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

Food Chemistry 136 (2013) 376–383

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Thermodynamic analysis of the thermal stability of sulphonamides in milk using 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, Spain Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, R.P.L. Kreder 2805, 3080 Esperanza, Argentina c Instituto de Ciencia y Tecnología Animal, Universidad Politécnica de Valencia, Camino de Vera 14, 46071 Valencia, Spain b

a r t i c l e

i n f o

Article history: Received 20 April 2012 Received in revised form 12 July 2012 Accepted 22 August 2012 Available online 31 August 2012 Keywords: Sulphonamides Milk Thermal stability First-order kinetic model Thermodynamic compensation

a b s t r a c t The present study investigates the kinetics of the degradation of eight sulphonamides in skimmed milk when heated at 60, 70, 80, 90 and 100 °C using an LC–MS/MS methodology. To determine the thermal stability 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 different heat treatments used in dairy processing indicates that sulphonamides are very stable during pasteurisation (63 °C; 30 min and 72 °C; 15 s) as well as UHT sterilisation (140 °C; 4 s). In contrast, the calculations 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 existence of thermodynamic compensation was also tested for sulphonamide degradation. Results show that enthalpy and entropy values displayed a good linear relationship, and thermodynamically we can establish that the thermal degradation of sulphonamides in skimmed milk exhibits enthalpy–entropy compensation. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Sulphonamides (SAs) have proven effective antimicrobial agents since their discovery at the beginning of the 20th century. They represent a class of synthetic compounds with a bacteriostatic mechanism of action based on the inhibition of nucleic acid synthesis in bacteria (Huovinen, 1999). Given their low cost and relative efficacy against many common bacterial infections, they are routinely used in veterinary medicine to treat a variety of bacterial and protozoan infections in dairy cattle (Biswas, Rao, Kondaiah, Anjaneyulu, & Malik, 2007; Knecht et al., 2004; Vargas, Reyes, & Rath, 2009). Improper use of SAs, such as excessive administration and an inadequate withdrawal period, may result in sulphonamide residues in milk. These residues are of great public health interest given the risk of developing the growth of an antibiotic-resistant bacteria strain, thus rendering inefficient this type of drug for therapeutic use (Chung, Lee, Chung, & Lee, 2009; Haagsma, Pluijmakers, Aets, & Beek, 1989). Moreover, SA residues are of particular concern given the potential carcinogenic character of some substances, such as sulfamethazine (Littelefield, Sheldon, Allen, & Gaylor, 1990). They may also pose a technological problem for

⇑ Corresponding author. Tel.: +34 963877431; fax: +34 963877436. E-mail addresses: [email protected], [email protected] (M. Roca). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.08.055

industrial production, and may affect bacterial fermentation processes in dairy products such as yogurt and cheese (Bradley & Green, 2009; Demoly & Romano, 2005; Packham, Broome, Limsowtin, & Roginskim, 2001). Therefore, the determination of SA residues in milk used for human consumption is of utmost importance. To prevent health problems 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 in edible animal tissues, including milk, for all the substances of the SAs group. On the other hand, the dairy industry subjects milk to different heat treatments, such as pasteurisation and sterilisation. Therefore, SA residues in milk may degrade depending on the times and temperatures used in heat treatments. The degradation kinetics of a chemical reaction as a result of temperature is defined by Arrhenius (Ash & Ash, 1995), relating the degradation reaction rate with increasing temperature. Moreover, the order of the reaction establishes a relationship between each compound’s concentration and degradation rate (Martin, 1993). From such kinetics, parameterbased prediction models can be developed to estimate the concentration losses of antimicrobial compounds in terms of temperature and time. In previous studies (Roca, Castillo, Marti, Althaus, & Molina, 2010; Roca, Villegas, Kortabitarte, Althaus, & Molina, 2011), kinetic models of degradation to study quinolones and beta-lactam antibi-

