美国农业食品化学文章

J. Agric. Food Chem. XXXX, XXX, 000–000

DOI:10.1021/jf9040518

A

Effect of Heating on the Stability of Quinolones in Milk

M. R OCA , †M. C ASTILLO , ‡P. M ARTI , ‡R. L. A LTHAUS , §AND M. P. M OLINA *,†

†

cnica de Valencia, Camino de Vera 14, 46071Instituto de Ciencia y Tecnologıa Animal, Universidad Polite

‡

blica de Valencia, Avenida de Catalunya 21, 46020Valencia, Valencia, Spain, Laboratorio de Salud Pu

§

tedra de Biofısica, Facultad de Ciencias Veterinaria, Universidad Nacional del Litoral, Spain, and Ca

RPL Kreder 2805, 3080Esperanza, Argentina

Nowadays, the possible public health risk associated with the presence of quinolone residues and other antibiotics in milk is well-known, but there is a lack of information about the effect milk pro-cessing temperatures have on the presence of antimicrobial residues. The aim of this work was to determine the effect of different temperatures and heating times on the concentration of quinolones in milk by employing liquid chromatographic equipment analysis with fluorescence detection. In order to determine the thermo-stability of these compounds, the first-order kinetic model was app-lied, and the activation energies, half-lives, and percentages of degradation of each compound were calculated. Results showed that quinolones are very resistant to different heat treatments with maximum losses of concentration of 12.71%for ciprofloxacin and 12.01%for norfloxacin at 120°C and 20min. The high stability of quinolones represents a significant risk to human health because the residues of these antibiotics can remain in milk after heat treatment and, therefore, can reach the dairy industry and consumers.

KEYWORDS:Quinolones; milk; heat treatments; first-order kinetic model; thermo-stability

INTRODUCTION

Quinolones and fluoroquinolones are a large, powerful, and expanding group of synthetic antimicrobial compounds whose main action is the inhibition of bacterial DNA-gyrase within the bacterial cell (3). In the last 10years, these compounds have been used in veterinary medicine to treat various diseases as they are highly active against a wide range of Gram-negative and Gram-positive bacteria, but mainly for the treatment and prevention of cattle mastitis (20).

The abuse or misuse of quinolones, and other antimicrobial agents in veterinary medicine, can constitute a serious public health risk associated with the presence of residues in foods of animal origin, and often induce problems in the manufacturing of products (17).

In recent years, some studies have shown that the excessive therapeutic use of quinolones in humans may be related with allergic reactions and the emergence of resistance to Campylo-bacter and Salmonella (1, 19, 22), indicating that the use of these antibiotics in food producing animals can have a direct impact on public health (8, 25). To ensure control over the presence of quinolone residues in foodstuffs of animal origin, Regulations 37/2010/CEof the European Union (6) sets maximum residue limits (MRLs)for some of these substances in milk (enrofloxacinand ciprofloxacin, 100μg kg -1; flumequine, 50μg kg -1; and marbofloxacin, 75μg kg -1).

Microbiological methods based on the inhibition of Geobacil-lus stearothermophilus are most frequently used for the screening

*Towhom correspondence should be addressed. Tel:(34)963877431. Fax:(34)963877436. E-mail:[email protected].

analysis of milk in farms and dairy industries. These methods detect β-lactam antibiotics efficiently and in some cases tetra-cyclines, but are not capable of detecting quinolones at or near MRL levels. Also, some other methods, such as protein receptor-binding, inmuno-enzimatic tests, etc. are employed routinely at the farm level and in the dairy industry because they are fast and simple to use, but the majority of them are specific and detect merely β-lactam and sometimes tetracycline residues.

Only a few methods for the detection of quinolones in milk are available, such as a bioassay based on the inhibition of Escherichia coli as part of multiresidue microbiological systems (9, 16) and some protein receptor-binding tests (e.g.,Rosa Charm Enroflox). These methods are not generally used in quality control schemes at the farm and dairy industry levels, which means that the presence of quinolones in milk often remains unchecked.

