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MATRIX SOLID-PHASE DISPERSION COMBINED WITH THIN-LAYERCHROMATOGRAPHY-DIRECT BIOAUTOGRAPHY FORDETERMINATION OF FLUMEQUINE RESIDUES IN MILK:IMPROVEMENT OF THE METHODWioleta Bka; Irena Maria Chomaa; Barbara Majer-Dziedzicb

aDepartment of Chromatographic Methods, M. Curie - Skodowska University, Lublin, Poland b

Department of Veterinary Microbiology, University of Life Sciences, Lublin, Poland

Online publication date: 17 May 2011

To cite this ArticleBk, Wioleta , Choma, Irena Maria and Majer-Dziedzic, Barbara(2011) 'MATRIX SOLID-PHASEDISPERSION COMBINED WITH THIN-LAYER CHROMATOGRAPHY-DIRECT BIOAUTOGRAPHY FORDETERMINATION OF FLUMEQUINE RESIDUES IN MILK: IMPROVEMENT OF THE METHOD', Journal of LiquidChromatography & Related Technologies, 34: 10, 920 927

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MATRIX SOLID-PHASE DISPERSION COMBINED WITH

THIN-LAYER CHROMATOGRAPHY-DIRECT BIOAUTOGRAPHY

FOR DETERMINATION OF FLUMEQUINE RESIDUES IN MILK:

IMPROVEMENT OF THE METHOD

Wioleta Bak,1 Irena Maria Choma,1 and Barbara Majer-Dziedzic2

1

Department of Chromatographic Methods, M. Curie Skodowska University,Lublin, Poland2Department of Veterinary Microbiology, University of Life Sciences, Lublin, Poland

& Flumequine is a fluoroquinolone antibiotic, mainly used in veterinary and, to some extent, inhuman treatment. It can be found as a residue in milk and in other food products. A new versionof matrix solid-phase dispersion of milk samples spiked with this antibiotic was developed. This

preseparation method was combined with thin-layer chromatographydirect bioautography to obtainsemi-quantitative results.Escherichia coli were used as test bacteria. The various modes of the

procedure were tested, and the one giving the best recovery of the antibiotics from milk was chosen.

Keywords flumequine, fluoroquinolones, matrix solid-phase dispersion, thin-layerchromatographydirect bioautography

INTRODUCTION

The matrix solid-phase dispersion (MSPD) procedure, introduced in1989 by S. A. Baker as a process for the disruption and extraction of viscous,semi-solid, or solid samples, involves blending the sample with a solid sup-port material, usually silica with bonded alkyl (e.g., C18) chains.[1,2] Ourprevious papers proved that bare siliceous sorbents with a low surface areacould be used for MSPD of milk samples before TLC or HPLC analysisinstead of expensive C18 sorbents.[3,4]

Fluoroquinolones are important synthetic antibacterial agents widelyused in human and veterinary treatment.[5] They are highly active againstGram-negative and moderately active against Gram-positive bacteria.

Address correspondence to Irena Maria Choma, Department of Chromatographic Methods,University of M. Curie-Skodowska, M. Skodowska Sq. 3, 20-031 Lublin, Poland. E-mail: irena.choma@

poczta.umcs.lublin.pl

Journal of Liquid Chromatography & Related Technologies, 34:920927, 2011Copyright# Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.571158

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Flumequine (9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[ij]quinolizine-2-carboxylic acid), belonging to fluoroquinolones, is often applied in farm-ing of cattle, poultry, and fish, both as a therapeutic agent and as a feed or

water additive to enhance weight gain. The residues of flumequine can bepresent in food causing harm to human health. The European Union hasset maximum residue limits (MRLs) for flumequine in various edible pro-ducts of animal origin. In the case of bovine milk, a very low MRL value(i.e., 50 ppb) has been established.[6,7]

