Food Chemistry 136 (2013) 376–383

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Food Chemistry

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Thermodynamic analysis of the thermal stability of sulphonamides in milkusing liquid chromatography tandem mass spectrometry detection

M. Roca a,⇑, R.L. Althaus b, M.P. Molina c

a Centro Superior de Investigación en Salud Pública, Avenida de Catalunya 21, 46020 Valencia, Spainb Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, R.P.L. Kreder 2805, 3080 Esperanza, Argentinac Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, Camino de Vera 14, 46071 Valencia, Spain

a r t i c l e i n f o

Article history:Received 20 April 2012Received in revised form 12 July 2012Accepted 22 August 2012Available online 31 August 2012

Keywords:SulphonamidesMilkThermal stabilityFirst-order kinetic modelThermodynamic compensation

0308-8146/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.foodchem.2012.08.055

⇑ Corresponding author. Tel.: +34 963877431; fax:E-mail addresses: roca_marmar@gva.es, martiroca

a b s t r a c t

The present study investigates the kinetics of the degradation of eight sulphonamides in skimmed milkwhen heated at 60, 70, 80, 90 and 100 �C using an LC–MS/MS methodology. To determine the thermalstability of these compounds, the first-order kinetic model was applied and the activation energies,half-lives and degradation percentages were calculated. Application of kinetic equations to the differentheat treatments used in dairy processing indicates that sulphonamides are very stable during pasteuri-sation (63 �C; 30 min and 72 �C; 15 s) as well as UHT sterilisation (140 �C; 4 s). In contrast, the calcula-tions performed with the kinetic model estimated losses in concentrations between 6.5%(sulfadimethoxine) and 85.1% (sulfamethazine) for the sterilisation at 120 �C for 20 min. The existenceof thermodynamic compensation was also tested for sulphonamide degradation. Results show thatenthalpy and entropy values displayed a good linear relationship, and thermodynamically we can estab-lish that the thermal degradation of sulphonamides in skimmed milk exhibits enthalpy–entropycompensation.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Sulphonamides (SAs) have proven effective antimicrobialagents since their discovery at the beginning of the 20th century.They represent a class of synthetic compounds with a bacterio-static mechanism of action based on the inhibition of nucleic acidsynthesis in bacteria (Huovinen, 1999). Given their low cost andrelative efficacy against many common bacterial infections, theyare routinely used in veterinary medicine to treat a variety of bac-terial and protozoan infections in dairy cattle (Biswas, Rao, Konda-iah, Anjaneyulu, & Malik, 2007; Knecht et al., 2004; Vargas, Reyes,& Rath, 2009).

Improper use of SAs, such as excessive administration and aninadequate withdrawal period, may result in sulphonamide resi-dues in milk. These residues are of great public health interest gi-ven the risk of developing the growth of an antibiotic-resistantbacteria strain, thus rendering inefficient this type of drug for ther-apeutic use (Chung, Lee, Chung, & Lee, 2009; Haagsma, Pluijmak-ers, Aets, & Beek, 1989). Moreover, SA residues are of particularconcern given the potential carcinogenic character of some sub-stances, such as sulfamethazine (Littelefield, Sheldon, Allen, & Gay-lor, 1990). They may also pose a technological problem for

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+34 963877436.@hotmail.com (M. Roca).

industrial production, and may affect bacterial fermentation pro-cesses in dairy products such as yogurt and cheese (Bradley &Green, 2009; Demoly & Romano, 2005; Packham, Broome, Limsow-tin, & Roginskim, 2001).

Therefore, the determination of SA residues in milk used for hu-man consumption is of utmost importance. To prevent healthproblems and to control the presence of SA residues in foodstuffs,the European Commission (EC) adopted Council Regulation 37/10/CE, stipulating a Maximum Residue Level (MRL) of 100 lg/kg inedible animal tissues, including milk, for all the substances of theSAs group.

On the other hand, the dairy industry subjects milk to differentheat treatments, such as pasteurisation and sterilisation. Therefore,SA residues in milk may degrade depending on the times and tem-peratures used in heat treatments. The degradation kinetics of achemical reaction as a result of temperature is defined by Arrhe-nius (Ash & Ash, 1995), relating the degradation reaction rate withincreasing temperature. Moreover, the order of the reaction estab-lishes a relationship between each compound’s concentration anddegradation rate (Martin, 1993). From such kinetics, parameter-based prediction models can be developed to estimate the concen-tration losses of antimicrobial compounds in terms of temperatureand time.

In previous studies (Roca, Castillo, Marti, Althaus, & Molina,2010; Roca, Villegas, Kortabitarte, Althaus, & Molina, 2011), kineticmodels of degradation to study quinolones and beta-lactam antibi-

M. Roca et al. / Food Chemistry 136 (2013) 376–383 377

otics in milk at various temperatures were established employingchromatographic methods. In both works, the results show thatthe analyzed substances are resistant to the pasteurisation(72 �C; 15 s) and Ultra High Temperature sterilisation (140 �C;4 s), except for some cephalosporins which substantially degradeunder the effect of these heat treatments.

