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From: Food Quality & Safety magazine, August/September 2009

Accurate Testing of Complicated Food Matrices

LC/MS technology avoids manual sample prep and increases throughout

by Yang Shi, PhD; Catherine Lafontaine; John A. Fink; and Francois A Espourteille

Worldwide food safety concerns have risen dramatically as the number of food contamination incidents and product recalls has increased. Accurately monitoring contaminant levels in food and agricultural products is essential to assure the safety of the food supply and to manage human health risks.

It is well-known that the basic analytical requirements in food analysis are high-resolution, high-throughput, high-sensitivity detection and quantification of contaminants at or below the maximum residue limit (MRL) or tolerance of the compound in a given food matrix. Liquid chromatography-mass spectrometry (LC/MS) as the central enabling technology has been recognized as an indispensable tool in the food safety and quality control fields.1 LC/MS provides high-speed, high-resolution, and high-sensitivity separation of various chemical compounds.

Every food analysis starts with sample preparation, widely accepted as one of the most critical steps of LC/MS. Increased demand for higher throughput and accuracy and lower matrix interference from food analysis laboratories has made sample preparation the largest bottleneck. Currently, solvent extraction and solid phase extraction (SPE) are two of the most commonly used methods to isolate and/or enrich target analytes from food matrices. Done manually, these offline techniques are often labor-intensive, time-consuming, and costly, resulting in low sample throughput. Turbulent flow chromatography technology can do away with the need for lengthy offline sample preparation steps, thereby eliminating these disadvantages.

Turbulent Flow Chromatography

This article will review a number of key applications in food safety using turbulent flow chromatography. All experiments used an LC/MS system (Thermo Scientific Aria TLX liquid chromatography system) powered by turbulent flow chromatography (Thermo Scientific TurboFlow technology) to separate analytes from various matrices prior to MS/MS analysis. The system injected the sample directly onto a narrow diameter (0.5 mm or 1.0 mm) chromatography column (the patented TurboFlow column) packed with large particles. High linear velocities are created inside the column, which force large molecules to flow quickly through to waste while retaining the small molecule analytes.

The technology is an improvement over traditional SPE because it uses reusable extraction columns in a closed system, reducing the time required for offline sample preparation from hours to minutes. It also allows automatic removal of proteins and larger molecules in complex mixtures by combining turbulence, diffusion, and chemistry. Shows the typical configuration of a single-channel TLX system.

By injecting food samples directly into the LC/MS system, which eliminates time-consuming and costly sample pre-paration steps, food safety and quality laboratories can achieve significant analytical improvements. Turbulent flow chromatography technology also allows the broad selection of stationary phases. These features make the technology a versatile and important tool in the food safety arena.

Applications in Food Safety

1) Veterinary drugs and chemicals: In recent years, there has been increased concern about the use of unauthorized veterinary drugs and other potentially hazardous chemicals in farming operations. The U.S., Canada, the European Union, and many other countries have either banned or limited the usage of many veterinary drugs involved in food production.

Four common chemical residues in fish, shrimp, and pig liver were analyzed using a triple stage quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Access).2 Malachite green (MG), a triphenylmethane dye, is an effective and inexpensive fungicide used in aquaculture, particularly in Asian countries. The principle metabolite, leucomalachite green (LMG), has been shown to accumulate in fatty fish tissues treated with MG due to its longer retention time.

Ciprofloxacin is a synthetic, broad-spectrum antibiotic belonging to the fluoroquinolone group that is used to treat severe bacterial infections. Tetracycline is a polyketide antibiotic that is highly effective against a number of gram-positive and gram-negative bacteria. The MRLs for these analytes range from 2 µg/kg for the sum of MG and LMG residues in fish muscle to 100 µg/kg for both ciprofloxacin and tetracycline in muscle for all food-producing species.

The total offline sample preparation time was approximately 30 to 40 minutes, including homogenization, centrifugation, and calibrator preparation. Compares representative standard high performance liquid chromatography (HPLC) and turbulent flow chromatography method chromatograms of 500 ng/kg (parts per trillion) tetracycline in a fish matrix. Using turbulent flow chromatography technology, the limits of quantitation (LOQs) were significantly lower (two- to 10-fold) for all four analytes using online extraction followed by LC/MS/MS compared to standard HPLC. This indicates that the LC/MS system can remove endogenous interferences, thus reducing ion suppression effects and improving detection limits.

2) Antibiotics in honey: Antibiotics are commonly used in beehives to control bacterial disease in honeybees, although caution is required to prevent persistent residues in food-grade honey. If antibiotic residues are present in high enough quantities, allergic reactions and bacterial resistance may develop. The conventional sample preparation for LC/MS/MS analysis of antibiotics in honey is time- and labor-intensive, often involving pH modification, hydrolysis, liquid-liquid extraction, SPE, solvent evaporation, and pre-concentration, and suffering, therefore, from low throughput. In addition, it is always an analytical challenge to deal with a large number of antibiotics belonging to different classes and often requiring multiple LC/MS methods.

