Streamlining the Use of High Resolution Mass Spectrometry Data to Fingerprint Adulterated Honey using Multivariate Data Analysis to Facilitate Food Product Quality Control

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The adulteration of food products with materials of lesser value or even potentially unsafe origins has been observed worldwide in foods diverse as olive oil, basmati rice, meats and honey [1]. At first glance approaching suspect products with high resolution mass spectrometry can seem intimidating due to the high complexity of the data, as it is not unusual to find evidence of several thousand components. Development for many years has focused on addressing these large mass spectrometric data sets with statistical analysis to rapidly produce results easy to interpret [2,3]. With the Progenesis QI software package Waters has sought to provide an easy to use, highly visual guided workflow for the statistical analysis of mass spectrometry data. In Progenesis QI the steps to the workflow are in a logical order and include everything from importing data, detection of peaks, normalisation of samples, multivariate statistical analysis to determine statistically relevant components of interest that differentiate sample groups, and tools for identifying components of interest. The Progenesis software has been successfully utilised for the statistical analysis of metabolites [4,5], as metabolomics research has pioneered the comparison of groups for finding key differences. In this study the goal was to demonstrate the utility of using high resolution mass spectrometer data derived from honey and two possible adulterants to show that mass fingerprints inclusive of hundreds of compounds, can quickly differentiate these foods and combinations of the foods.

Data Acquisition Methods
To facilitate the comparison of several samples methods must be selected carefully and maintained while collecting data from all samples. The data set for statistical analysis is composed of Exact Mass Retention Time (EMRT) information for all components detected in the samples. Considerations are appropriate chromatography and mass spectrometer methods. Data was acquired using a Waters Acquity UPLC (classic model) and a Waters Xevo G2 QTof mass spectrometer (Melbourne, Australia). 
Methods were carried out using both positive and negative ion modes, with LC columns, gradients and buffers selected for appropriate screening with the specified ion modes. For positive ion mode data acquisition reverse phase chromatography was selected using a Waters UPLC BEH C18 1.7 µm particle, 2.1 mm x 100 mm column. Mobile phase A was water containing 0.1% (v/v) formic acid, mobile phase B was acetonitrile containing 0.1% (v/v) formic acid. Mobile phase B was held at 2% (v/v) for 15 seconds followed by a gradient to 99% (v/v) B over 12 minutes. The flow rate was maintained at 0.450 mL/min and column temperature was set at 45°C. For negative ion mode data acquisition HILIC chromatography was performed using a Waters BEH Amide 1.7 µm particle, 2.1 mm x 100 mm column, as this chromatography and ion mode are complimentary for simple sugars and some polar metabolites (5). Mobile phase A was 100% acetonitrile, mobile phase B was water containing 10 mM ammonium formate pH 8. Mobile phase B was held at 2% for 15 seconds followed by a gradient to 90% B over 12 minutes. The flow rate was maintained at 0.450 mL/min and column temperature was set at 60°C. Total acquisition times were 15 minutes. The mass spectrometry methods for both ion modes were MSE - a simple, patented method of unbiased data acquisition that comprehensively catalogues complex samples in a single analysis [6,7]. A mass range of 50 to 1200 m/z was scanned, with alternating low and elevated collision energy scans of 0.3 seconds. For elevated energy scans (the high energy fragment channel) the collision energy was ramped from 10 to 45 eV. Leucine-enkephalin was used as a lockspray [6], acquired for 0.5 seconds at 20 seconds intervals. The mass spectra of the lockspray are recorded in a separate channel that is used to recalibrate acquired data improving mass accuracy to within 5 ppm.

Samples used were honey (a major domestic Australian brand), a supermarket brand golden syrup (major ingredient was cane sugar syrup, referred to as ‘golden’), and a brand name maple flavoured syrup (major ingredient was wheat glucose syrup, referred to as ‘wheat’). As corn syrup based products were unavailable they were not included, but would make sense to include in some markets where it is a likely adulterant. Additional samples analysed were 1:1 mixtures of honey and the same golden syrup, and also of honey and the wheat glucose syrup. A QC mixture was prepared that was equal parts of each product. Each product was diluted 20x with water, mixed until homogeneous and passed through 0.2 micron microcentrifuge filters. A negative control of extraction solvent was the same dilution water passed through an identical filter. An injection volume of 5 µL was used for each sample.