Moreover, in the past 15years, the online combination of chro-matography with different detectors has developed into a widely applicable detection system for antimicrobial residue analysis in foodstuffs. Current methods for detecting quinolones in biologi-cal matrixes are based on liquid chromatography (LC),mainly with fluorescence, ultraviolet, mass spectrometry, and tandem mass spectrometry (MS/MS)detection (4, 5, 7, 11, 12, 14, 23, 26). These methods allow for the detection and quantification of antimicrobial agents with high selectivity and sensitivity. How-ever, they are slow and costly and also require highly qualified personnel for their use. That is why these methodologies are gene-rally only used by quality control and public health laboratories but not for screening in farms and the dairy industry.

Milk in the dairy industry is subjected to different thermal treat-ments before marketing to ensure its quality and preservation.

pubs.acs.org/JAFC

XXXX American Chemical Society

B J. Agric. Food Chem., Vol. XXX, No. XX, XXXX However, there is a lack of scientific research regarding the effect milk processing temperatures can have on the presence of anti-microbial residues (3).

The relevance of the probable heat inactivation of quinolone residues in milk for food safety makes it necessary to carry out studies that evaluate the effect of processing of these compounds. Therefore, in a previous study we analyzed heat treatments in different groups of antibiotics employing bioassays with various microorganism tests, and we studied certain time -temperature combinations (28-30). However, we considered whether it is necessary to continue with this study applying quantitative tech-niques such as HPLC to establish kinetic models of degradation concerning the most frequently used antibiotics in cows.

Therefore, the aim of this work is to establish a kinetic model of degradation of quinolones by heating and estimate the effect of different heat treatments on the concentrations of ciprofloxacin, enrofloxacin, norfloxacin, flumequine, and oxolinic acid in milk.

MATERIALS AND METHODS

Chemicals and Reagents. Acetonitrile and methanol were of HPLC-grade; 10M potassium hydroxide, 0.05M potassium phosphate buffer at pH 7.4, 0.02M potassium phosphate buffer at pH 3, 2M sodium hydro-xide, and 25%liquid ammonia were of analytical-reagent grade (MerckKGaA, Darmstadt, Germany). Ultrahigh purity water was obtained from a Milli-Q system (MilliporeCorp., Bedford, MA).

Standard Solutions. Five quinolones were analyzed:ciprofloxacin, enrofloxacin, norfloxacin, flumequine, and oxolinic acid purchased from Sigma (Sigma;European Pharmacopeia, Strasbourg Cedex, France). Individual -quinolone 1stock standard solutions were prepared in metha-nol at 1ng kg after correcting for purity and stored at 4°C in the dark for no longer than one month. Two standard working solutions were prepared every day before analysis which were detected in two separate HPLC runs by means of their fluorescence, one with ciprofloxacin, enrofloxacin, and norfloxacin, and another with flumequine and oxolinic acid.

Calibration standards -1were prepared at concentrations of 20, 50, 100, 300, and 600ng kg for ciprofloxacin, enrofloxacin, and norfloxacin, and 30, 150, 300, 600, and 1200ng kg -1for flumequine and oxolinic acid. Sample Preparation and Heat Treatments. Spiked milk samples were prepared by fortifying commercial UHT milk with a quinolone standard working solution in order to obtain samples with 1500μg kg -1. This concentration was chosen due to the sensitivity and linearity of the detector to respond to quinolones ranging from 10to 5000μg kg -1which permits the detection of losses of concentration above the detection limit. Then, milk samples were heated for 0, 30, 60, 90, 120, 150, and 180min at 80and 100°C in a water bath and for 0, 10, 20, 30, and 40min at 120°C in an autoclave. The samples were allowed to stand for 15min at room temperature before extraction as described below.