Regarding SAs, the only studies on thermal stability carry outassessed the loss of antimicrobial activity employing microbiolog-ical methods and considering different time–temperature combi-nations Hasset, Patey, & Shearer, 1990; Malik, Duncan, Taylor, &Bishop, 1994; Rose, Farrington, & Shearer, 1996), or thermal degra-dation through UV-HPLC techniques (Papanagiotou, Fletouris, &Psomas, 2005). However these works did not establish quantitativemodels based on kinetic and thermodynamic parameters whichcan be used for predictive purposes.

Therefore, the aim of this study was to analyse the effect of thetemperature and time applied in the heat treatments on the con-centration of eight SAs in skimmed milk; this was done using a li-quid chromatographic tandem MS/MS method to determinedegradation by means of kinetic models and thermodynamicparameters to subsequently estimate losses of concentrationthrough conventional milk processing.

2. Materials and methods

2.1. Chemicals and reagents

Reference standards: Sulfadiazine (SDZ), sulfathiazole (STZ), sul-fapyridine (SPD), sulfamerazine (SMR), sulfamethazine (SMZ), sul-fachloropyridazine (SCP), sulfadimethoxine (SDM) andsulfaquinoxaline (SQX) were purchased from Sigma Chemical Co(St. Louis, MO, USA). Stock solutions of standards were preparedin methanol at 1 mg/ml after correcting for purity. Solutions wereprepared daily and stored at 4 �C until spiked samples were pre-pared. All organic reagents and other materials were of the highestpurity or HPLC grade (Sigma Chemical Co.). Water was obtainedfrom a Milli-Q system (Millipore Corp., Bedford, MA, USA). In orderto model the kinetics of degradation of SAs in a semi-synthetic ma-trix, skimmed milk powder for microbiology was used(Ref:115363, Merck, Darmstadt, Germany).

2.2. Spiked milk samples and heat treatment

Spiked samples were prepared by fortifying skimmed milkpowder for microbiology reconstituted to 10% with stock sulph-onamide solution in order to obtain samples with 200 lg/l (twotimes MRL). After spiking and mixing, fortified milk was allowedto stand for at least 30 min at room temperature to allow equilibra-tion. Thereafter, samples were divided into aliquots to examine theeffects of the temperatures of 60, 70, 80, 90 and 100 �C during incu-bation in a thermostatic water bath at different times. Table 1 pro-vides the experimental design used in this study.

Table 1Temperature–time combinations used in the experimental study.

Sulphonamides Temperature ±2 �C Time (minutes)

SulfachloropyridazineSulfadiazineSulfadimethoxineSulfamerazineSulfamethazineSulfapyridineSulphaquinoxalineSulfathiazole

60 0, 30, 60, 90, 120, 150,18070 0, 30, 60, 90, 120, 150,18080 0, 30, 60, 90, 120, 150,18090 0, 15, 30, 45, 60, 75, 90100 0, 15, 30, 45, 60, 75, 90

2.3. Extraction procedure

The analytical techniques, described as follows, were carriedout in accordance with protocols established and validated at theInstituto Lactológico de Lekunberri, using ISO standard 17025(ISO/IEC, 2005) and Commission Decision 657/2002/EC, 2002. Themethod was validated for a linear working range between 5–250 lg/kg for all sulphonamides, and CCa (decision limit) wasset at 10 lg/kg level for each one.

After spiking and mixing, 10 g of spiked milk were subsequentlymixed with 40 ml of acetonitrile into a polypropylene centrifugeflask and centrifuged for 15 min at 10,000 rpm to achieve milkdeproteinisation. The supernatant liquid was decanted and filteredinto a flask through a funnel with glass wool. Then, 2 ml of recov-ered extract was cleaned up using an Oasis HLB column (60 mg,3 ml; Waters, Milford, USA) previously conditioned with 1 ml ofmethanol and 1 ml of ultrapure water. Next, the column waswashed with 2.5 ml of ultrapure water, and the extract was elutedwith 2 ml of methanol. The eluate was evaporated to dryness un-der a stream of nitrogen. The residue was finally reconstituted in0.5 ml of 0.1% water/formic acid and filtered through a PVDF filter(25 mm, 0.2 lm); it was then added into a chromatographic vial foranalysis.