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otics in milk at various temperatures were established employing chromatographic methods. In both works, the results show that the 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 degrade under the effect of these heat treatments. Regarding SAs, the only studies on thermal stability carry out assessed the loss of antimicrobial activity employing microbiological methods and considering different time–temperature combinations Hasset, Patey, & Shearer, 1990; Malik, Duncan, Taylor, & Bishop, 1994; Rose, Farrington, & Shearer, 1996), or thermal degradation through UV-HPLC techniques (Papanagiotou, Fletouris, & Psomas, 2005). However these works did not establish quantitative models based on kinetic and thermodynamic parameters which can be used for predictive purposes. Therefore, the aim of this study was to analyse the effect of the temperature and time applied in the heat treatments on the concentration of eight SAs in skimmed milk; this was done using a liquid chromatographic tandem MS/MS method to determine degradation by means of kinetic models and thermodynamic parameters to subsequently estimate losses of concentration through conventional milk processing.

2.3. Extraction procedure

2. Materials and methods

2.4. Chromatographic analysis

2.1. Chemicals and reagents

Sulphonamides (SAs) were analysed using a liquid chromatographic-mass spectrometry system consisting of an Alliance Waters™ 2695 LC module with a Micromass model Quattro Premier triple-quadruple mass spectrometer (Milford, MA, USA). The analytical column was a 100  2.1 mm, internal diameter 3.5 lm, C18 X-Bridge (Waters, Milford, MA, USA). The two mobile phases used consisted of 0.1% water/formic acid (solvent A) and acetonitrile (solvent B), and the flow rate was 0.2 ml/min. The mobile phase gradient profile (where t refers to time in min) was as follows: t0, A = 95%; t8, A = 25%; t14, A = 5%; t15, A = 95%. The eluent from the LC column was directed into the electrospray source of the tandem quadrupole mass spectrometer, which was operated in the positive ionisation mode (ESI+). Two multiple reaction monitoring (MRM) transitions were monitored for each compound. Table 2 provides details of the source cone and the collision cell voltages for each transition.

Reference standards: Sulfadiazine (SDZ), sulfathiazole (STZ), sulfapyridine (SPD), sulfamerazine (SMR), sulfamethazine (SMZ), sulfachloropyridazine (SCP), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX) were purchased from Sigma Chemical Co (St. Louis, MO, USA). Stock solutions of standards were prepared in methanol at 1 mg/ml after correcting for purity. Solutions were prepared daily and stored at 4 °C until spiked samples were prepared. All organic reagents and other materials were of the highest purity or HPLC grade (Sigma Chemical Co.). Water was obtained from a Milli-Q system (Millipore Corp., Bedford, MA, USA). In order to model the kinetics of degradation of SAs in a semi-synthetic matrix, skimmed milk powder for microbiology was used (Ref:115363, Merck, Darmstadt, Germany).

The analytical techniques, described as follows, were carried out in accordance with protocols established and validated at the Instituto Lactológico de Lekunberri, using ISO standard 17025 (ISO/IEC, 2005) and Commission Decision 657/2002/EC, 2002. The method was validated for a linear working range between 5– 250 lg/kg for all sulphonamides, and CCa (decision limit) was set at 10 lg/kg level for each one. After spiking and mixing, 10 g of spiked milk were subsequently mixed with 40 ml of acetonitrile into a polypropylene centrifuge flask and centrifuged for 15 min at 10,000 rpm to achieve milk deproteinisation. The supernatant liquid was decanted and filtered into a flask through a funnel with glass wool. Then, 2 ml of recovered extract was cleaned up using an Oasis HLB column (60 mg, 3 ml; Waters, Milford, USA) previously conditioned with 1 ml of methanol and 1 ml of ultrapure water. Next, the column was washed with 2.5 ml of ultrapure water, and the extract was eluted with 2 ml of methanol. The eluate was evaporated to dryness under a stream of nitrogen. The residue was finally reconstituted in 0.5 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 for analysis.

2.2. Spiked milk samples and heat treatment

2.5. Statistical analysis

Spiked samples were prepared by fortifying skimmed milk powder for microbiology reconstituted to 10% with stock sulphonamide solution in order to obtain samples with 200 lg/l (two times MRL). After spiking and mixing, fortified milk was allowed to stand for at least 30 min at room temperature to allow equilibration. Thereafter, samples were divided into aliquots to examine the effects of the temperatures of 60, 70, 80, 90 and 100 °C during incubation in a thermostatic water bath at different times. Table 1 provides the experimental design used in this study.

