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Ultra-Fast Analysis of Contaminant Residue from Propolis by LC/MS/MS Using SPE

Matthew Trass, Philip J. Koerner and Jeff Layne

Phenomenex, Inc., 411 Madrid Ave.,Torrance, CA 90501 USA

The rapid growth in the global economy has seen a dramatic increase in food imports from China, which has become a major exporter and consumer of agricultural products. Recent outbreaks of foodborne illnesses and the discovery of contaminated food and feed have generated significant media attention and highlighted the need for improved testing methods and procedures.

Potentially harmful contaminants such as fluoroquinolone antibiotics have been found in a number of products intended for human consumption. In some instances the

matrix of the contaminated product can make analysis very problematic. One such problematic matrix is propolis, a wax-like substance found in beehives that is used as a traditional medicine, for skin care and for a multitude of other purposes.

We describe here a method for the analysis of contaminant residues in propolis, which utilizes liquid-liquid extraction of the resinous propolis matrix followed by SPE to further clean up and concentrate the sample prior to LC/MS/MS analysis.

Introduction

Compound Structure

Ofloxacin

Norfloxacin

Ciprofloxacin

Enrofloxacin

Difloxacin

Sparfloxacin

Gemini®-NX

Ethane cross-linking sta-bilizes the silica particle, providing resistance to high pH attack while maintaining high efficiency and mechani-cal strength.

Sample Preparation

Weigh out 1 g of beeswax propolis and transfer to a 50 mL conical-bottom centrifuge tube.

Add 40 mL of methyl tert-butyl ether (MTBE). Shake contents vigorously by hand to mix, and then ultrasonicate for 30 minutes. Invert the tube at 5 minute

intervals during the ultrasonication to assist in sample dispersion.

Transfer 5 mL of the dissolved beeswax propolis solution to a 15 mL centrifuge tube. Add 5 mL of 10 mM monopotassium phosphate with 2 % trichloroacetic acid

(TCA) buffer. Shake the samples for 20 minutes using a mechanical shaker.

Centrifuge the suspension for 10 minutes at 4700 rpm at 15 °C.

Using a Pasteur pipette, transfer the lower fraction to a 15 mL test tube. Reserve the upper organic fraction.

Add 5 mL of the monopotassium phosphate with 2 % TCA buffer to the reserved organic fraction from step 5. Shake samples using mechanical shaker for 20 minutes.

Centrifuge the suspension for 10 minutes at 4700 rpm at 15 °C.

Discard the supernatant (including waxy substance in the bi-layer) and combine the aqueous fraction with the aqueous fraction from step 5. The beeswax propolis extract is now ready for SPE.

Solid Phase Extraction

The extracted beeswax propolis is cleaned up and concentrated using solid phase extraction (SPE)

Cartridge: Strata™-X 200 mg/6 mL

Part No.: 8B-S100-FCH

Condition: 4 mL Methanol (1-2 mL/min)

Equilibrate: 2 x 4 mL Water (1-2 mL/min) Note: Do not let sorbent run dry

Load: 10 mL extracted beeswax propolis sample (1-2 drops/sec)

Dry: >10” Hg for 5-10 minutes to remove residual water

Elute: 2 x 3 mL 49/49/2 Acetonitile/Methanol/Acetic acid (ca. 1 drop/sec)

Dry down: Nitrogen gas at 55 ºC

Reconstitute: 0.5 mL of initial mobile phase

LC/MS/MS Conditions

MRM conditions:

AnalyteRetention Time, min Q1 Q3 DP, V CE, V

Ofloxacin 1.6 362.2 318.2 71 25

Norfloxacin 1.69 320.2 276.2 61 23

Norfloxacin-d5 (IS) 1.69 325.2 281.2 70 25

Ciprofloxacin 1.78 332.2 288.2 71 31

Ciprofloxacin-d8 (IS) 1.78 340.2 296.2 71 25

Enrofloxacin 1.87 360.2 316.2 66 27

Difloxacin 1.99 400.2 356.2 66 29

Sparfloxacin 2.33 393.2 349.2 66 29

HPLC conditions:Column: Gemini-NX 3 μm C18

Dimensions: 50 x 2.0 mmPart No.: 00B-4453-B0

Mobile Phase: A: 0.1 % Formic acid in Water B: Methanol

Gradient:

Flow Rate: 0.4 mL/minInjection: 25 μL

Temperature: 40 °C

Time (min) B (%)

0.00 15

5.00 90

6.00 90

6.01 15

9.01 15

AB SCIEX™ API 4000™

Ion source conditions:IS: 4500

TEM: 550 ˚CGas1: 50Gas2: 50

Scan Type: MRM

Results

Figure 2. Norfloxacin extracted calibration curve ranging from 0.5 - 10 µg/L

Figure 1. Representative chromatogram of a 0.5 µg/L extract

19321

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2,34,5

6

7

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1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e5

1.4e5

1.5e5

1.6e5

1.7e5

1.8e5

1.9e5

2.0e5

2.1e52.2e5

Inte

nsity

, cps

1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 min

Ap

p ID

193

21

Analyte list: 1. Ofloxacin2. Norfloxacin3. Norfloxacin-D54. Ciprofloxacin 5. Ciprofloxacin-D8 6. Enrofloxacin 7. Difloxacin 8. Sparfloxacin

Analyte Conc. / IS Conc.