2.4. Chromatographic analysis

Sulphonamides (SAs) were analysed using a liquid chromato-graphic-mass spectrometry system consisting of an AllianceWaters™ 2695 LC module with a Micromass model Quattro Pre-mier triple-quadruple mass spectrometer (Milford, MA, USA). Theanalytical column was a 100 � 2.1 mm, internal diameter 3.5 lm,C18 X-Bridge (Waters, Milford, MA, USA). The two mobile phasesused consisted of 0.1% water/formic acid (solvent A) and acetoni-trile (solvent B), and the flow rate was 0.2 ml/min. The mobilephase gradient profile (where t refers to time in min) was as fol-lows: t0, A = 95%; t8, A = 25%; t14, A = 5%; t15, A = 95%. The eluentfrom the LC column was directed into the electrospray source ofthe tandem quadrupole mass spectrometer, which was operatedin the positive ionisation mode (ESI+). Two multiple reaction mon-itoring (MRM) transitions were monitored for each compound. Ta-ble 2 provides details of the source cone and the collision cellvoltages for each transition.

2.5. Statistical analysis

2.5.1. Kinetic studyThe adjusted kinetic first-order model (Martin, 1993) based on

the target compound’s decreasing availability was developed to fitthe thermal degradation of b-lactams in the following way:

@½C�@t¼ �k � ½C� ð1Þ

where o[C]/ot derives from the sulphonamides’ concentration inrelation to time (t), k is the degradation rate constant, and [C] isthe concentration of each compound in the milk sample at differenttime lengths. By integrating Eq. (1), we obtain:

ln ½C� ¼ ln ½C0� � k � t ð2Þ

where [C0] is the initial concentration of each compound at t = 0.To study the variations in antimicrobial concentrates along

time, the simple linear regression model was applied by followingthe PROC REG procedure for the SAS� statistics package (SAS,2001).

For each temperature, half-lives (t1/2) were calculated as thetime required for antimicrobial activity to decrease to half its ini-tial value (Chen, Ahn, & Tsong, 1997), as shown by this equation:

Table 2Multiple Reaction Monitoring (MRM) conditions and Retention Time (RT) for detection of sulphonamides in MS/MS mode.

Sulphonamides Parent ion (m/z) Cone voltaje (V) Collision energy (eV) Fragment ions (m/z) RT

Sulfachloropyridazine 285 28 28–15 156 > 92 8.64Sulfadiazine 251 30 26–16 156 > 92 5.34Sulfadimethoxine 311 40 32–20 156 > 92 9.69Sulfamerazine 265 32 28–18 156 > 92 7.01Sulfamethazine 279 30 30–17 186 > 92 7.70Sulfapyridine 250 30 27–16 156 > 92 6.75Sulfaquinoxaline 301 35 30–18 156 > 92 9.69Sulfathiazole 256 28 28–15 156 > 92 6.68

378 M. Roca et al. / Food Chemistry 136 (2013) 376–383

t1=2 ¼ln 2

kð3Þ

According to Arrhenius (Ash & Ash, 1995), the degradation rateconstants (k) depend on temperature and can be expressed as:

k ¼ A � e� EaR�T ð4Þ

where A is the frequency factor, e is the base of natural logarithms(e = 2.7182), Ea is the activation energy, R is the universal gas con-stant (R = 8.315 J/mol K) and T is the absolute temperature (K). Bylogarithmically transforming Eq. (4), the following equation isobtained:

ln k ¼ ln A� EaR � T ð5Þ

Application of the linear regression model to the logarithmictransformations of the degradation rate constant based on the in-verse values of the absolute temperatures makes it possible to cal-culate the lnA (collision frequency) and Ea (activation energy)values. For linear regression, the PROC REG procedure of theSAS� statistical package was used.

Finally, by using Eq. (2) and Eq. (5), we can estimate the degrada-tion percentages of each SA for all the different heat treatments by:

%Degradation ¼ C0 � CC0

� 100 ¼ 1� e½A�eð� Ea

R�TÞ ��t� �

� 100 ð6Þ

2.6. Thermodynamic molecular stability study

Using the degradation rate constants (k) of Eq. (3), the standardmolar Gibbs free energy of activation (DG0⁄) was calculated foreach temperature value according to the Eyring Polanyi equation:

DG0� ¼ �R:T: lnk:h

T:KBð7Þ

where the new terms in the equation are: h: Planck’s constant(6.6260 � 10�34 J/s) and KB: Boltzmann constant (1.3806 � 10�23 J/K).

For each SA, the DG0⁄ values as a function of the absolute tem-peratures (T) were represented by the following equation:

DG0� ¼ DH0� � T:DS0� ð8Þ

The linear representation for each sulphonamide was carriedout to calculate the thermodynamic variables DH0⁄ (standard mo-lar enthalpy of activation) and DS0⁄ (standard molar entropy ofactivation) by determining the y-axis intercept and the slope,respectively. The REG procedure contained in the SAS� statisticspackage (SAS, 2001) was used to determine these thermodynamicparameters. Furthermore, the quadratic regression coefficient wasdetermined.

