Determination of the presence of 10 antimicrobial residues in mexican pasteurized milk



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DETERMINATION OF THE PRESENCE OF 10 ANTIMICROBIAL RESIDUES IN MEXICAN PASTEURIZED MILK

Rey Gutiérrez Tolentino, Mario Noa Pérez, Gilberto Díaz González, Salvador Vega y León, Magdalena González López and Guadalupe Prado Flores


Rey Gutiérrez Tolentino. Dr. in Biological Sciences, Univer­sidad Autónoma Metro­politana (UAM), Mexico. Professor-Researcher, Department of Agricultural and Animal Production, UAM, Unidad Xochimilco, Mexico. Address: Calzada del Hueso 1100. Coyoacán, Distrito Federal. CP. 04960, México. e-mail: reygut@correo.xoc.uam.mx.

Mario Noa Pérez. Dr. in Veterinary Sciences, Instituto Superior en Ciencias Agropecuarias, Cuba. Professor-Researcher, Department of Public Health, CUCBA, Universidad de Guadalajara, Mexico. Address: Apartado 1-140, Guadalajara, Jal. C.P. 44100, Mexico. e-mail: mnoa@yahoo.com

Gilberto Díaz González. Dr. in Marine Sciences, Universidad Nacional Autónoma de Méxi­co. Professor-Researcher, Department of Agricultural and Animal Production, UAM, Unidad Xochimilco, Mexico.

Salvador Vega y León. Dr. in Veterinary Sciences, Centro Na­cional de Sanidad Agro­pe­cuaria, Cuba. Professor-Researcher, Department of Agricultural and Animal Production, UAM, Unidad Xochimilco, Mexico. e-mail: svega@correo.xoc.uam.mx.

Magdalena González López. Engineer, Universidad Juárez Autónoma de Tabasco. Professor-Researcher, Department of Agricultural and Animal Production, UAM, Unidad Xochi­milco, Mexico.

Guadalupe Prado Flores. Dr. in Biological Sciences, UAM, Mexico. Professor-Researcher, Department of Agricultural and Animal Production, UAM, Unidad Xochimilco, Mexico.


Resumen
Los residuos de 9 agentes antimicrobianos aprobados en México para su uso veterinario en el ganado lechero (sul­fa­tiazol, sulfamerazina, sulfametazina, sulfacloropiridazina, sulfa­mo­nometoxina, sulfametoxazol, nitrofurazona, furaltadona y fura­zo­lidona), así como de cloranfenicol, cuyo uso está prohibido en la producción pecuaria, fueron determinados en leche entera pasteurizada y comercializada en la Ciudad de México. Cuatro industrias de leche (A, B, C y D) fueron estudiadas quin­cenalmente durante un año (n=4×24=96). Los residuos fueron analizados por cromatografía de líquidos de alta presión (HPLC) con detector UV. El porcentaje de muestras positivas a residuos de sulfonamidas, fueron 47,2% (leche A); 58,3% (leche B); 44,7% (leche C) y 50% (leche D). Los residuos de sulfonamidas estuvieron en el intervalo de 1,9 a 180µg/kg. Residuos de sulfatiazol y sulfamerazina fueron los más encontrados. Sólo tres muestras (leche B) excedieron los niveles de residuo máximo establecidos para sulfonamidas. No se detectaron residuos de nitrofuranos en ninguna de las muestras de leche analizadas. Residuos de cloranfenicol fueron detectados en una muestra de leche (leche A) con un nivel de 27,2µg/g.
Summary
Residues of 9 antimicrobial agents approved in México for veterinary use in dairy cattle (sulfathiazole, sulfamerazine, sulfamethazine, sulfachloropyridazine, sulfamonomethoxine, sulfa­meth­oxazole, nitrofurazone, furaltadone, and furazolidone) and non-approved chloramphenicol residues were studied every two weeks in four commercial brands of Mexican pasteurized milk (A, B, C and D) during one year (n=4×24=96). Drug residues were analyzed by high pressure liquid chromatography HPLC with UV detection. Percentage of positive samples to sulfonamide residues were 47.2% (Milk A), 58.3% (Milk B), 44.7% (Milk C) and 50% (Milk D). Sulfonamide residues ranged between 1.9 and 180µg/kg. sulfathiazole and sulfamerazine were the most common residues found. Only three samples (Milk B) exceeded maximum residue levels established for sulfonamides. Nitrofuran residues were not detected in any milk sample. Illegal Chloramphenicol residues were detected in one milk sample (Milk A) in levels of 27.2µg/kg.
Resumo
Os resíduos de 9 agentes antimicrobianos aprovados no México para seu uso veterinário no gado leiteiro (sulfatiazol, sulfamerazina, sulfametazina, sulfacloropiridazina, sulfa­mo­no­metoxina, sulfametoxazol, nitrofurazona, furaltadona e furazolidona), assim como de cloranfenicol, cujo uso está proibido na produção pecuária, foram determinados no leite integral pasteurizado e comercializado na Cidade do México. Quatro indústrias de leite (A, B, C e D) foram estudadas quinzenalmente durante um ano (n=4×24=96). Os resíduos foram analisados por cromatografia de líquidos de alta pressão (HPLC) com detector UV. A porcentagem de amostras positivas a resíduos de sulfonamidas foi de 47,2% (leite A); 58,3% (leite B); 44,7% (leite C) e 50% (leite D). Os resíduos de sulfonamidas estiveram no intervalo de 1,9 a 180µg/kg. Resíduos de sulfatiazol e sulfamerazina foram os mais encontrados. Somente três amostras (leite B) excederam os níveis de resíduo máximo estabelecidos para sulfonamidas. Não se detectaram resíduos de nitrofuranos em nenhuma das amostras de leite analisadas. Resíduos de cloranfenicol foram detectados numa amostra de leite (leite A) com um nível de 27,2µg/g.
KEYWORDS: Antimicrobial Residues / Chloramphenicol / Nitrofurans / Pasteurized Milk / Sulfonamides /

