Using LC-MS to Analyse Sulfonamides in Food Grade Honey

Honey manufacturers are faced with a range of stringent food safety regulations, which are subject to change over time. Here Joe Anacleto, VP for Bruker Daltonics Applied Markets Business Group, explores the challenges surrounding the detection of sulfonamides in honey according to international standards and how liquid chromatography mass spectrometry with multiple reaction monitoring can offer a fast, robust and simple solution for food safety testing.

What are the key food safety challenges facing honey manufacturers?

Joe Anacleto (JA): Honey manufacturers need to consider a range of contested environmental, ecological and personal health issues. Regulations for the content, labeling and even the very nature of components within honey are under almost constant debate and vary worldwide. This means that honey manufacturers must meet a whole range of regulations, which are subject to change, in order to ensure consumer protection. It is therefore particularly important for honey producers to have access to sophisticated food analysis methods to exact control over their products and meet regulatory criteria.

What are sulfonamides and why do they present particular risks in honey?

JA: Sulfonamides are a key group of broad spectrum antibacterial drugs, frequently employed in the production of honey to prevent and treat bacterial growth, as well as to fight disease in honey bees. Consequently, residues of sulfonamides are frequently detected in honey products. However, sulfonamides are a common allergen and can provoke a variety of reactions in hypersensitive humans, with effects ranging from mild irritation to severe dermatological disorders, as well as liver and kidney damage. It has also been suggested that the use of sulfonamides in honey may contribute to the development of antibiotic-resistant pathogenic bacteria, a problem of increasing global concern.

How is the presence of sulfonamides in honey regulated?

JA: Regulation of sulfonamides is complicated, since the risk to consumers relates to potential allergic reactions. As the varied effects of allergens are dependent on the response of the individual consumer, it is technically impossible to quantify an exact concentration at which food can be classified as safe for consumption. Similarly, there is no scientific consensus on the level of contribution that sulfonamides in honey makes to the development of antibiotic-resistant bacteria, nor as to how best to mitigate the effects. For this reason, legislation related to the maximum residue allowance varies considerably between regions. Many regions set particularly low minimum required performance levels (MRPLs) for residue concentration, with the European Union limiting sulfonamide content in honey to 10 μg/kg. 

What challenges are presented by the detection of sulfonamides in honey?

JA: Regulation in some regions is very strict; EU legislation defines the required levels of specificity, sensitivity and reproducibility needed to maintain high level consumer protection, with more specific rules to ensure effective quantitation. Low level sulfonamide residues are notoriously difficult to analyze. The complex interactions that arise from biological matrices such as honey substantially obfuscate analysis; traditional techniques suffer extensive preparation times, limited selectivity and low throughput. European Union Directive 2002/657/EC lists a number of potential analytical techniques, of which mass spectrometry with ultra high performance liquid chromatography (UHPLC) is inarguably the most powerful and accessible for the identification of organic compounds within a variety of matrices.

How can multiple reaction monitoring with liquid chromatography mass spectrometry benefit food safety analysis?

JA: Of the range of mass spectrometry techniques available, multiple reaction monitoring (MRM) with triple quadrupole liquid chromatography mass spectrometry (LC-MS TQ) has emerged as the most effective for food analysis, favored for its high sensitivity and selectivity in complex matrices with minimal sample preparation. MRM mass spectrometry methods employ multiple mass filtering functions to enhance selectivity during analyte quantitation. Firstly, specific ions of interest are pre-selected as ‘pre-cursor’ ions, before undergoing fragmentation within a collision cell. By then filtering only the compound-specific fragment ions for analysis, MRM mass spectrometry enables highly sensitive and straightforward multiplexing analysis, with the specificity required for low analyte levels even within complex biological matrices. 

How can LC-MS TQ be used to meet EU regulations on sulfonamides in honey?

JA: A case study by Bruker demonstrates how MRM LC-MS TQ can be used for the analysis of sulfonamides in honey according to EU standards, while ensuring accuracy and substantially decreasing routine analysis time. Nine commonly used sulfonamide antibiotics were analyzed using the EVOQ Elite with Advance UHPLC, following a simple sample preparation procedure of thirty minutes. The method’s repeatability was tested over three separate days, by spiking blank honey matrices with 0.1, 1 and 10 ppb of sulfonamides. The S/N ratios for all nine sulfonamides under study at LOQ (0.1 ng/g) level concentration were found to be well within the acceptable range of accuracy and precision. The method was also found to be extremely repeatable (intra-day and inter-day) with relative standard deviations below 12%, again satisfying an important specification of the EU directive.

The European Union (EU) specifies several parameters within the method, to provide greater stringency and more precaution against false positives. Group A substances, such as sulfonamides, require a minimum of four identification points (IP) to confirm detection of the compounds of interest. This requirement is satisfied through the acquisition of two MRM transitions where 1.0 is assigned for the ‘precursor ion’ and 1.5 IPs are assigned for each target analyte. The ion ratio of a pair of MRM transitions acquired for each target analyte must match to those of the certified reference material within specified tolerance. These were found to be consistent for all the analyte samples taken for this study, demonstrating that this method me EU guidelines.

How are advances in triple quadrupole liquid chromatography mass spectrometry meeting the needs of food safety more broadly?

JA: Food safety testing requires a high level of sensitivity, to meet increasing regulatory demands and provide complete user confidence. Certain MRM mass spectrometry systems deliver even higher levels of specificity and selectivity for food testing, through innovative advances in instrument design. The EVOQ range from Bruker includes Vacuum Insulated Probe HESI (VIP-HESI), which prevents the degradation of thermally fragile molecules for improved sensitivity. Speed and robustness are also crucial in the high throughput environment of a food testing laboratory. Advance UHPLC reduces chromatographic run time, while PACER software offers exception-based review, which eliminates the need for non-experts to review chromatograms, to accelerate sample-to-report times and improve efficiency. The EVOQ also features the unique Active Exhaust in the ion source, which eliminates gas recirculation within the instrument to reduce chemical noise over time and significantly reduce cleaning and downtime.

Details of Bruker's range of liquid chromatography mass spectrometry products can be found below.