Subsequently, according to Fisher, Purnell, and Kang (2010), alinear behaviour between the DS0⁄ and DH0⁄ values was postu-lated, as shown in the following equation:

DS0� ¼ aþ b:DH0� ð9Þ

3. Results and discussion

3.1. Kinetic study of the thermal stability of sulphonamides inskimmed milk

Table 3 shows the parameters calculated by applying a first-or-der kinetic model to the logarithmic transformation of the concen-tration of SAs in skimmed milk at different temperatures (60, 70,80, 90 and 100 �C) and heating times (0–180 min.) (Eq. (2)). Thestatistical values obtained show that the effect of heating timewas significant (p < 0.05) for all the temperatures and SAs tested.In addition, the regression coefficients were good in all cases sincethey ranged between 0.7089 (sulfadiazine, 70 �C) and 0.9998 (sul-famerazine, 100 �C) showing that the first-order kinetic model issuitable to study the thermal degradation of these substances atthe temperature–time combinations employed.

In terms of slope values (rate of degradation ‘‘k1’’), these coeffi-cients rose when the heating temperature of the samples of milkcontaining the different sulphonamide analytes increased, indicat-ing a drop in the concentration of the molecules under prolongedheating times. However, the increase of these coefficients (k1) doesnot follow the same pattern for all analytes; sulfadimethoxine(�0.00112, �0.00125, �0.00153, �0.00212, �0.00239) and sulfat-hiazole (�0.00055, �0.00076, �0.00084, �0.00204, �0.00272)had the lowest values and the least variation of these slopes withincreasing temperature. This shows the greater stability of thesesubstances with a slow and steady degradation behaviour if com-pared with other sulphonamides.

The sharpest drops in concentrations of SAs occurred when milkwas heated at 90 and 100 �C (elevated ‘‘k1’’). Moreover, this ther-mal inactivation was greater for sulfamerazine, sulfamethazineand sulfaquinoxaline molecules. This rapid degradation is probablyrelated to the high activation energy (Ea) of these substances, aswell as a high probability of effective collisions between the molesin the transition state of each molecule.

The values of the ‘‘k1’’ parameters obtained (Table 3) wereslightly higher than those reported by Roca et al. (2010) when study-ing the thermal stability of quinolones (ciprofloxacin, enrofloxacin,flumequine, norfloxacin and oxolinic acid) in skimmed milk at dif-ferent temperatures (80, 90 and 100 �C). Nevertheless, these coeffi-cients were similar to those calculated by Roca et al. (2011) in astudy into thermal degradation of penicillins (amoxicillin, ampicil-lin, cloxacillin and penicillin), but lower than those determined forcephalosporins (cefoperazone, cefquinome, cephalexin, cephaloni-um, cephapirin and cephuroxime) at the same temperatures.

From these ‘‘k1’’ values, Eq. (3) was applied to calculate the half-life (t1/2) of each sulphonamide at the different temperaturestested (Table 4). In addition, the model developed by Arrhenius(Eq. (5)) was employed, and the kinetic parameters of activationenergy (Ea) and collision frequency (lnA) were calculated (Table 5).

The half-life times (t1/2) obtained were longer when sulfadime-thoxine (t1/2 = 327, t1/2 = 290 min) and sulfathiazole (t1/2 = 340, t1/

2 = 255 min) were heated to 90 and 100 �C (Table 3), demonstrat-ing the persistence of both molecules and the difficulty of reducing

Table 3First-order degradation kinetic model of sulphonamides in milk at differenttemperatures.

T (�C) Intercept (a) Slope (k1) Standard error p-value R

a k

Sulfachloropyridazine60 5.1515 �0.00119 0.0627 0.00058 0.0415 0.823570 5.1780 �0.00453 0.0713 0.00066 0.0102 0.975280 5.2782 �0.00619 0.0137 0.00105 0.0201 0.966990 5.0419 �0.00781 0.1097 0.00203 0.0126 0.9299100 5.3633 �0.01098 0.1061 0.00378 0.0324 0.9482

Sulfadiazine60 5.2136 �0.00049 0.0389 0.00036 0.0251 0.720870 5.0752 �0.00111 0.0929 0.00086 0.0204 0.708980 5.3523 �0.00267 0.0485 0.00045 0.0019 0.967690 5.2083 �0.00563 0.0431 0.00079 0.0009 0.9764100 5.2331 �0.00813 0.0714 0.00254 0.0002 0.9562

Sulfadimethoxine60 5.2137 �0.00112 0.0384 0.00035 0.0246 0.904470 5.2709 �0.00125 0.0147 0.00013 0.0003 0.985780 5.2414 �0.00153 0.0247 0.00023 0.0011 0.973890 5.2595 �0.00212 0.0178 0.00033 0.0014 0.9719100 5.2917 �0.00239 0.0095 0.00033 0.0192 0.9904