Extraction Procedure. The extraction and purification of quinolones from milk samples was conducted using a procedure similar to that repor-ted by Delepine et al. (7). In this procedure, 2g of spiked milk sample was mixed with 20mL of 0.05M potassium phosphate buffer at pH 7.4. The prepared solutions were homogenized for 10min in an ultrasonic bath and centrifuged for 10min at 4000rpm and 15°C. The supernatant liquid was then filtered through a 0.80μm Millex filter (MilliporeCorporation, USA). Aliquots of 15mL of each filter solution were purified with SPE Discovery DSC-18cartridges (Waters,Mildford, MA), previously condi-tioned with 3mL of methanol and 3mL of water. Quinolones were eluted with 5mL of 25%methanol/ammonia(75:25).The collected solutions were evaporated at 40°C under a nitrogen stream until dry, and finally, residues were resuspended in 1mL of phosphate buffer at pH 7.4.

HPLC Analysis. The quinolones in milk samples were determined by means of a liquid chromatographic equipment analysis system made up of a separation module Alliance Waters 2695equipped with a Waters 2475fluorescent detector using a Luna C 18column (5μm, 250Â4.6mm) and a Phenomenex C 18precolumn (5μm; 4Â3mm). The system and acquisition of data were controlled by the software Empower Pro Millennium 40(Waters,Mildford, MA). The isocratic mobile phase consisted of a mix-ture of 0.02M potassium phosphate buffer, pH 3(83%A), and acetonitrile (17%B) at a flow rate of 0.3mL min -1. The fluorescence detector

Roca et al.

operated at an excitation of 294nm and an emission wavelength of 514nm for 0-11.75min for the detection of enrofloxacin, ciprofloxacin, and norfloxacin. An excitation of 280and an emission wavelength of 450nm for 11.75and 30min were used for flumequine and oxolinic acid.

Statistical Analysis. The first-order kinetic model was applied for the statistical analysis of the thermal degradation of quinolones in the following way (2):

D ½C

¼-k 13½C ðeq 1Þ

where ∂[C ]/∂t is derived from the concentration of quinolones related to time, k 1is the degradation rate constant, and [C ]is the concentration of each compound in the milk sample. By integrating equation (eq1), we get:

ln ½C ¼ln ½C 0 -k 13t

ðeq 2Þ

For each temperature, the effect heating has on the logarithmic transformations of the concentration of quinolones in milk is adjusted by means of the linear regression model using the PROC REG procedure of the SAS statistical program (21).

According to the theory postulated by Arrhenius, the degradation rate constant (k 1) depends on temperature and can be expressed as follows:

k 1¼A 3e -Ea =R 3T

ðeq 3Þ

where A is the frequency factor, e is the base of the natural logarithms (e=2.7182), Ea is the activation energy, R is the universal gas constant (R =8.315J mol -1K), and T is absolute temperature (K).Using a loga-rithmic transformation of the expression (eq3), the following is obtained:

ln k 1¼ln A -Ea 3ðeq 4Þ

The application of the linear regression model to the logarithmic transformations of the degradation rate constant based on the inverse values of the absolute temperatures allows the values of A and Ea to be calculated. To do this, the PROC REG procedure of SAS was used (21).

Finally, using equations (eq2) and (eq3) we can estimate the percentages of degradation of each quinolone for the dairy heat treatments by the following equation:"

#

%degradation ¼

C 0-C

100¼ð1-e ÞA 3e ð-Ea Þ03

3t 3100

ðeq 5Þ

RESULTS AND DISCUSSION

Table 1shows the equations calculated by applying the first

kinetic model (eq2). For each quinolone, it can be seen that parameter k 1(degradationrate constant) increases as the tem-perature rises. This demonstrates that at higher temperatures these molecules show greater heat inactivation.