High-performance liquid chromatography (HPLC) is the most com-mon method used in the analysis of flumequine as well as other fluoroqui-nolones in feed and food samples.[711] However, thin-layer chromatography(TLC) seems to be very convenient, especially when many samplesought to be analyzed at the same time.[11] Thin-layer chromatography-

direct bioautography (TLC-DB) combines planar chromatography withmicrobiological detection. The developed TLC plates are dipped in a bac-terial growth medium seeded with an appropriate bacterial strain. Visualiza-tion of antibacterial agents is usually carried out using tetrazolium salts,

which are reduced into intensely colored formazan by dehydrogenase of liv-ing microorganisms. Cream-white spots against a purple background pointto the presence of antibacterial agents on the TLC plate surface. Thelocation and size of growth inhibition zones allow for the information aboutthe kind and quantity of antibiotics.[12,13]

The conditions for semi-quantitative TLC-DB of flumequine were estab-

lished in a recent paper.[6] The MSPD procedure was combined withmicrobial detection using Chrom Biodip1 Antibiotics Test Kit (producedby Merck) based onBacillus subtilis.[14] Because this test is not commerciallyavailable any more, we had to develop our own bioautographic test. Wechose Gram-negative Escherichia colias the test bacteria. E. coliseem to bemore suitable thenBacillusfor bioautography of flumequine, which as men-tioned previously, is active mainly against Gram-negative bacteria.

In the present paper, MSPD based on Chromaton N-AW was used as apre-separation method for TLC-DB of flumequine residues in milk using as

test bacteria E. coli.

EXPERIMENTAL

Equipment and Reagents

DS sandwich chambers were purchased from Chromdes, Lublin,Poland.[15] Pre-coated silica gel TLC plates Si60 F

254were purchased from

Merck (Darmstadt, Germany).Flumequine was supplied by Sigma (St. Louis, MO, U.S.A.). Acetoni-

trile, hexane, dichloromethane, and methanol HPLC grade were from

Improved Determination Method for Flumequine Residues in Milk 921

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Merck (Darmstadt, Germany). Ammonia (25%) and 2-propanol were pur-chased from P.O.Ch. (Gliwice, Poland). Chromaton N-AW 0.160.2 mm

was from Lachema Brno, Chemapol (Praha, Czech Republic). Planimeter

was obtained from PZO (Warsaw, Poland). Reprostar 3 Video Camera wasfrom Camag (Muttenz, Switzerland). The MTT dye [3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyltetrazolium bromide] used to visualize antimicrobialactivity as well as Hepes and Triton X-100, laboratory grade, were obtainedfrom Sigma (St. Louis, MO, USA).

The test strain E. coli(ATCC 25922) was obtained from the AmericanType Culture Collections. Mueller-Hinton broth, Mueller-Hinton agar,and agarose were purchased from Biocorp (Warsaw, Poland).

Preparation of Standards

The stock solution of flumequine was prepared in 0.03 M NaOH at1mgmL1. It was stored at18C. The standard solution was yet preparedby diluting the stock solution with methanol to obtain concentrations at0.0005, 0.001, and 0.01 mg mL1. The milk samples were spiked with thestock solution at 10 ppm and 0.05 ppm level.

Matrix Solid-Phase Dispersion

Chromaton N-AW was blended with the spiked milk sample and aceto-

nitrile in proportion 2:1:1 (w=v=v) and packed into the syringe. In someexperiments, an additional layer of Chromaton N-AW (about 1 mL) wasput at the bottom of the syringe. The sample in the cartridge was defatted

with 10 or 20 mL of hexane (aspirated by the water pump) and the syringewas centrifuged for 5 min (8400 g). The hexane eluates were rejected.Then, 10 mL of dichloromethane was used to elute the antibiotics. The syr-inge was centrifuged again and both CH2Cl2 eluates were combined. Thesample was then evaporated to dryness; the test tube was rinsed with1 mL of dichloromethane, which was evaporated to dryness again and the

residue at the bottom of the tube was dissolved in 100m

L of methanol.