Sulfamerazine60 5.1360 �0.00063 0.0684 0.00083 0.0366 0.710970 5.1716 �0.00166 0.0648 0.00060 0.0105 0.933880 5.1796 �0.00239 0.0705 0.00065 0.0123 0.866390 5.1413 �0.00774 0.0702 0.00129 0.0019 0.9677100 5.2869 �0.02457 0.0122 0.00043 0.0003 0.9998

Sulfamethazine60 5.0982 �0.00105 0.0840 0.00078 0.0494 0.719770 5.1395 �0.00203 0.1088 0.00100 0.0233 0.877780 5.1980 �0.00256 0.0168 0.00043 0.0020 0.967190 5.0758 �0.01092 0.0944 0.00175 0.0015 0.9704100 5.2522 �0.03092 0.0996 0.00355 0.0129 0.9935

Sulfapyridine60 5.1941 �0.00134 0.0441 0.00041 0.0221 0.908770 5.1441 �0.00204 0.0548 0.00051 0.0101 0.934880 5.2663 �0.00313 0.0289 0.00027 0.0001 0.991190 5.1353 �0.00527 0.0444 0.00082 0.0014 0.9717100 5.3517 �0.00882 0.0862 0.00307 0.0129 0.9472

Sulfaquinoxaline60 5.1600 �0.00235 0.0594 0.00055 0.0079 0.941370 5.1213 �0.00238 0.1198 0.00111 0.0344 0.832480 5.1802 �0.00290 0.0564 0.00052 0.0026 0.963390 5.1298 �0.01115 0.0735 0.00136 0.0004 0.9822100 5.1807 �0.01725 0.1268 0.00452 0.0223 0.9684

Sulfathiazole60 5.2761 �0.00055 0.0116 0.00010 0.0037 0.957470 5.2263 �0.00076 0.0300 0.00028 0.0408 0.880280 5.2439 �0.00084 0.0229 0.00021 0.0106 0.933690 5.2425 �0.00204 0.0242 0.00046 0.0068 0.9449100 5.2951 �0.00272 0.0039 0.00014 0.0027 0.9986

a Log[C0]; k: degradation rate constant.

Table 4Half-lives (t1/2) for sulphonamides in milk at different temperatures.

Sulphonamides t1/2 = ln2/k (min)

60 �C 70 �C 80 �C 90 �C 100 �C

Sulfachloropyridazine 582 153 112 89 63Sulfadiazine 1415 624 260 123 85Sulfadimethoxine 619 555 453 327 290Sulfamerazine 620 341 271 63 22Sulfamethazine 835 418 290 90 28Sulfapyridine 517 340 221 132 79Sulphaquinoxaline 295 291 239 62 40Sulfathiazole 1260 912 825 340 255

t1/2: half-lives (min.)

Table 5Statistical parameters calculated using the Arrhenius model for the degradation ofsulphonamides in milk.

Sulphonamide lnA Ea F value p value R

Sulfachloropyridazine 12.466 52.10 19.79 0.0211 0.9317Sulfadiazine 19.539 75.03 266.28 0.0005 0.9944Sulfadimethoxine 0.754 21.05 85.85 0.0027 0.9830Sulfamerazine 24.152 86.60 23.32 0.0169 0.9413Sulfamethazine 24.121 87.30 39.36 0.0082 0.9640Sulfapyridine 10.878 48.61 409.19 0.0003 0.9964Sulphaquinoxaline 13.979 56.49 14.91 0.0307 0.9124Sulfathiazole 7.890 42.97 34.17 0.0100 0.9588

A, frequency factor for the reaction; Ea, activation energy (kJ/mol); F value and pvalue, F and probability values after applying ANOVA; R, determination coefficientof adjustment of the first order.

M. Roca et al. / Food Chemistry 136 (2013) 376–383 379

their concentrations by heating milk. In contrast, sulfamerazine (t1/

2 = 63, t1/2 = 22 min) and sulfamethazine (t1/2 = 90, t1/2 = 28 min)

presented shorter half-life times for these temperatures, indicatinga more unstable nature. This thermal degradation can be explainedby the kinetic parameters obtained by applying Arrheniusequation.

The kinetic parameters presented the highest values for sulfa-merazine (lnA = 24.152, Ea = 86.60 kJ/mol) and sulfamethazine(lnA = 24.121, Ea = 87.30 kJ/ mol) showing that these molecules re-quire a high temperature to reach their activation energy and bringabout the transition state, where the successful collisions betweenmoles have enough energy (Ea) to break the pre-existing bonds andform the reaction products. The elevated collision frequency (lnA)and the high temperature induce more successful collisions, whichincrease the reaction rate. This explains why the degradation ofsulfamerazine and sulfamethazine is slower even when milk isheated at 80 �C, and, furthermore, the reaction is acceleratedquickly upon reaching higher temperatures, obtaining the lowesthalf-life values (Table 3).