2.5.1. Kinetic study The adjusted kinetic first-order model (Martin, 1993) based on the target compound’s decreasing availability was developed to fit the thermal degradation of b-lactams in the following way:

Table 1 Temperature–time combinations used in the experimental study. Sulphonamides Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulphaquinoxaline Sulfathiazole

Time (minutes)

60 70 80 90 100

0, 0, 0, 0, 0,

60, 60, 60, 30, 30,

90, 90, 90, 45, 45,

120, 150,180 120, 150,180 120, 150,180 60, 75, 90 60, 75, 90

ð1Þ

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

ln ½C ¼ ln ½C 0   k  t

Temperature ±2 °C

30, 30, 30, 15, 15,

@½C ¼ k  ½C @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 following the PROC REG procedure for the SASÒ statistics package (SAS, 2001). For each temperature, half-lives (t1/2) were calculated as the time required for antimicrobial activity to decrease to half its initial value (Chen, Ahn, & Tsong, 1997), as shown by this equation:

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Table 2 Multiple 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 Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulfaquinoxaline Sulfathiazole

285 251 311 265 279 250 301 256

28 30 40 32 30 30 35 28

28–15 26–16 32–20 28–18 30–17 27–16 30–18 28–15

156 > 92 156 > 92 156 > 92 156 > 92 186 > 92 156 > 92 156 > 92 156 > 92

8.64 5.34 9.69 7.01 7.70 6.75 9.69 6.68

t 1=2 ¼

ln 2 k

ð3Þ

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

k ¼ A  eRT

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 constant (R = 8.315 J/mol K) and T is the absolute temperature (K). By logarithmically transforming Eq. (4), the following equation is obtained:

ln k ¼ ln A 

Ea RT

ð5Þ

Application of the linear regression model to the logarithmic transformations of the degradation rate constant based on the inverse values of the absolute temperatures makes it possible to calculate the ln A (collision frequency) and Ea (activation energy) values. For linear regression, the PROC REG procedure of the SASÒ statistical package was used. Finally, by using Eq. (2) and Eq. (5), we can estimate the degradation percentages of each SA for all the different heat treatments by:

%Degradation ¼

  ð Ea Þ C0  C  100 ¼ 1  e½Ae RT t  100 C0

ð6Þ

2.6. Thermodynamic molecular stability study Using the degradation rate constants (k) of Eq. (3), the standard molar Gibbs free energy of activation (DG0⁄) was calculated for each temperature value according to the Eyring Polanyi equation:

DG0 ¼ R:T: ln

k:h T:K B

ð7Þ

where the new terms in the equation are: h: Planck’s constant (6.6260  1034 J/s) and KB: Boltzmann constant (1.380 6  1023 J/K). For each SA, the DG0⁄ values as a function of the absolute temperatures (T) were represented by the following equation:

DG0 ¼ DH0  T:DS0

ð8Þ

The linear representation for each sulphonamide was carried out to calculate the thermodynamic variables DH0⁄ (standard molar enthalpy of activation) and DS0⁄ (standard molar entropy of activation) by determining the y-axis intercept and the slope, respectively. The REG procedure contained in the SASÒ statistics package (SAS, 2001) was used to determine these thermodynamic parameters. Furthermore, the quadratic regression coefficient was determined. Subsequently, according to Fisher, Purnell, and Kang (2010), a linear behaviour between the DS0⁄ and DH0⁄ values was postulated, as shown in the following equation:

