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Molecularly Imprinted Polymer SPE for the Highly Selective Extraction of Fluoroquinolones from Bovine Kidney

By: Anna-Karin Wihlborg1, Olga Shimelis2, and An Trinh2, Reporter US Vol 27.1

Contributed Article

Anna-Karin Wihlborg1, Olga Shimelis2,and An Trinh2

1. MIP Technologies AB, Scheelevägen 22, 220 07, Lund, Sweden
2. Supelco, a division of Sigma-Aldrich, 595 N. Harrison Rd., Bellefonte, PA 16823, USA


Fluoroquinolones (FQLs) are a class of broad-spectrum antibiotics heavily used in veterinary and human medicine. Widespread usage of this class of antibiotics has resulted in the emergence of resistant bacterial strains (1). The presence of this antibiotic class in the environment at sub-therapeutic levels can lead to multiple FQL resistant bacterial strains. In addition, when used to treat animals, FQLs may end up in the human food change causing potential allergic reactions when consumed. The surveillance of FQLs is mandated by law. The EU has set strict Maximum Residue Limits (MRLs), the values of which depend on the particular compound and matrix. For example, the MRL for enrofloxacin in bovine kidney is set at 200 μg/kg. The US, Canada and Japan have also set MRLs but for a more limited range of fluoroquinolones.

In this report, we discuss the extraction of FQLs from bovine kidney using molecularly imprinted polymer SPE technology (SupelMIP™ SPE – Fluoroquinolones); and compared the technique against a tandem mixed mode SPE method. Note that SupelMIP FQL procedures have also been developed for honey and milk (2). The specific FQLs examined in this report include: sarafloxacin, norfloxacin, enrofloxacin, and ciprofloxacin (Figure 1).

Figure1. Structures of Fluoroquinolones

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What are SupelMIPs

SupelMIPs are based on molecularly imprinted polymers (MIPs). MIPs are a class of highly cross-linked polymer-based molecular recognition elements engineered to bind one target compound or a class of structurally related compounds with high selectivity. Selectivity is introduced during MIP synthesis in which a template molecule, designed to mimic the analyte, guides the formation of specific cavities that are sterically and chemically complementary to the target analyte(s). As a result, multiple interactions (e.g., hydrogen bonding, ionic, Van der Waals, hydrophobic) can take place between the MIP cavity and analyte functional groups. The strong retention offered between a MIP phase and its target analyte(s) allows for the use of exhaustive wash procedures during solid phase extraction that results in superior sample cleanup prior to analysis.

Extraction of FQLs from Bovine Kidney

Sarafloxacin, norfloxacin, enrofloxacin, and ciprofloxacin were spiked into bovine kidney at the levels of 0-75 μg/kg and I.S. d5 – norfloxacin 75 μg/kg. 2 g of spiked kidney sample was homogenized with 30 mL 50 mM sodium hydrophosphate, pH 7.4 and centrifuged for 10 min. at 5000 rpm. The resulting supernatant was filtered using a 0.45 μm filter and processed using the SupelMIP procedure described in Table 1 and the tandem polymer MAX and MCX procedure described in Table 2 (3). LC-MS/MS analysis was conducted using the method described in Table 3.

Table 1. SupelMIP SPE – Fluoroquinolones SPE Procedure(53269-U)

Table 2. Tandem Polymer MAX/MCX SPE Procedure (3)

Table 3. LC-MS/MS Conditions for Fluoroquinolones Analysis(581307-U)

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SupelMIP SPE Offers Low Background

Spiked bovine kidney was spiked with FQLs and extracted and analyzed using the procedures described in Tables 1-3. LC-MS/MS chromatograms of both blank and spiked (3 μg/kg) kidney using the SupelMIP approach are described in Figures 2 and 3, respectively. The SupelMIP approach provided low background and good analyte response at the mass transitions monitored.

Figure 2. LC-MS/MS Chromatogram of Blank Kidney Extracted with SupelMIP SPE (colored lines represent m/z transitions monitored)

Figure 3. LC-MS/MS Chromatogram of Fluoroquinolone Spiked Kidney (3 μg/kg) Extracted with SupelMIP SPE (colored lines represent m/z transitions monitored)

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Recovery & Decision/Detection Limit

Absolute and relative recovery values for both the SupelMIP and tandem polymer mixed-mode SPE procedures across a range of spike levels tested were comparable. Absolute and relative recovery values for the SupelMIP approach are described in Table 4. Recovery values for polymer MAX/MCX SPE methods are not shown. Note that although the recovery values were comparable, the SupelMIP procedure only required one SPE cartridge. In contrast, the tandem polymer MAX/ MCX SPE approach required two separate SPE procedures for sample cleanup.

Table 4. Absolute & Relative Recovery of FQLs in Bovine Kidney using SupelMIP SPE

The decision and detection limits for both the SupelMIP and tandem polymer MAX/MCX approach were calculated. The CCα(decision limit where alpha error is 1%) and CCβ(detection capability where beta error is 5%) were calculated (Table 5). Note that both decision and detection limits, CCα and CCβ values, were lower than the Polymer MAX/MCX SPE approach.

Table 5. Detection Limits for FQLs in Bovine Kidney using SupelMIP and Tandem Polymer MAX / MCX SPE

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In this report, we discussed the use of a molecularly imprinted polymer SPE procedure engineered and optimized for the selective extraction of fluoroquinolones in difficult sample matrices such as bovine kidney. The SupelMIP SPE – FQL procedure was compared against a method using tandem polymer MAX/MCX SPE. For the comparison study, recovery values were comparable across the two techniques; however, the SupelMIP approach only required one SPE method whereas the tandem polymer MAX/MCX approach required two SPE methods thereby significantly reducing overall assay time. In addition, decision (CCα) and detection limits (CCβ) were lower on the SupelMIP method relative to the tandem polymer MAX/MCX procedure.

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  1. Fluoroquinolone resistance – Overuse of fluoroquinolones in human and veterinary medicine can breed resistance, Piddock L, BMJ 1998; 317:1029-1030.
  2. SupelMIP SPE – Fluoroquinolones Data/Instruction Sheet – Available at http://www.
  3. Fluoroquinolone Antibiotics in Beef Kidney – Tandem Oasis MAX-MCX Method (Excerpt from Oasis Applications Notebook) – Available at http:// waters/library.htm?locale=en_US&lid=1534786.
  4. European Commission Council Directive 2002/657/EC on implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results.

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