Moreover, sulfadimethoxine (Ea = 21.05 kJ/mol, lnA = 0.754)and sulfathiazole (Ea = 42.97 kJ/mol, lnA = 7.890) presented lowerkinetic parameters. Low activation energy means that these sub-stances quickly reach the transition state. However, the low prob-ability of successful collisions (low values of lnA) significantlyreduced the reaction, and therefore these molecules become moreresistant at high temperatures (90 and 100 �C).

The activation energy values of SAs in skimmed milk (Table 5)were similar than those calculated by Roca et al. (2010) for thethermal degradation of quinolones (between 27.90 kJ/mol forflumequine, and 63.42 kJ/mol for norfloxacin) and beta-lactamantibiotics, with values ranging from 50.30 kJ/mol for cefquinometo 88.84 kJ/mol for cefoperazone (Roca et al., 2011). It should beemphasised that there are no values for the kinetic parameters(Ea and lnA) of SAs in the reviewed literature, even in aqueoussolutions.

On the other hand, Fig. 1 shows the plot of the liberalised Arrhe-nius’ equation (Eq. (5)) for the eight SAs analysed. This figure illus-trates again how sulfadimethoxine and sulfathiazole have lowerslopes than the other SAs tested, indicating the greater stabilityof both molecules to heating; therefore, they present lower ther-mal degradation with higher temperatures and longer heatingtimes.

Later, Eq. (6) was used to estimate the degradation percentages(Table 6) of the SAs in pasteurised milk at high temperature-shorttime (72 �C – 15 s), and UHT sterilisation (ultra-high temperature,140 �C for 4 s), which are the most frequently used treatments inthe dairy industry. Additionally, degradation percentages of SAsin sterilised milk at 120 �C for 20 min, and pasteurisation at lowtemperature-long time (63 �C – 30 min) were investigated, sincethese treatments are also frequent in the dairy industry in Spain.The estimated degradations show that SAs are very resistant tothe heat treatments used in pasteurisation (63 �C, 30 min and

2.65 2.7 2.75 2.8 2.85 2.9 2.95 3.0 3.05

1000/T (ºK)

ln [k

]

Sulfachloropiradizine Sulfadiazine Sulfadimethoxine Sulfamerazine

-8

-7

-6

-5

-4

-3

-2

2.65 2.7 2.75 2.8 2.85 2.9 2.95 3 3.05

1000/T (ºK)

ln [k

]

Sulfamethazine SulfaquinoxalineSulfapyridine Sulfathiazole

Fig. 1. Arrhenius equation for thermal degradation of sulphonamides in milk.

Table 6Degradation percentages of sulphonamides in milk for different heat treatments.

Sulphonamides Pasteurisation Sterilisation

63 �C – 30 min 72 �C – 15 s 120 �C – 20 min 140 �C – 4 s

Sulfachloropyridazine 6.0 0.1 46.0 0.4Sulfadiazine 2.0 0.0 47.9 1.7Sulfadimethoxine 3.3 0.0 6.5 0.0Sulfamerazine 2.4 0.0 77.4 1.8Sulfamethazine 3.1 0.1 85.1 2.3Sulfapyridine 4.3 0.1 30.7 0.3Sulfaquinoxaline 5.7 0.1 51.9 0.6Sulfathiazole 1.7 0.0 9.9 0.1

Table 7Thermodynamic parameters (standard molar enthalpy and entropy of activation) ofsulphonamides in milk.

Sulphonamide DG0⁄ = DH0⁄ � T.DS0⁄ R

Sulfachloropiridazine DG0⁄Sulfacloropidazine = 48096 � (�196) � T 0.9614

Sulfadiazine DG0⁄Sulfadiazine = 71800 � (�135) � T 0.9855

Sulfadimethoxine DG0⁄Sulfadimethoxine = 18272 � (�290) � T 0.9999

Sulfamerazine DG0⁄Sulfamerazine = 84303 � (�94) � T 0.7968

Sulfamethazine DG0⁄Sulfamethazine = 83776 � (�97) � T 0.8618

Sulfapyridine DG0⁄Sulfapyridine = 45830 � (�205) � T 0.9985

Sulfaquinoxaline DG0⁄Sulfaquinoxaline = 55025 � (�176) � T 0.9266

Sulfathiazole DG0⁄Sulfathiazole = 40560 � (�229) � T 0.9878

DH0⁄, standard molar enthalpy of activation; DS0⁄, standard molar entropy ofactivation.

