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From: Food Quality & Safety magazine, February/March 2010

Sweet Science

LC-MS/MS technology improves honey screening

by Dipankar Ghosh

Throughout history, people have used honey to sweeten and add flavor. Although its sweetness is similar to that of granulated sugar, honey has a distinctive flavor that is largely determined by the flower type from which the nectar is gathered.

According to the Food and Agriculture Organization of the United Nations (FAO), current world honey production is estimated at 1.3 million tons annually.1 The majority of honey produced each year is designated as table honey and intended for direct consumption. The remainder is used as an ingredient in a wide range of products. The food industry uses honey extensively to sweeten and flavor baked goods, cereals, sauces, and beverages. It is also used as a coloring and an emollient in cosmetics such as soap, shampoos, and lotions, as well as in the pharmaceutical industry, primarily to flavor cough remedies and throat lozenges and to soothe and coat the throat.

In total, about 300,000 tons of honey is traded internationally each year. The European Union, the United States, and Japan, which all depend heavily on imported honey to meet consumer demand, together account for 70% of all imports. Given the global patterns that exist in the movement of honey between consuming and producing countries, there is a great international need for analysis to prevent honey that has been contaminated by pesticides, insecticides, or antibiotics from reaching the market.

Environmental contaminants and antibiotics are the most common residues found in honey. For instance, nectar and pollen collected from pesticide-treated flowers can result in contaminated honey. Persistent residues from the antibiotics used to control bacterial diseases in bees can also be a contaminant. Because of extensive honey exporting and importing, analyzing for these contaminants is challenging. One country may approve certain pesticides or antibiotics, while another may ban them. Approved compounds may have varying restrictions on permissible exposure levels.

The Challenge

Increasing concern over the presence of antibiotic and pesticide residues in honey and the related potential health threats to humans has led food quality control laboratories to develop fast and efficient detection methods. The complex honey matrix and the large number and variety of potential contaminants mean that analysis is extremely challenging.

Fundamentally, honey is a highly concentrated solution of two invert sugars, dextrose and levulose, in water with small amounts of numerous complex sugars. In addition to these sugars, which are responsible for the principal physical characteristics and behavior of honey, it also contains aromatic volatile oils, which give it flavor, along with minerals, various enzymes, vitamins, and pigments. These minor constituents, largely responsible for the differences among individual honey types, contribute to the complexity of the honey matrix.

The basic analytical requirements for food analysis are high-resolution, high-throughput, high-sensitivity detection and the quantitation of contaminants at or below the maximum residue limit (MRL) of the compound in a given food matrix.2 Professionals in the food safety and quality control fields recognize liquid chromatography-tandem mass spectrometry (LC-MS/MS) as the central analytical technology. LC-MS/MS provides high-speed, high-resolution, and high-sensitivity separation and quantitation of various chemical compounds. An LC-MS/MS-based technique is also useful as a simultaneous screening method for the multiple classes of contaminants at trace levels in honey.

Honey analysis, like every food analysis, starts with sample preparation. Sample preparation is widely accepted as one of the most critical steps of the LC-MS/MS analysis. The increased demand from food analysis laboratories for higher throughput, higher accuracy, and lower matrix interference has made sample preparation the bottleneck step in the analysis.

Conventional sample preparation for LC-MS/MS analysis of honey is time and labor intensive and often involves pH modification, hydrolysis, liquid-liquid extraction (LLE), solid phase extraction (SPE), solvent evaporation, and pre-concentration steps to isolate and enrich target analytes from the honey matrix. When manually undertaken, these offline techniques are often costly and can result in low sample throughput.

One Solution

Food quality control laboratories are challenged by their need for multi-component quantitation, their desire for limited or no sample preparation, and their requirement to make quality control screening cost effective. New automated, online sample extraction techniques, such as Thermo Scientific TurboFlow technology coupled with LC-MS/MS, can reduce sample preparation and eliminate the disadvantages of conventional techniques.

This new technology allows for direct injection of the honey samples into the MS/MS system, which eliminates time-consuming and costly steps, simplifying the sample preparation process and increasing sample throughput. The technology reduces sample preparation time from hours to minutes and significantly decreases analytical costs. This patented technique also enables automatic removal of proteins and larger molecules from the complex honey mixture. When combined with a triple-stage quadrupole mass spectrometer, efficient quantitative results are possible with reduced levels of ion suppression and chemical noise compared to traditional techniques.

Representative selected reaction monitoring chromatogram (20 µg/kg) showing the selected ion transitions and retention times for the studied analyte.
click for large version
Representative selected reaction monitoring chromatogram (20 µg/kg) showing the selected ion transitions and retention times for the studied analyte.

Automated online sample extraction technology is based on turbulent flow chromatography, an innovative approach to sample preparation based on chromatographic principles. This process combines principles of diffusion, chemistry, and size exclusion to eliminate matrix interferences while capturing analytes of interest. When the mobile phase flows through the turbulent flow column, high linear velocities are created that are 100 times greater than those typically seen in high-pressure LC columns. This high linear mobile phase velocity and the large interstitial spaces between the column particles create turbulence within the column, which quickly flushes the large sample compounds through the column to waste before they have an opportunity to diffuse into the particle pores, while smaller molecular weight molecules are able to diffuse into the particle pores.

