, the trend is towards their increasing use in macrolides analysis.
Some recent developments in MS technology, in the form of the triple quadrupole linear ion trap (QqLIT), time-of-flight (TOF) and quadrupole time-of-flight (QqTOF) mass analysers, promise to deliver powerful new tools for macrolide analysis [4]. The QqLIT combines the components and advantages of a QqQ MS and QIT MS in one instrument without compromising the performance of either. Although QqLIT has not been widely used for macrolide analysis, it is likely to become a key technique because it can perform SRM and acquire product-ion scan spectra for analytes at low concentrations in a single analysis. The TOF mass analyser is the simplest mass spectrometer design, where ions that have the same kinetic energy but different m/z values reach a detector after different flight times. These instruments are configured as either a TOF alone or combined with quadrupole mass filters in a QqTOF configuration. Both TOF and QqTOF offer medium range high-resolution, accurate mass measurement, excellent full-scan sensitivity and complete mass spectral information. When combined with UHPLC, these instruments are likely to become important tools to screen, quantitate and confirm macrolide residues in food [4].
The new UHPLC-MS technologies will complement conventional LC-MS/MS techniques. One can use UHPLC-QqTOF to screen for unknown and targeted chemical residues; any incurred residues detected can be quantitated using LC-QqQ. Alternately, residues can be detected and quantitated by LC-QqQ and confirmed by UHPLC-QqTOF based on accurate mass measurements [5].
References
1. Stolker AAM, Brinkman UATh. Analytical strategies for residue analysis of veterinary drugs and growth promoting agents in food producing agents–a review. J Chromatogr A 2005; 1067: 15-53.
2. Blasca C, Torres CM, Picó Y. Progress in analysis of residual antibacterials in food. Trends Anal Chem 2007; 26: 895-913.
3. McGlinchey TA, Rafter PA, Regan F, McMahan GP. A review of analytical methods for the determination of aminoglycoside and macrolide residues in food matrices. Anal. Chim Acta 2008; 624: 1-15.
4. Wang J. Analysis of macrolide antibiotics, using liquid chromatography-mass spectrometry, in food, biological and environmental matrices. Mass Spectrom Rev 2009; 28: 60-92.
5. Wang J, Leung D. Analyses of macrolide antibiotic residues in eggs, raw milk, and honey using both ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry and high performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2007; 21: 3213-3222.
The authors
Dr. Leslie C. Dickson
Canadian Food Inspection Agency Saskatoon Laboratory, Centre for Veterinary Drug Residues, Saskatoon, Saskatchewan, Canada
&
Dr. Jian Wang
Canadian Food Inspection Agency Calgary Laboratory, Calgary, Alberta, Canada