DS0 ¼ a þ b:DH0

ð9Þ

3. Results and discussion 3.1. Kinetic study of the thermal stability of sulphonamides in skimmed milk Table 3 shows the parameters calculated by applying a first-order kinetic model to the logarithmic transformation of the concentration of SAs in skimmed milk at different temperatures (60, 70, 80, 90 and 100 °C) and heating times (0–180 min.) (Eq. (2)). The statistical values obtained show that the effect of heating time was significant (p < 0.05) for all the temperatures and SAs tested. In addition, the regression coefficients were good in all cases since they ranged between 0.7089 (sulfadiazine, 70 °C) and 0.9998 (sulfamerazine, 100 °C) showing that the first-order kinetic model is suitable to study the thermal degradation of these substances at the temperature–time combinations employed. In terms of slope values (rate of degradation ‘‘k1’’), these coefficients rose when the heating temperature of the samples of milk containing the different sulphonamide analytes increased, indicating a drop in the concentration of the molecules under prolonged heating times. However, the increase of these coefficients (k1) does not follow the same pattern for all analytes; sulfadimethoxine (0.00112, 0.00125, 0.00153, 0.00212, 0.00239) and sulfathiazole (0.00055, 0.00076, 0.00084, 0.00204, 0.00272) had the lowest values and the least variation of these slopes with increasing temperature. This shows the greater stability of these substances with a slow and steady degradation behaviour if compared with other sulphonamides. The sharpest drops in concentrations of SAs occurred when milk was heated at 90 and 100 °C (elevated ‘‘k1’’). Moreover, this thermal inactivation was greater for sulfamerazine, sulfamethazine and sulfaquinoxaline molecules. This rapid degradation is probably related to the high activation energy (Ea) of these substances, as well as a high probability of effective collisions between the moles in the transition state of each molecule. The values of the ‘‘k1’’ parameters obtained (Table 3) were slightly higher than those reported by Roca et al. (2010) when studying the thermal stability of quinolones (ciprofloxacin, enrofloxacin, flumequine, norfloxacin and oxolinic acid) in skimmed milk at different temperatures (80, 90 and 100 °C). Nevertheless, these coefficients were similar to those calculated by Roca et al. (2011) in a study into thermal degradation of penicillins (amoxicillin, ampicillin, cloxacillin and penicillin), but lower than those determined for cephalosporins (cefoperazone, cefquinome, cephalexin, cephalonium, cephapirin and cephuroxime) at the same temperatures. From these ‘‘k1’’ values, Eq. (3) was applied to calculate the halflife (t1/2) of each sulphonamide at the different temperatures tested (Table 4). In addition, the model developed by Arrhenius (Eq. (5)) was employed, and the kinetic parameters of activation energy (Ea) and collision frequency (ln A) were calculated (Table 5). The half-life times (t1/2) obtained were longer when sulfadimethoxine (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), demonstrating the persistence of both molecules and the difficulty of reducing

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M. Roca et al. / Food Chemistry 136 (2013) 376–383 Table 3 First-order degradation kinetic model of sulphonamides in milk at different temperatures. T (°C)

Intercept (a)

Slope (k1)