380 M. Roca et al. / Food Chemistry 136 (2013) 376–383

72 �C, 15 s) and that sterilisation at 140 �C for 4 s obtained very lowdegradation rates (below 6% in all cases). Conversely, sterilisationat 120 �C produced greater losses, which reached 77.4% for sulfa-merazine and 85.1% for sulfamethazine. These molecules displayhigh activation energy (Ea) and collision frequency values (lnA),in addition to having low half-life times at 100 and 120 �C (Tables4 and 5). However, this heat treatment (120 �C for 20 min) led to aslight inactivation of sulfadimethoxine and sulfathiazole since theyhad longer half-life times at 100 and 120 �C.

3.2. Thermodynamic molecular stability study

Table 7 shows the mathematical equations obtained by apply-ing the linear regression model (Eq. (8)) and Gibbs free energy val-ues (Eq. (7)) as a function of absolute temperature. For the eightSAs examined, the specific molar entropy of activation valueswas negative, indicating that the thermal stability of these mole-cules is a thermodynamically unfavourable phenomenon.

The plot of the specific Gibbs free energy as a function of abso-lute temperature is provided in Fig. 2, where we can observe a lin-ear behaviour between the two variables as established by Eq. (8).

-350

-300

-250

-200

-150

-100

-50

020 40 60 80 100

H0* (KJ/mol)

SulfathiazoleSulfapyridine

Sulfachloropiridazine Sulfaquinoxaline

SulfadiazineSulfamethazine

Sulfamerazine

Sulfadimethoxine

S0*(K

J/m

ol)

Fig. 3. Enthalpy and entropy of activation of thermal degradation for sulphona-mides in milk.

M. Roca et al. / Food Chemistry 136 (2013) 376–383 381

To assess the relationship between specific molar entropy(DS0⁄) and enthalpy (DH0⁄) of activation, Fig. 3 was constructedby applying the linear regression model. The linear behaviour be-tween the two thermodynamic variables (F value = 1755.88, p va-lue = 0.001) can be clearly seen according to the followingequation:

DH0� ¼ 2:939þ 342DS0� R ¼ 0:9980

Other authors also observed a strong linear regression betweenthese two thermodynamic parameters with a good statistical fit(Fisher et al., 2010; Kang & Warren, 2007; Rudra, Singh, Basu, &Shivhare, 2008).

As the eight SAs have similar basic structures, it has been sug-gested that the stability of these molecules is due to, not onlythe enthalpy and entropy values, but also to a combination of boththermodynamic variables (Fisher et al., 2010).

Indeed, while the specific molar Gibbs energy (DG0⁄) of SAs pre-sents slight variations between 113 and 126 kJ/mol (approximately11.5%, Fig. 2), the DH0⁄ values vary from 18.3 kJ/mol (sulfadime-thoxine) to 84.3 kJ/mol (sulfamerazine), while the DS0⁄ valuesrange from �94 J/mol K (sulfamerazine) to �290 J/mol K (sulfadi-methoxine), with wide ranges of variations of next to four (DH0⁄)and three (DS0⁄) times in magnitude.

112000

114000

116000

118000

120000

122000

124000

126000

330 340 350

Temp

Sulfachloropiridazine Sulfadiazine

112000

114000

116000

118000

120000

122000

124000

126000

330 340 350

Temp

Sulfamethazine Sulfapyridine

G0*

(J/m

ol)

G0*

(J/m

ol)

Fig. 2. Temperature-dependent standard molar free energy of ac

Therefore, it can be established that SAs have great thermal sta-bility, but that each molecule has different thermodynamic param-eters. Thus, the stability of sulfamerazine and sulfamethazine isdue to high DH0⁄ values since the DS0⁄ values are very low, whilesulfadimethoxine and sulfathiazole present negative DS0⁄ valuesand low DH0⁄ values.

360 370 380

erature (ºK)

Sulfadimethoxine Sulfamerazine

360 370 380

erature (ºK)

Sulfaquinoxaline Sulfathiazole

tivation of thermal degradation for sulphonamides in milk.

382 M. Roca et al. / Food Chemistry 136 (2013) 376–383

In summary, the structural differences of each sulphonamidemolecule can undergo significant changes in the statistical DH0⁄

and DS0⁄ parameters due to thermal degradation, but they do notcause significant changes in the DG0⁄ values. This is due to a com-pensatory effect between the DH0⁄ and DS0⁄ values, as shown inFig. 3. In this Figure an increase in the values of DH0⁄ and a de-crease in the values of DS0⁄ can be appreciate as we move in thedirection of sulfadimethoxine, sultathiazole, sulfapyridine, sulfa-cloropiridazine, sulphaquinoxaline, sulfadiazine, sulfamerazineand sulfamethazine. This fact explains the higher thermal stabilityof the first sulphonamides compared with the minor stability ofthe last molecules of the series.

This compensatory effect was also reported by Fisher et al.(2010) in their study into the thermodynamic stability of quino-lones in milk. These authors suggest that the enthalpy–entropycompensation of very stable molecules is responsible for the rela-tively slight variations in DG0⁄ in the thermal degradation of quin-olones in milk.