Chemistry also separates analytes from other sample molecules. Those sample molecules that have an affinity to the chemistry inside the pores bind to the column particles’ internal surface. The small sample molecules that have a lower binding affinity quickly diffuse out of the pores and are flushed to waste. A mobile phase change then elutes the small molecules that were bound by the turbulent flow column to the mass spectrometer or to a second analytical column for further separation.

Applications in Screening

A broad, generic, automated LC-MS/MS method has been developed for screening multi-class antibiotics in honey using dual online turbulent flow extraction columns with different chemistries.3 Ten representative antibiotics used in honey, belonging to four different structural classes, were selected: sulfonamides, tetracyclines, aminoglycosides, and macrolides. Sample preparation time was minimal, requiring only the addition of a buffer to reduce sample viscosity. The total LC-MS/MS method run time was less than 18 minutes. This design facilitates the separation and quantification of all of the representative compounds in the complex honey matrix in a single analysis.

Chromatography comparison of CAP selected reaction monitoring m/z 257 transition (upper traces) and CAP-d5 (lower traces) in pre-blank honey matrix (panel A), at lower limit of quantitation of 0.047 µg/kg (panel B).
click for large version
Chromatography comparison of CAP selected reaction monitoring m/z 257 transition (upper traces) and CAP-d5 (lower traces) in pre-blank honey matrix (panel A), at lower limit of quantitation of 0.047 µg/kg (panel B).

For the quantitation of 12 fluoroquinolones and four quinolones in honey, a sensitive and reproducible LC-MS/MS method has been developed.4 An online extraction method using turbulent flow chromatography was employed instead of a traditional SPE method. The sample preparation time decreased from five hours to 40 minutes. The limits of quantitation (LOQ) for the majority of analytes were one µg/kg (parts per billion) with no matrix interference. This online extraction, coupled with a triple-stage quadrupole mass spectrometer, is an excellent total solution for the quantification of a large number of compounds in honey.

A quick, automated sample preparation using the LC-MS/MS method was also developed for the screening of chloramphenicol in honey.5 The only pretreatment required was dilution with water to reduce sample viscosity. The method is sensitive enough to detect 0.023 µg/kg and quantify 0.047 µg/kg of chloramphenicol in honey, significantly lower than the minimum required performance limit of 0.3 µg/kg set by the European Union. Compared to offline detection such as SPE, QuEChERS (quick, easy, cheap, effective, and safe), and LLE, sample preparation with the TurboFlow method was between seven and 24 times faster. The LC-MS method run time was equal to or as much as four times faster than offline detection. Finally, the limit of detection (LOD) was between 5.7 and 20 times lower, and the lower limit of quantitation was between 3.7 and 27 times lower.

Chromatogram of five fluoroquinolone antibiotic standards at five parts per billion using online clean-up of the Aria TLX-1 system. The system provided a sensitive and reliable analytical method of detecting a full range of antibiotics.
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Chromatogram of five fluoroquinolone antibiotic standards at five parts per billion using online clean-up of the Aria TLX-1 system. The system provided a sensitive and reliable analytical method of detecting a full range of antibiotics.

Case Study

The Korea Beekeeping Association (KBA) needed to find an analytical solution that could detect multi-component antibiotics simultaneously and at low levels in honey analysis. They determined that an LC-MS/MS method using a Thermo Scientific Aria system powered by TurboFlow technology was superior to offline sample preparation techniques for antibiotic residue analysis in honey.

The large number and variety of potential contaminants in honey presented a challenge to the KBA. The online sample extraction method on the Aria TLX-1 system provided both increased analysis throughput and higher reproducibility. The system virtually eliminated pre-injection sample preparation, saving labor costs as well as increasing productivity. In addition, matrix effects, which are typically a challenge in LC-MS/MS analysis, decreased. The performance of the method was excellent. The LOD of one ng/mL achieved are well below the maximum residue limits of the antibiotics tested. ■

 

Ghosh is strategic marketing manager, Food Safety Solutions, Thermo Fisher Scientific. For more information, call (800) 246-4550, e-mail turboflow@thermo.com, or go to www.thermo.com/turboflow.

References

  1. Food and Agriculture Organization of the United Nations. FAO Web site. Available at: www.fao.org. Accessed January 26, 2010.
  2. 2. Soler C, Mañes J, Picó Y. The role of the liquid chromatography-mass spectrometry in pesticide residue determination in food. Crit Rev Anal Chem. 2008;38(2):93-117.
  3. 3. Lafontaine C, Shi Y, Espourteille FA. Multi-class antibiotic screening of honey using online extraction with LC-MS/MS. Thermo Scientific Application Note 464. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_51570.pdf. Accessed January 26, 2010.
  4. 4. Hammel Y-A, Schoutsen F, Martins CPB. Analysis of (fluoro)quinolones in honey with online sample extraction and LC-MS/MS. Thermo Fisher Scientific Application Note 465. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_51980.pdf. Accessed January 26, 2010.
  5. 5. Lafontaine C, Shi Y, Espourteille F. Measurement of chloramphenicol in honey using automated sample preparation with LC-MS/MS. Thermo Scientific Application Note 473. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_52958.pdf. Accessed January 26, 2010.

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