It can also be seen that enrofloxacin (0.00067,0.00081, and 0.00338), flumequine (0.00050,0.00064, and 0.00170), and ox-olinic acid (0.00051,0.00083, and 0.00184) are more heat stable than ciprofloxacin (0.00113,0.00158, and 0.00880) and norflox-acin (0.00092,0.00129, and 0.00855) as they present lower values of their k 120°1coefficients after the same heat treatments at 80, 100, and C, respectively. The fit obtained by applying the first kinetic model is good, as the determination coefficient ranges between 73.03%(fornorfloxacin at 80°C) and 98.84%(fornorfloxacin at 120°C).

Figure 1shows the effect the heating time has on the concen-tration of quinolones in milk at different temperatures. When compared to the other temperatures tested, a greater heat inactivation of the quinolones was observed every time they were heated at 120°C. A greater degradation of ciprofloxacin and norfloxacin was also observed, compared to that of the rest of the quinolones.

Article

Table 1. First-Order Equations of Quinolone Concentrations at Different Temperatures a

temperature (°C ) kinetic model of first order R 2ciprofloxacin 80ln [ciprofloxacin ]=7.2129-0.001130.73390[ciprofloxacin ]=7.2880-0.001583t ln 0.944100

[ciprofloxacin ]=7.2911-0.008803t ln 0.988enrofloxacin 3t 80ln [enrofloxacin ]=7.2778-0.000670.88490enrofloxacin ]=7.3166-0.000813t ln [0.926100

enrofloxacin ]=7.3128-0.003383t ln [0.985flumequine 3t 80ln [flumequine ]=7.3166-0.000500.95590flumequine ]=7.2893-0.000643t ln [0.900100

ln [flumequine ]=7.3254-0.001703t 0.907norfloxacin 3t 80ln [norfloxacin ]=7.2268-0.000920.73090ln [norfloxacin ]=7.2929-0.001293t 100

ln [norfloxacin ]=7.2914-0.008553t t 0.9080.988oxolinic acid 380ln [oxalinic acid ]=7.2829-0.000510.81490ln [oxalinic acid ]=7.3190-0.000533t 0.959100

oxalinic acid ]=7.3298-0.001843t ln [3t

0.906

a

t :time (min ) . R 2:determination coefficient.

Applying the linear regression model to the logarithmic trans-formations of the Arrhenius expression (eq4) allows us to calculate the coefficients ln A and Ea /R , which are shown in Table 2. These coefficients present greater values for ciprofloxacin and norfloxacin, which points to the more unstable nature of these molecules when compared to flumequine and oxolinic acid, as may be seen in Table 2and Figure 1.

From the previous linear regression and the first-order kinetic model (Tables 1and 2), the values of activation energy (Ea ) and half-lives (t 1/2) of the quinolones were estimated, as shown in Table 3. Once again, it can be seen that ciprofloxacin and norfloxacin are of a more unstable nature when compared to flumequine and oxolinic acid, as they have shorter half-lives. In the case of quinolones, no activation energy and half-life values have been calculated by other authors; therefore, the results obtained in this study cannot be compared. Nevertheless, studies have been carried out into the thermo-stability of other antimicrobials, especially β-lactam antibiotics, which demon-strate that these compounds are more unstable than most anti-microbial substances such as quinolones and tetracyclines, with shorter half-lives (10, 28) and higher activation energy values (15, 18, 24, 27).

Equation 5was used to calculate degradation percentages of quinolones in HTST pasteurized milk (72°C, 15s, high temperature -short time), sterilized milk at 120°C, 20min, and UHT sterilization (140°C, 4s, ultrahigh temperature), most frequently used treatments in the dairy industry. The coefficients from Tables 1and 2were used to this end. The estimated degradations (Table 4) show that quinolones are very resistant to the different heat treatments used in the dairy industry with very low degradation rates (

In an earlier study by the same group of researchers (28), higher degradation percentages were obtained when they used the microbiological method based on the inhibition of E. coli ATCC 11303, with a loss of antimicrobial activity in milk of 18%(enrofloxacin),17%(flumequine),and 32%(norfloxacin)for treatment at 120°C and 20min. The differences found between the two studies are probably caused by the different analytical techniques employed in each case. We must remember that results

J. Agric. Food Chem., Vol. XXX, No. XX, XXXX C

Figure 1. Variation of the concentration of quinolones at different tem-peratures and heating

times.