Similarly, Rudra et al. (2008) emphasised the existence of en-thalpy–entropy compensation when studying the thermal degra-dation of chlorophyll. The enthalpy of activation (DH0⁄) variesfrom 2.36 to 91.99 kJ/mol, while entropy of activation (DS0⁄)ranges from �0.047 to �0.713 kJ/mol K, with a high correlationcoefficient (R2 = 0.96) between the two thermodynamicparameters.

In addition, in another study, Kang and Warren (2007) carriedout a thermodynamic analysis of the transition of mono-specificantibodies (native and urea-treated) to show that this antibodytransition exhibits enthalpy–entropy compensation. These authorsobserved vast changes in the variations of DH0⁄ and TDH0⁄,whereas the changes in the DG0⁄ levels were only very slight.

The study into the dissociation of four peptides from the com-plexes at 37 �C conducted by Kang and Auerbach (2009) revealeda linear behaviour between the specific molar entropy and the en-thalpy of activation, with a high regression coefficient (R2 = 0.957).The disassociation of the complexes underwent changes of 23.3times in the DH0⁄ values and of 18.5 times in the DS0⁄ values, whilethe DG0⁄ variations were lower (5.7 times). This fact also indicatesan enthalpy–entropy compensation phenomenon.

These thermodynamic results are consistent with the structuralcharacteristics of sulphonamides. For the SAs with a pyrimidinering in their molecule structure (sulfadiazine, sulfamethazine, sul-famerazine and sulfadimethoxine) it is appreciated that the incor-poration of one (sulfamerazine) or two methyl functional groups(sulfamethazine) decreases their stability due to their higher en-tropy activation values, compared with sulfadiazine. Whereas,the incorporation of two methoxy functional groups (sulfadime-thoxine) increases the stability of the pyrimidine ring linked to adecrease in the entropy activation (Fig. 1, Supplementary data).

Moreover, in the case of the sulfachloropyridazine which pos-sesses a pyridazine ring (similar to the pyrimidine ring), the chlo-rine substituent produces a similar effect to that of the methoxyfunctional group, increasing the stability of this molecule. For theother SAs the different heterocyclic structures (quinoxaline, pyri-dine and thiazole rings) give them intermediate stabilities.

4. Conclusions

The present study investigated the thermal stability of sulph-onamides in skimmed milk during heating, applying the first-orderkinetic model and calculating the kinetic parameters of activationenergies, half-lives and degradation percentages. The results ob-tained show that sulphonamides are very stable molecules whichcan resist even the most common heat treatments performed inthe dairy industry without degrading significantly.

The differences of stability obtained between the sulphona-mides evaluated were explained by thermodynamic evaluation.In this study we concluded that the chemical structure of each sub-stance contributes to the specific molar entropy and enthalpy ofactivation during the thermal degradation of these molecules indifferent ways. However, the free energy of activation is almostconstant due to the enthalpy–entropy compensation, given thehigh stability of the sulphonamides.

To summarize, the high thermo-stability of sulphonamidesdemonstrate that the heat treatments used in the dairy industrycould be insufficient to completely inactivate sulphonamide resi-dues in milk. In addition, the dairy industry currently uses specificmethods to detect only beta-lactams and tetracyclines as well asslow-response and low sensitive microbiological methods to de-tect other antimicrobial agents. Therefore, sulphonamide residuesin milk may represent a potential health risk to consumers if thecontrol of the presence of these residues is not adequate to preventthem from reaching the food chain.

Thus, it is also interesting to note that the high stability of sul-phonamides can also provoke serious problems if their residuesreach the environment. Regarding this aspect, some authors havefound sulphonamide residues in rivers and streams in the U.S (Gar-cía-Galán, Díaz-Cruz, & Barceló, 2008), in surface waters in France(Vulliet & Cren-Olivé, 2011) and natural mineral water in Italy(Perret, Gentili, Marchese, Greco, & Curini, 2006). Moreover, otherauthors point out that small amounts of sulphonamides from agri-cultural activity, cause changes in the population of microorgan-isms in the environment and can be potentially hazardous tohuman health (Baran, Sochacka, & Wardas, 2006). Therefore, thehigh thermal stability of sulphonamides and the potential risk thatthey might have on the microorganism population invites futureresearch strategies for the treatment of waste and effluents con-taining these compounds.

Acknowledgements

This research has been supported financially by the SpanishMinistry of Education and Science (AGL2003-03663 project, Ma-drid, Spain) and has been carried out with the help of the Vice-rec-torate of Research, Development and Innovation at the PolytechnicUniversity of Valencia (Reference 6567). Moreover, the authorswish to thank the Polytechnic University of Valencia for fundingthe collaboration of Dr. Rafael Althaus with the Institute for AnimalScience and Technology.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2012.08.055.

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