Preparations of the MSPD Cartridges at 10 ppm Level10 mL of milk was spiked with flumequine at 10 ppm level, and then

10 mL of acetonitrile was added to denaturize milk proteins. The milkspiked in this way was used to prepare four different types of cartridge:

A: 2 g (about 4 mL) of Chromaton were mixed with 1 mL of milk spikedand deprotoinized as described previously and put into the cartridge. A vol-ume of 10 mL of hexane was used to elute lipids. Then, flumequine waseluted from the cartridge with 10 mL of dichloromethane. The procedure

was exactly the same as in Ref. 6.

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B: 2 g (about 4 mL) of Chromaton were mixed with 1 mL of milk spikedand deprotoinized as described previously and put into the cartridge. Anadditional layer of about 1 mL of pure Chromaton, put at the bottom of

the cartridge, worked as a filter. The volume of 10 mL of hexane was usedto elute lipids. Then, flumequine was eluted from the cartridge with 10 mLof dichloromethane.

C: 2 g (about 4 mL) of Chromaton were mixed with 1 mL of milk spikedand deprotoinized as described previously and put into the cartridge. A vol-ume of 20 mL of hexane was used to elute lipids. Then, flumequine waseluted from the cartridge with 10 mL of dichloromethane.

D: 2 g (about 4 mL) of Chromaton were mixed with 1 mL of milk spikedand deprotoinized as described previously and put into the cartridge. Anadditional layer of about 1 mL of pure Chromaton, put at the bottom of

the cartridge, worked as a filter. A volume of 20 mL of hexane was usedto elute lipids. Then, flumequine was eluted from the cartridge with10 mL of dichloromethane.

Preparations of the MSPD Cartridges at 0.05 ppm LevelThe procedure D was repeated five times for milk spiked at MRL level,

that is, 0.05 ppm.

Sample Spotting and Development

The samples and the standards were applied to the TLC plate at 5 mLvolumes using a Hamilton microsyringe (Bonaduz, Switzerland) for10 ppm level or at 40mL volumes using a Linomat 5 Camag applicator (Mut-tenz, Switzerland) for 0.05 ppm level. The plates were developed to the dis-tance of about 9 cm. The mobile phase was dichloromethane=methanol=2-propanol=25%aqueous ammonia (3:3:5:2).

Bioautography

Pre-incubationA bacterial colony was taken from E. coli culture, put into 10 mL of

Mueller-Hinton (M-H) broth (pH 7.2 0.2, adjusted with Hepes) and incu-bated at 37C for 20 hr, the first hour in a shaker incubator and then on amagnetic stirrer. After that time, the bacterial mean concentration equaled4 109 c.f.u.=mL.

Incubation20 mL of M-H broth (pH 7.2 0.2) was inoculated with 1 mL of bac-

terial suspension obtained directly from the pre-incubation and placed


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on the magnetic stirrer for 2hr at 37C. Finally, bacterial concentrationequaled 8 107(c.f.u.=mL).

The developed TLC plates were dried successively in air and in a vac-uum desiccator. They were then immersed briefly in the microorganism sol-ution and incubated for 5hr at 37C. After incubation, the plates weresprayed with 0.2%MTT aqueous solution and incubated for about 30 minat 37C. Cream-white inhibition zones were observed against a purple back-ground. The plates were dried in air and scanned for documentation. Theinhibition zone areas were then measured with a planimeter.

RESULTS AND DISCUSSION

The MSPD procedure applied before the TLC-DB was established inrecent research for the determination of residues of flumequine in milk.[6]

The proportions of sorbent (Chromaton N-AW), milk, and acetonitrile

FIGURE 1 TLC-DB of the eluates obtained from milk spiked with flumequine at 10 ppm level (car-

tridges A and B) and of standards at 100 ppm (S1). The applied volumes equaled 5mL. (Figure availablein color online.)

FIGURE 2 TLC-DB of the eluates obtained from milk spiked with flumequine at 10 ppm level (car-tridges C and D) and of standards at 100 ppm (S2). The applied volumes equaled 5 mL. (Figure available

in color online.)