Standard error a

k

p-value

R

Sulfachloropyridazine 60 5.1515 70 5.1780 80 5.2782 90 5.0419 100 5.3633

0.00119 0.00453 0.00619 0.00781 0.01098

0.0627 0.0713 0.0137 0.1097 0.1061

0.00058 0.00066 0.00105 0.00203 0.00378

0.0415 0.0102 0.0201 0.0126 0.0324

0.8235 0.9752 0.9669 0.9299 0.9482

Sulfadiazine 60 5.2136 70 5.0752 80 5.3523 90 5.2083 100 5.2331

0.00049 0.00111 0.00267 0.00563 0.00813

0.0389 0.0929 0.0485 0.0431 0.0714

0.00036 0.00086 0.00045 0.00079 0.00254

0.0251 0.0204 0.0019 0.0009 0.0002

0.7208 0.7089 0.9676 0.9764 0.9562

Sulfadimethoxine 60 5.2137 70 5.2709 80 5.2414 90 5.2595 100 5.2917

0.00112 0.00125 0.00153 0.00212 0.00239

0.0384 0.0147 0.0247 0.0178 0.0095

0.00035 0.00013 0.00023 0.00033 0.00033

0.0246 0.0003 0.0011 0.0014 0.0192

0.9044 0.9857 0.9738 0.9719 0.9904

Sulfamerazine 60 5.1360 70 5.1716 80 5.1796 90 5.1413 100 5.2869

0.00063 0.00166 0.00239 0.00774 0.02457

0.0684 0.0648 0.0705 0.0702 0.0122

0.00083 0.00060 0.00065 0.00129 0.00043

0.0366 0.0105 0.0123 0.0019 0.0003

0.7109 0.9338 0.8663 0.9677 0.9998

Sulfamethazine 60 5.0982 70 5.1395 80 5.1980 90 5.0758 100 5.2522

0.00105 0.00203 0.00256 0.01092 0.03092

0.0840 0.1088 0.0168 0.0944 0.0996

0.00078 0.00100 0.00043 0.00175 0.00355

0.0494 0.0233 0.0020 0.0015 0.0129

0.7197 0.8777 0.9671 0.9704 0.9935

Sulfapyridine 60 5.1941 70 5.1441 80 5.2663 90 5.1353 100 5.3517

0.00134 0.00204 0.00313 0.00527 0.00882

0.0441 0.0548 0.0289 0.0444 0.0862

0.00041 0.00051 0.00027 0.00082 0.00307

0.0221 0.0101 0.0001 0.0014 0.0129

0.9087 0.9348 0.9911 0.9717 0.9472

Sulfaquinoxaline 60 5.1600 70 5.1213 80 5.1802 90 5.1298 100 5.1807

0.00235 0.00238 0.00290 0.01115 0.01725

0.0594 0.1198 0.0564 0.0735 0.1268

0.00055 0.00111 0.00052 0.00136 0.00452

0.0079 0.0344 0.0026 0.0004 0.0223

0.9413 0.8324 0.9633 0.9822 0.9684

Sulfathiazole 60 5.2761 70 5.2263 80 5.2439 90 5.2425 100 5.2951

0.00055 0.00076 0.00084 0.00204 0.00272

0.0116 0.0300 0.0229 0.0242 0.0039

0.00010 0.00028 0.00021 0.00046 0.00014

0.0037 0.0408 0.0106 0.0068 0.0027

0.9574 0.8802 0.9336 0.9449 0.9986

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

presented shorter half-life times for these temperatures, indicating a more unstable nature. This thermal degradation can be explained by the kinetic parameters obtained by applying Arrhenius equation. The kinetic parameters presented the highest values for sulfamerazine (ln A = 24.152, Ea = 86.60 kJ/mol) and sulfamethazine (ln A = 24.121, Ea = 87.30 kJ/ mol) showing that these molecules require a high temperature to reach their activation energy and bring about the transition state, where the successful collisions between moles have enough energy (Ea) to break the pre-existing bonds and form the reaction products. The elevated collision frequency (ln A) and the high temperature induce more successful collisions, which increase the reaction rate. This explains why the degradation of sulfamerazine and sulfamethazine is slower even when milk is heated at 80 °C, and, furthermore, the reaction is accelerated quickly upon reaching higher temperatures, obtaining the lowest half-life values (Table 3). Moreover, sulfadimethoxine (Ea = 21.05 kJ/mol, ln A = 0.754) and sulfathiazole (Ea = 42.97 kJ/mol, ln A = 7.890) presented lower kinetic parameters. Low activation energy means that these substances quickly reach the transition state. However, the low probability of successful collisions (low values of ln A) significantly reduced the reaction, and therefore these molecules become more resistant 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 the thermal degradation of quinolones (between 27.90 kJ/mol for flumequine, and 63.42 kJ/mol for norfloxacin) and beta-lactam antibiotics, with values ranging from 50.30 kJ/mol for cefquinome to 88.84 kJ/mol for cefoperazone (Roca et al., 2011). It should be emphasised that there are no values for the kinetic parameters (Ea and ln A) of SAs in the reviewed literature, even in aqueous solutions. On the other hand, Fig. 1 shows the plot of the liberalised Arrhenius’ equation (Eq. (5)) for the eight SAs analysed. This figure illustrates again how sulfadimethoxine and sulfathiazole have lower slopes than the other SAs tested, indicating the greater stability of both molecules to heating; therefore, they present lower thermal degradation with higher temperatures and longer heating times. Later, Eq. (6) was used to estimate the degradation percentages (Table 6) of the SAs in pasteurised milk at high temperature-short time (72 °C – 15 s), and UHT sterilisation (ultra-high temperature, 140 °C for 4 s), which are the most frequently used treatments in the dairy industry. Additionally, degradation percentages of SAs in sterilised milk at 120 °C for 20 min, and pasteurisation at low temperature-long time (63 °C – 30 min) were investigated, since these treatments are also frequent in the dairy industry in Spain. The estimated degradations show that SAs are very resistant to the heat treatments used in pasteurisation (63 °C, 30 min and