Ten representative antibiotics used in honey, belonging to four different structural classes, were selected: sulfonamides, tetracyclines, aminoglycosides, and macrolides.3 The only offline sample preparation step required was the aqueous buffer dilution of raw honey to reduce the sample viscosity, which took less than 10 minutes. The online extraction clean-up was accomplished using a turbulent flow chromatography method involving two TurboFlow columns placed in tandem—a mixed mode anion exchange column and a polar-capped polymer-based column. Simple sugars were un-retained and moved to waste during the loading step while the analytes of interest were retained on the extraction column set. This was followed by organic elution to an end-capped silica-based mixed mode reversed-phase analytical column and gradient elution to a triple stage quadrupole mass spectrometer (Thermo Scientific TSQ Quantum Ultra). The total LC/MS/MS method run time was less than 18 minutes. A representative chromatogram of the 10 analytes at 100 ng/mL in 1:1 honey/ buffer was developed.

The results indicated that using two online turbulent flow chromatography extraction columns with different chem-istries extended the affinity range—further facilitating the separation and quantification of all of the representative compounds that have different chemical properties—in a complex honey matrix in a single analysis.

Quinolones, including fluoroquino-lones, in honey were also investigated.4 Instead of an SPE method, an online extraction method using turbulent flow chromatography was developed. The sample preparation time for the entire batch, including 16 compounds, dropped from five hours to 40 minutes, eliminating 80% of sample preparation time. The LOQs for the majority of analytes were 1 µg/kg (ppb) with no matrix interference. Representative selective reaction monitoring chromatograms at 20 µg/kg were developed and showed the selected ion transitions and retention times for the studied analytes.

3) Melamine in dairy products: The most notorious food safety incident in 2008 was the Chinese milk scandal involving melamine-tainted milk and infant formula. This incident triggered the largest global recall of Chinese-made diary products to date and prompted much stricter food safety regulations worldwide.

Consequently, scientists have developed many methods to analyze melamine residues in dairy-based products. Most of these approaches employ offline, disposable, cation exchange, SPE cartridges to prepare samples, coupled with LC/MS (MS/MS) analysis.

The goal in this experiment was to measure melamine with minimal sample preparation and high sample throughput. Using an LC/MS system, no offline sample extraction was required. Liquid or powdered dairy products were mixed with a solution of ammonium acetate in water and acetonitrile. After centrifugation, the supernatants were injected into the system. The quick-elute method took four minutes, with a one-minute data window. The lower detection limit was at least 50 ppb. Ion suppression caused by the co-elution of matrix components was minimal due to use of an atmospheric pressure chemical ionization source instead of the commonly used electrospray ionization source. The carryover level was also well-controlled and below 1%.5

Besides these representative studies, Thermo Fisher Scientific is actively studying an array of other contaminants (pesticides, mycotoxins, beta-agonists) in a variety of food matrices (fruit juice, wine, meat, and animal organs) using the TLX system with TurboFlow technology to minimize sample preparation, enhance analysis accuracy and reliability, and improve sample throughput.

Multiplexing capabilities of certain LC/MS systems such as the Thermo Scientific Transcend system allow up to four independent, parallel HPLC systems to run into a single MS, quadrupling the throughput of a traditional LC/MS. In addition, this unique multiplexing technology has attracted a great deal of interest because of its capability to run multiple methods simultaneously, one of the most important features desired by food safety and quality laboratories.

Online sample extraction utilizing turbulent flow chromatography coupled with LC/MS/MS and complementary techniques has gained popularity in the food safety arena. The objective of this technology is to provide automated, high-resolution, high-sensitivity, and high-specificity separation of target analytes from extremely complex food matrices, removing the need for manual sample preparation and increasing sample throughput. Turbulent flow chromatography also facilitates mass spectrometry detection and quantitative measurement and minimizes ion suppression and matrix effects. In addition, the multiplexing capability of the Aria TLX system can quadruple the throughput of a turbulent flow chromatography method, providing unmatched productivity and cost savings.

Dr. Shi is a senior scientist, Lafontaine is an applications chemist, Fink is a product manager, and Dr. Espourteille is manager, applications, at Thermo Fisher Scientific. For more information, contact Dr. Shi at yang.shi@thermofisher.com or at (508) 520-5575.

References

  1. Soler C, Manes J, Pico Y. The role of the liquid chromatography-mass spectrometry in pesticide residue determination in food. CRC CR Rev Anal Chem. 2008;38(2):93-117.
  2. Yang C, Ghosh D. LC-MS/MS analysis of malachite green, leucomalachite green, ciprofloxacin, and tetracycline in food samples using a TurboFlow method. Thermo Fisher Scientific Application Note 442. Available at: www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile_9061.pdf. Accessed August 2009.
  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 August 4, 2009.
  4. Hammel Y, 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 August 4, 2009.
  5. Di Bussolo J, Rohm R. Comparing ESI and APCI sources to screen dairy-based foods for melamine by rapid on-line extraction with LC-MS/MS. Paper presented at: 57th ASMS Conference on Mass Spectrometry and Allied Topics; June 3, 2009; Philadelphia.

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