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were 2:1:1 (w=v=v). Acetonitrile was added to cause deproteinization ofmilk and to provide better recoveries of flumequine from milk. TheMSPD-TLC-DB procedure led to good recoveries, which equaled, indepen-dently, on the concentration of flumequine (10, 1, 0.1 and 0.05 ppm),about 75 percent. The bioautography was based on Chrom Biodip1 Anti-biotics Test Kit (Merck). Because this test is no longer available, we pre-

pared another bioautographic procedure based on E. coli. Bettersensitivity of our test, compared to the previously achieved, was expected,because flumequine is active mostly against Gram-negative bacteria. Inthe present paper, we made some additional experiments to obtain higherrecovery. We compared the previous procedure denoted as A with three

various modifications of the method denoted B, C, and D. Finally,four types of cartridges were prepared (each one in duplicates):

A: 10 mL of hexane without additional Chromaton bed; Figure 1;B: 10 mL of hexane with additional Chromaton bed, Figure 1;C: 20 mL of hexane without additional Chromaton bed, Figure 2; andD: 20 mL of hexane with additional Chromaton bed, Figure 2.See detailed descriptions of the cartridges in the Experimental section.The eluates obtained in the MSPD procedures were spotted on four TLC

plates: 2 2 plates spotted as in Figure 1 and Figure 2. Additionally, the stan-dards at 100 ppm level were applied. It is worthwhile to mention that eluatescoming from the MSPD procedure are about 10 fold concentrated

TABLE 1 Recoveries of Flumequine at 10 ppm Level (Mean From the Four Values) and StandardDeviations

MSPD A MSPD B MSPD C MSPD D

Recovery (%) 67.87 0.19 78.55 0.01 79.56 0.06 84.470.09

The results related to chromatograms described as Figures 1 and 2.

FIGURE 3 TLC-DB of the eluates obtained from milk spiked with flumequine at 0.05 ppm level (car-tridges D1-D5) and of standards: S3 0.0005 mgmL1 (0.5 ppm), S40.001mgmL1 (1ppm) and

S5 0.01mgmL1

(10 ppm). The applied volume equaled 40mL. (Figure available in color online.)


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compared to initial antibiotic level in milk (the volume is reduced by a factorof 10 when going from 1 mL sample of milk to 100 microliters of final eluatein methanol). As a consequence, the recoveries should be calculated inrelation to 100 pm, and not to 10 ppm, standards. Next to the spotting,the plates were developed with the mobile phase and dried at roomtemperature. After microbiological procedure, zones of inhibitions were

visualized, scanned, and measured. The recoveries of flumequine are shownin Table 1. As can be seen from the Table, the best recovery was obtained forprocedure D, which involved both additional filter and additional amount

of hexane as compared to procedure described in Ref. 6.The second part of our analysis concerned determination of flume-

quine residues in milk on MRL level, that is, at 0.05 ppm. The best MSPDvariant was chosen, that is, procedure D; according to it, five D car-tridges were prepared, denoted D1, . . ., D5. The eluates obtained fromD1-D5 cartridges were spotted on TLC plate together with three standards,0.0005 (S3), 0.001 (S4), and 0.01 (S5) mg mL1 (i.e., 0.5, 1, and 10 ppm)(Figure 3). The areas of inhibition zones were measured and the concentra-tions of flumequine in eluates from series D were calculated using the cali-bration curve constructed on the basis of standards inhibition zones

(Figure 4). Table 2 contains inhibition zone areas for eluates coming from

FIGURE 4 Calibration curve for the standards: A inhibition zone areas, c concentration ofstandards.

TABLE 2 Recoveries of Flumequine From Milk Spiked at MRL Level

Numberof Cartridge

Zone InhibitionArea A [cm2]

Concentrationc [mgmL1] Log c Recovery (%)

MeanRecoverySD

D1 0.47 0.000452 3.34511 90.35D2 0.45 0.000429 3.3672 85.87

D3 0.48 0.000463 3.33407 92.67 88.57 2.95D4 0.45 0.000429 3.3672 85.87

D5 0.46 0.00044

3.35616 88.08

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the D1-D5 cartridges, the related flumequine concentration in eluates fromthe cartridges, and the recoveries. The mean recovery obtained for five Dcartridges at 0.05 ppm equals 88.57 2.95% and is about 13% higher than

that obtained in Ref. 6.