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

Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulphaquinoxaline Sulfathiazole

t1/2 = ln 2/k (min) 60 °C

70 °C

80 °C

90 °C

100 °C

582 1415 619 620 835 517 295 1260

153 624 555 341 418 340 291 912

112 260 453 271 290 221 239 825

89 123 327 63 90 132 62 340

63 85 290 22 28 79 40 255

t1/2: half-lives (min.)

their concentrations by heating milk. In contrast, sulfamerazine (t1/ 2 = 63, t1/2 = 22 min) and sulfamethazine (t1/2 = 90, t1/2 = 28 min)

Table 5 Statistical parameters calculated using the Arrhenius model for the degradation of sulphonamides in milk. Sulphonamide

ln A

Ea

F value

p value

R

Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulphaquinoxaline Sulfathiazole

12.466 19.539 0.754 24.152 24.121 10.878 13.979 7.890

52.10 75.03 21.05 86.60 87.30 48.61 56.49 42.97

19.79 266.28 85.85 23.32 39.36 409.19 14.91 34.17

0.0211 0.0005 0.0027 0.0169 0.0082 0.0003 0.0307 0.0100

0.9317 0.9944 0.9830 0.9413 0.9640 0.9964 0.9124 0.9588

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

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Sulfadiazine

Sulfadimethoxine

Sulfamerazine

ln [k]

Sulfachloropiradizine

2.65

2.7

2.75

2.8

2.85

2.9

2.95

3.0

3.05

1000/T (ºK) Sulfamethazine

Sulfapyridine

Sulfaquinoxaline

Sulfathiazole

-2 -3

ln [k]

-4 -5 -6 -7 -8 2.65

2.7

2.75

2.8

2.85

2.9

2.95

3

3.05

1000/T (ºK) Fig. 1. Arrhenius equation for thermal degradation of sulphonamides in milk.

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

Sulfachloropyridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulfaquinoxaline Sulfathiazole

Pasteurisation

Sterilisation

63 °C – 30 min

72 °C – 15 s

120 °C – 20 min

140 °C – 4 s

6.0 2.0 3.3 2.4 3.1 4.3 5.7 1.7

0.1 0.0 0.0 0.0 0.1 0.1 0.1 0.0

46.0 47.9 6.5 77.4 85.1 30.7 51.9 9.9

0.4 1.7 0.0 1.8 2.3 0.3 0.6 0.1

72 °C, 15 s) and that sterilisation at 140 °C for 4 s obtained very low degradation rates (below 6% in all cases). Conversely, sterilisation at 120 °C produced greater losses, which reached 77.4% for sulfamerazine and 85.1% for sulfamethazine. These molecules display high activation energy (Ea) and collision frequency values (ln A), in addition to having low half-life times at 100 and 120 °C (Tables 4 and 5). However, this heat treatment (120 °C for 20 min) led to a slight inactivation of sulfadimethoxine and sulfathiazole since they had longer half-life times at 100 and 120 °C. 3.2. Thermodynamic molecular stability study Table 7 shows the mathematical equations obtained by applying the linear regression model (Eq. (8)) and Gibbs free energy values (Eq. (7)) as a function of absolute temperature. For the eight SAs examined, the specific molar entropy of activation values was negative, indicating that the thermal stability of these molecules is a thermodynamically unfavourable phenomenon.

