Sweet Science

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.

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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.

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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.