CONCLUSIONS

The previously established MSPD-TLC-DB procedure was improved byenlarging the volume of hexane used for elution of lipids and by using anadditional layer of sorbent put at the bottom of the cartridge. The new pro-cedure gives better recoveries of flumequine residues from milk comparingto the old one (about 88%versus 75%) at very low MRL level that is at 0.05ppm.

REFERENCES

1. Barker, S. A. Applications of Matrix Solid-Phase Dispersion in Food Analysis.J. Chromatogr. A2000,880, 6368.

2. Barker, S. A. Matrix Solid-Phase Dispersion (MSPD). InSolid-Phase Extraction: Principles, Techniques,and Applications; Simpson, N. J. K., Ed.; Marcel Dekker, Inc.: New York, 2000, pp 361380.

3. Choma, I.; Pilorz, K. A Novel Application of Matrix Solid-Phase Dispersion for Determination of

Doxycycline and Flumequine Residues in Milk. J. Liq. Chromatogr. & Rel. Technol. 2004, 27,21432151.

4. Choma, I.; Komaniecka, I. Matrix Solid-Phase Dispersion Combined with Thin-Layer Chromato-graphy-Direct Bioautography for determination of Enrofloxacin and Ciprofloxacin Residues in

Milk. J. Liq. Chromatogr. & Rel. Technol. 2005, 28, 24672478.5. Lambert, H. P.; OGrady, F. W.Antibiotics and Chemotherapy, 6th Ed.; Longman: London, 1992.6. Choma, I. Thin-Layer Chromatography-Direct Bioautography of Flumequine Residues in Milk. J.

Liq. Chromatogr. & Rel. Technol. 2006, 29, 20832093.7. Hernandez-Arteseros, J. A.; Barbosa, J.; Compano, R.; Prat, M. D. Analysis of Quinolone Residues in

Edible Animal Products. Review. J. Chromatogr. A2002, 945, 124.8. Cinquina, A. L.; Roberti, P.; Giannetti, L.; Longo, F.; Draisci, R.; Fagiolo, A.; Brizioli, N. R. Determi-

nation of Enrofloxacin and Its Metabolite Ciprofloxacin in Goat Milk by High-Performance Liquid

Chromatography with Diode-Array Detection. Optimization and Validation. J. Chromatogr. A2003,987, 221226.

9. Tyczkowska, K. L.; Voyksner, R. D.; Kevin, L. A.; Papich, M. G. Simultaneous Determination of Enro-floxacin and Its Primary Metabolite Ciprofloxacin in Bovine Milk and Plasma by Ion-Pairing LiquidChromatography.J. Chromatogr. B1994, 658, 341348.

10. Corcia, A. D.; Nazzari, M. Liquid Chromatographic-Mass Spectrometric Methods for Analyzing Anti-biotics and Antibacterial Agents in Animal Food Products A Review. J. Chromatogr. A2002, 974,

5389.11. Choma, I. Screening of Enrofloxacin and Ciprofloxacin Residues in Milk by HPLC and by TLC with

Direct Bioautography.J. Planar Chromatogr. Modern TLC2006, 19, 104108.12. Botz, L.; Nagy, S.; Kocsis, B. Detection of Microbiologically Active Compounds. InPlanar Chromato-

graphy, 1st Ed.; Nyiredy, Sz., Ed.; Springer: Budapest, 2001, pp 489516.

13. Choma, I. The Use of Thin-Layer Chromatography with Direct Bioautography for AntimicrobialAnalysis. LC-GC Europe2005, 18, 482488.

14. Eymann, R.; Hauck, H. E. Chrom Biodip1 Antibiotics A High-Performance Bioautographic TestKit. In Proceedings Planar Chromatography, 2000; Lillafured: Hungary, 2000, pp 6775.

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