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

Table 7 Thermodynamic parameters (standard molar enthalpy and entropy of activation) of sulphonamides in milk. Sulphonamide Sulfachloropiridazine Sulfadiazine Sulfadimethoxine Sulfamerazine Sulfamethazine Sulfapyridine Sulfaquinoxaline Sulfathiazole

DG0⁄ = DH0⁄  T.DS0⁄ 0⁄

DG Sulfacloropidazine = 48096  (196)  T DG0⁄Sulfadiazine = 71800  (135)  T DG0⁄Sulfadimethoxine = 18272  (290)  T DG0⁄Sulfamerazine = 84303  (94)  T DG0⁄Sulfamethazine = 83776  (97)  T DG0⁄Sulfapyridine = 45830  (205)  T DG0⁄Sulfaquinoxaline = 55025  (176)  T DG0⁄Sulfathiazole = 40560  (229)  T

R 0.9614 0.9855 0.9999 0.7968 0.8618 0.9985 0.9266 0.9878

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

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

DH0 ¼ 2:939 þ 342DS0

R ¼ 0:9980

Other authors also observed a strong linear regression between these 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 suggested that the stability of these molecules is due to, not only the enthalpy and entropy values, but also to a combination of both thermodynamic variables (Fisher et al., 2010). Indeed, while the specific molar Gibbs energy (DG0⁄) of SAs presents slight variations between 113 and 126 kJ/mol (approximately 11.5%, Fig. 2), the DH0⁄ values vary from 18.3 kJ/mol (sulfadimethoxine) to 84.3 kJ/mol (sulfamerazine), while the DS0⁄ values range from 94 J/mol K (sulfamerazine) to 290 J/mol K (sulfadimethoxine), with wide ranges of variations of next to four (DH0⁄) and three (DS0⁄) times in magnitude.

Sulfachloropiridazine

Sulfadiazine

H 0* (KJ/mol) 0 -50

20

40

60

Sulfachloropiridazine -200 -250 -300

Sulfaquinoxaline

Sulfapyridine Sulfathiazole Sulfadimethoxine

-350 Fig. 3. Enthalpy and entropy of activation of thermal degradation for sulphonamides in milk.

Therefore, it can be established that SAs have great thermal stability, but that each molecule has different thermodynamic parameters. Thus, the stability of sulfamerazine and sulfamethazine is due to high DH0⁄ values since the DS0⁄ values are very low, while sulfadimethoxine and sulfathiazole present negative DS0⁄ values and low DH0⁄ values.

Sulfadimethoxine

Sulfamerazine

124000

G0* (J/mol)

122000 120000 118000 116000 114000 112000 340

350

360

370

380

Temperature (ºK) Sulfamethazine

Sulfapyridine

Sulfaquinoxaline

Sulfathiazole

126000 124000

G0* (J/mol)

122000 120000 118000 116000 114000 112000 330

340

350

100

Sulfamethazine Sulfadiazine

-150

126000

330

80

Sulfamerazine -100

S0* (KJ/mol)