Table 2. Summary of Estimates of Parameters in the Arrhenius Equation a

quinolones ln [k Quinolne ]=ln A -Ea /R Â(1/T ) R 2ciprofloxacin ln [k Ciprofloxacin ]=12.882-7024Â(1/T ) 0.919enrofloxacin ln [k Enrofloxacin ]=8.135-5524Â(1/T ) 0.901flumequine ln [k Flumequine ]=2.065-3362Â(1/T ) 0.934norfloxacin ln [k Norfloxacin ]=14.391-7641Â(1/T ) 0.858oxolinic acid

ln [k Oxalinic Acid ]=4.446-4310Â(1/T )

0.858

a

energy; k , first order kinetic constant; A , frecuency factor for the reaction; Ea , activation 2R , universal gas constant (R =8,315J/molK ) ; T , absolute temperature in K; R , determination coefficient.

obtained by the HPLC confirmation technique allow us to quantify the losses of concentration, whereas with microbiologi-cal methods, antimicrobial activity losses are calculated. Despite the differences found, if one takes into account the uncertainties in each of the techniques employed, the results are quite similar. Another study carried out on the effect of cooking on enro-floxacin residues in chicken tissue using a validated LC-MS (13)

D J. Agric. Food Chem., Vol. XXX, No. XX, XXXX Table 3. Activation Energy (Ea ) and Half-Lives (t 1/2) of Quinolones in Milk at Different Temperatures a

t 1/2(min )

quinolones Ea (kJ mol -1)

80°C 90°C 100°C ciprofloxacin 58.[1**********]enrofloxacin 45.[1**********]8flumequine 27.[1**********]6norfloxacin 63.[1**********]oxalinic acid

35.77

1631

848

471

a

Ea , activation energy; t 1/2, half-life.

Table 4. Percentages of Degradation of Quinolones in Milk:Estimates for Different Dairy Industry Treatments

quinolones 72°C, 15s 120°C, 20min

140°C, 4s ciprofloxacin 0.0112.710.11enrofloxacin 0.015.220.04flumequine 0.012.990.02norfloxacin 0.0112.010.11oxolinic acid

0.01

2.90

0.02

concluded that cooking procedures did not affect enrofloxacin residues, which remained stable during heating at different cooking processes (microwaving,roasting, boiling, grilling, and frying). These results show the thermal stability of enrofloxacin, as shown in this work. However, the results are not comparable because foodstuff and heat treatment used in both studies are different, as well as the analysis of data obtained.

In conclusion, the high stability of quinolones represents a significant human health risk as the residues of these anti-biotics can remain in milk after dairy processing and, therefore, can reach consumers. Current control systems use specific and microbiological methods to detect β-lactam and tetracycline antibiotics with little sensitivity to quinolones. Only some countries include in milk monitoring schemes a screening test for the detection of quinolones at MRL levels (e.g.,based on E. coli ). For this reason, it would be desirable to improve the control screening system used for the detection of these molecules in foodstuffs.

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Received for review November 18, 2009. Revised manuscript received March 31, 2010. Accepted April 2, 2010. This research was supported by

n y Ciencia financial assistance from the Ministerio de Educacio

(AGL2003-03663project, Madrid, Spain ) and carried out with the help

n, Desarrollo e Innovacio n of the of the Vicerrectorado de Investigacio

Polytechnic University of Valencia (Reference 6567) . Moreover, we thank

cnica de Valencia and Institute for Animal Science the Universidad Polite

and Technology by Instituto de Ciencia y Tecnologia Animal for funding the collaboration of Dr. Rafael Althaus with the Institute for Animal Science and Technology.