Ana

lyte

Are

a /

IS A

rea

5 10

23

22

21

20

1.9

1.8

1.7

1.6

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0.9

0.8

0.7

0.6

0.5

0.4

0.3

10

0.1

0.0

15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Figure 3. Ciprofloxacin extracted calibration curve ranging from 0.5 - 10 µg/L

Analyte Conc. / IS Conc.

5 10

27

26

24

22

20

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.015 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Ana

lyte

Are

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IS A

rea

Experimental Conditions for LC/MS/MS Analysis

LC/MS/MS Condition (acid, base, and neutral probes):Column: Kinetex® 2.6 µm C18

Dimensions: 50 x 2.1 mmMobile Phase: A: 0.1 % Formic acid in Water

B: 0.1 % Formic acid in AcetonitrileGradient: A/B (95:5) to (5:95) in 2 min, hold for 0.5

min, equilibrate for 1.5 minFlow Rate: 0.5 mL/min

Analysis condition for testosterone:Column: Kinetex® 2.6 µm C18

Dimensions: 50 x 2.1 mm Mobile Phase: A: 0.1 % Formic acid in Water

B: 0.1 % Formic acid in AcetonitrileA/B (30:70)

Flow Rate: 0.5 mL/min

Table 2. Absolute recovery for entire extraction process

Analyte Average recovery (n=3)

Norfloxacin 82 %

Ciprofloxacin 85 %

Difloxacin 75 %

Enrofloxacin 60 %

Ofloxacin 61 %

Sparfloxacin 72 %

Table 3. Quality Control Summary

AnalyteExpected

ConcentrationNumber of

Values % CV % Accuracy

Norfloxacin 1.5 7 5.79 110.8

Norfloxacin 7.5 8 5.84 104.4

Ciprofloxacin 1.5 7 3.59 112.4

Ciprofloxacin 7.5 8 5.6 109.4

Sparfloxacin 1.5 7 7.3 116.7

Sparfloxacin 7.5 8 7.33 124.9

Difloxacin 1.5 7 5.71 107.7

Difloxacin 7.5 8 8.44 114.6

Ofloxacin 1.5 7 16.5 128.4

Ofloxacin 7.5 8 6.96 94.1

Enrofloxacin 1.5 7 3.47 109.5

Enrofloxacin 7.5 8 5.5 112.7

Analyzing fluoroquinolones in propolis is problematic since both the analytes and the sample matrix are very hydrophobic. Because both the analyte and matrix are similar, an extended sample preparation procedure is required. Often, additional sample preparation steps can lead to decreased recovery. However, the described protocol acquired very acceptable recoveries ranging from 60-85 % (Table 2).

Following sample preparation, chromatographic separation was achieved using a Gemini-NX column (Figure 1). The run time is less than 3 minutes with baseline or nearly baseline resolution between each of the analytes. MS/MS acquisition in multiple reaction

monitoring (MRM) mode provided unique parent/daughter ion combinations for each analyte, allowing for accurate quantitation for any slightly co-eluting peaks.

Calibration curves for each of the fluoroquinolones were generated over the concentration range 0.5 – 10 µg/L (Figures 2 & 3). To investigate the accuracy and reproducibility of the method, QC samples were prepared at two concentrations, 1.5 and 7.5 µg/L, in dissolved propolis and multiple replicates analyzed (n=7-8). The results are summarized in Table 3 and demonstrate good reproducibility (% CV) and accuracy.

Results and Discussion

Conclusions

Due to the increasing use of antibiotics worldwide, there is a significant risk that products intended for human consumption have been contaminated with compounds such as fluoroquinolones. Therefore, testing for fluoroquinolones and related compounds is becoming more prevalent.

When analyzing compounds in a complex matrix such as propolis, a sample cleanup method is important as it reduces the amount of contamination that the LC/MS/MS system is exposed to.

SPE with Strata™-X in conjunction with the outlined sample preparation method is an effective way of concentrating and cleaning up propolis samples for fluoroquinolones analysis by LC/MS/MS.

Chromatographically, the Gemini-NX reversed phase HPLC column gives adequate resolution, allowing for an easily integrated peak with low detection limits.

TrademarksGemini is a registered trademark of Phenomenex, Inc. Strata-X is a trademark of Phenomenex, Inc. API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license.

© 2011 Phenomenex, Inc. All rights reserved.