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

360

370

380

Temperature (ºK) Fig. 2. Temperature-dependent standard molar free energy of activation 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 sulphonamide molecule can undergo significant changes in the statistical DH0⁄ and DS0⁄ parameters due to thermal degradation, but they do not cause significant changes in the DG0⁄ values. This is due to a compensatory effect between the DH0⁄ and DS0⁄ values, as shown in Fig. 3. In this Figure an increase in the values of DH0⁄ and a decrease in the values of DS0⁄ can be appreciate as we move in the direction of sulfadimethoxine, sultathiazole, sulfapyridine, sulfacloropiridazine, sulphaquinoxaline, sulfadiazine, sulfamerazine and sulfamethazine. This fact explains the higher thermal stability of the first sulphonamides compared with the minor stability of the last molecules of the series. This compensatory effect was also reported by Fisher et al. (2010) in their study into the thermodynamic stability of quinolones in milk. These authors suggest that the enthalpy–entropy compensation of very stable molecules is responsible for the relatively slight variations in DG0⁄ in the thermal degradation of quinolones in milk. Similarly, Rudra et al. (2008) emphasised the existence of enthalpy–entropy compensation when studying the thermal degradation of chlorophyll. The enthalpy of activation (DH0⁄) varies from 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 correlation coefficient (R2 = 0.96) between the two thermodynamic parameters. In addition, in another study, Kang and Warren (2007) carried out a thermodynamic analysis of the transition of mono-specific antibodies (native and urea-treated) to show that this antibody transition exhibits enthalpy–entropy compensation. These authors observed 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 complexes at 37 °C conducted by Kang and Auerbach (2009) revealed a linear behaviour between the specific molar entropy and the enthalpy of activation, with a high regression coefficient (R2 = 0.957). The disassociation of the complexes underwent changes of 23.3 times in the DH0⁄ values and of 18.5 times in the DS0⁄ values, while the DG0⁄ variations were lower (5.7 times). This fact also indicates an enthalpy–entropy compensation phenomenon. These thermodynamic results are consistent with the structural characteristics of sulphonamides. For the SAs with a pyrimidine ring in their molecule structure (sulfadiazine, sulfamethazine, sulfamerazine and sulfadimethoxine) it is appreciated that the incorporation of one (sulfamerazine) or two methyl functional groups (sulfamethazine) decreases their stability due to their higher entropy activation values, compared with sulfadiazine. Whereas, the incorporation of two methoxy functional groups (sulfadimethoxine) increases the stability of the pyrimidine ring linked to a decrease in the entropy activation (Fig. 1, Supplementary data). Moreover, in the case of the sulfachloropyridazine which possesses a pyridazine ring (similar to the pyrimidine ring), the chlorine substituent produces a similar effect to that of the methoxy functional group, increasing the stability of this molecule. For the other SAs the different heterocyclic structures (quinoxaline, pyridine and thiazole rings) give them intermediate stabilities.

4. Conclusions The present study investigated the thermal stability of sulphonamides in skimmed milk during heating, applying the first-order kinetic model and calculating the kinetic parameters of activation energies, half-lives and degradation percentages. The results obtained show that sulphonamides are very stable molecules which can resist even the most common heat treatments performed in the dairy industry without degrading significantly.

The differences of stability obtained between the sulphonamides evaluated were explained by thermodynamic evaluation. In this study we concluded that the chemical structure of each substance contributes to the specific molar entropy and enthalpy of activation during the thermal degradation of these molecules in different ways. However, the free energy of activation is almost constant due to the enthalpy–entropy compensation, given the high stability of the sulphonamides. To summarize, the high thermo-stability of sulphonamides demonstrate that the heat treatments used in the dairy industry could be insufficient to completely inactivate sulphonamide residues in milk. In addition, the dairy industry currently uses specific methods to detect only beta-lactams and tetracyclines as well as slow-response and low sensitive microbiological methods to detect other antimicrobial agents. Therefore, sulphonamide residues in milk may represent a potential health risk to consumers if the control of the presence of these residues is not adequate to prevent them from reaching the food chain. Thus, it is also interesting to note that the high stability of sulphonamides can also provoke serious problems if their residues reach the environment. Regarding this aspect, some authors have found sulphonamide residues in rivers and streams in the U.S (García-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, other authors point out that small amounts of sulphonamides from agricultural activity, cause changes in the population of microorganisms in the environment and can be potentially hazardous to human health (Baran, Sochacka, & Wardas, 2006). Therefore, the high thermal stability of sulphonamides and the potential risk that they might have on the microorganism population invites future research strategies for the treatment of waste and effluents containing these compounds.

Acknowledgements This research has been supported financially by the Spanish Ministry of Education and Science (AGL2003-03663 project, Madrid, Spain) and has been carried out with the help of the Vice-rectorate of Research, Development and Innovation at the Polytechnic University of Valencia (Reference 6567). Moreover, the authors wish to thank the Polytechnic University of Valencia for funding the collaboration of Dr. Rafael Althaus with the Institute for Animal Science and Technology.

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

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