Aminoglycosides are a well-known class of antibiotics that are routinely monitored in animal-derived foods. Many countries have instituted regulatory limits for aminoglycosides. There is growing concern about the impact these microbial resistant compounds have on human health and development. A sensitive and robust analytical method is required in order to enforce the regulations and ensure the safety and quality of the food supply.
The focus of this study is the extraction and analysis of ten aminoglycosides from porcine tissues using molecular imprinted polymer (MIP) solid phase extraction with LC/MS/MS detection. The current USDA method does not reliably quantify levels of these analytes in porcine tissues1. This study utilized the unique extraction capabilities of MIPs to successfully quantitate ten aminoglycosides by LC/MS/MS at 100 ng/g (400 ng/g for neomycin) with recoveries ≥ 70%.
Molecularly imprinted polymers (MIPs) are solid phase extraction phases that are prepared by polymerizing either preformed or self-assembled monomer template complexes together with a cross-linking monomer. After removal of the template molecule, a polymer with binding sites for the template is obtained. MIPs exhibit selective target recognition and can be described as artificial receptors. Selectivity is predetermined by the template for a particular analyte or group of analytes. Figure 1 illustrates a generalized synthesis and MIP interaction.
Figure 1. Generalized Diagram of MIP Synthesis and Selective Analyte Interaction
Locally obtained pork muscle was homogenized with a commercially purchased food processor. Samples (2 g) were weighed into 50 mL polypropylene centrifuge tubes. Since aminoglycosides can bind to untreated glass surfaces, polypropylene vessels were used throughout the procedure. Samples were fortified using a mixed aminoglycoside standard prepared at 10 µg/mL from 1 mg/mL individual stock solution standards. The stock solutions were prepared from neat materials obtained from Sigma-Aldrich. Ten milliliters of the extraction solvent, 10 mM of potassium phosphate monobasic (KH2PO4) with 0.4 mM ethylenediaminetetraacetic acid (EDTA) and 2% trichloroacetic acid (TCA) were added, and samples shaken for 2 min. Samples were then centrifuged for 5 min. at 3200 rpm, and the supernatant was decanted into a clean 50 mL polypropylene centrifuge tube. An additional 10 mL of extraction solvent was added to the pork muscle sample, and the shaking and centrifuge steps were repeated. The supernatant was combined with the previous supernatant, and then 20 mL of 50 mM potassium phosphate in water (pH = 7.8) was added. The pH of the sample was adjusted to 7.5 with concentrated ammonium hydroxide.
The SupelMIP® SPE-Aminoglycoside cartridge was conditioned with 1 mL of methanol followed by 1 mL of 50 mM potassium phosphate in water (pH = 7.8). Three milliliters of extracted pork sample were passed through the cartridge. The cartridge was then washed with 3 mL of water, dried on high vacuum for 2-3 min, washed with 1 mL of 60:40 water:acetonitrile (v/v) and dried with slight vacuum for 10 sec. The cartridge was finally washed with 1 mL of 50:50 dichloromethane:methanol (v/v) and dried with slight vacuum for 10 sec. Analytes were eluted with 1 mL of 1% formic acid containing 5 mM heptafluorobutyric acid (HFBA) in 80:20 acetonitrile:water (v/v).
Eluted samples were vortexed and transferred to 750 µL polypropylene HPLC vials. Analytical separation was performed using an Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 µm HPLC column (53823-U). Figure 2 depicts chromatograms of the analytes in pork muscle extracts. Quantitation was performed using matrix matched calibration standards, ranging from concentrations of 10 ng/mL to 1000 ng/mL.
Figure 2.LC-MS/MS Analysis of Aminoglycosides after SupelMIP SPE Cleanup
column: Ascentis Express C18, 10 cm x 2.1 mm I.D., 2.7 µm (53823-U); mobile phase: (A) 5mM HFBA in water, (B) 5 mM HFBA in acetonitrile; gradient: 20 to 90% B in 3.0 min; held at 90% B for 1 min; 90 to 20% B in 0.1 min; held at 20% B for 5.9 min; flow rate: 0.4 mL/min; temp.: 40 °C; injection: 10 µL; det.: MS/MS, ESI(+), MRM
Recoveries for the 10 aminoglycosides are given in Figure 3. Most of the analyte recoveries were ≥ 70%, except for neomycin and tobramycin. Low recoveries for neomycin and tobramycin may be attributed to insufficient elution of the analyte from the SPE material. Both of these analytes have a higher number of amino groups which could lead to stronger binding of the analyte to the MIP sorbent.
The use of the SupelMIP SPE-Aminoglycoside cartridge kept the analytes bound to the sorbent while a series of aggressive washes were applied to the sorbent to eliminate matrix interferences. The absence of matrix effects may be an indication of superior sample cleanup. The resulting matrix effects were evaluated by comparing solvent prepared standards to matrix-matched standards. The matrix factor was calculated for each analyte in Figure 4. Matrix factors close to 1.0 indicate little to no matrix influence on analyte detection. Values significantly greater than 1.0 suggest matrix enhancement on the analyte and values less than 1.0 are considered to be the result of matrix suppression. For these analytes, significant matrix enhancement was observed for neomycin and gentamicin with matrix factors greater than 2.
Figure 3. Aminoglycoside Recoveries in Pork Muscle Fortified at 100 ppb each (except for Neomycin at 400 ppb)
Figure 4. Matrix Factor for Aminoglycosides in Pork Muscle
A simple and sensitive method for the cleanup, analysis and quantification of aminoglycosides in pork muscle has been developed using SupelMIP SPE-Aminoglycosides and LC/MS/MS analysis. This method was able to successfully obtain recoveries ≥ 70% for most analytes including amikacin; whereas, the current USDA method does not reliably confirm amikacin in porcine tissues1. The unique features of the MIP material afforded the ability to wash additional matrix interferences off of the cartridge prior to eluting the analytes of interest.