Received: 01/12/2005. Modified: 04/13/2005. Accepted: 04/15/2005.


Introduction
Antimicrobial drugs, such as sulfonamides, nitrofurans and antibiotics, are widely used for treatment and prevention of disease in dairy cattle. Residues of these drugs may therefore be present in milk and can be a health hazard to consumers, considering that they may cause allergic reactions in sensitive persons (Thomson and Sporns, 1995), increase in the number of antibiotic resistant pathogenic bacteria (Honkanen and Reybroeck, 1997) and some of them, like sulfamethazine and nitrofurazone are considered to have carcinogenic properties (Paulson et al., 1992).

The monitoring of antimicrobial residues in commercial milk is essential for ensuring the safety and adequacy of food, as well as providing an indication of the proper usage of these chemotherapeutics at the producer level (Zomer et al., 1992).

The purpose of this work was to evaluate the residual levels of 9 antimicrobial drugs (6 sulfonamides and 3 nitrofurans) approved for veterinary treatments in dairy cattle, as well as illegal chloramphenicol residues in whole pasteurized commercial milk.
Materials and Methods
Procurement of milk
Three commercial brands with wide national distribution (A,B,C) and one brand (D) produced and distributed by the government stores for the low income population, were selected. Sampling was carried out in supermarkets (A,B,C) and in government stores (D) every 2 weeks over the course of a year (n=24, for each milk brand).
Reagents
Anhydrous sodium acetate, glacial acetic acid (ACS, reagent grade). Water, acetonitrile, hexane, methanol (HPLC grade). Acetone, chloroform (residue grade). 0.02M sodium acetate buffer solution, pH 4.8.

Mobile phases.-1) Sodium acetate buffer solution 0.02M - Acetonitrile (95:5 v/v). 2) Sodium acetate buffer solution 0.02M - Acetonitrile (80:20 v/v).

Extraction solution. Chloroform - Acetone (2:1 v/v). Prepared daily

Sulfonamide standards. Sulfathiazole (Alldrich), sulfamerazine, sulfamethazine, sulfachloropyridazine and sulfamonomethoxine (all from Sigma), and sulfamethoxazole (Mexican Pharmaceutical Standard).

Nitrofuran standards. Nitrofurazone, furaltadone (both from Sigma) and furazolidone (Mexican Pharmaceutical Standard).

Chloramphenicol standard (Mexican Pharmaceutical Standard).


Preparation of Standard Solutions
Stock solutions. Accurately weigh 25mg, to nearest 0.1mg of each one of the standards into separate weighing vessels, transfer to separate 25ml volumetric flasks (1mg/ml). Dissolve in methanol.

Fortification solution (10µg/ml of each substance under study). Transfer 250µl of each standard solution (1mg/ml) into a single 25ml volumetric flask, dilute to volume with methanol, and mix thoroughly. Prepare daily.
Sample extraction
Place a 10ml milk sample in a 125ml separation funnel. Add 50ml of extraction solution and place stopper. Shake vigorously for 1min; carefully vent through stopper. Do not vent through stopcock because milk solids clogging may result in sample loss.

Shake for an additional 1min, vent, and let phases separate during 1min. Repeat shaking sequence and let phases separate 5min. Draw off extraction solution, filtering through filter paper washed with the extraction solution, and collect in a 100ml round bottom flask, taking great care to prevent milk from entering stopcock. Add 25ml extraction solution to separation funnel and repeat the procedure exactly as before. Add this extract to the one previously drawn. Rinse filter with extraction solution (2×5ml) and add to the other extracts.


Sample preparation
Carefully evaporate the extracts collected in the 100ml round bottom flask, until dryness on a rotary evaporator at 32°C. Dissolve the residue in 1ml 0.02M sodium acetate buffer solution, agitating for 1min on a vortex mixer. Add 5ml hexane and vortex for 1min. Using a Pasteur pipette, transfer phases to 15ml conical tubes and let phases separate during 2min. Immediately vortex for another 1min. Let the phases separate for >15min. Remove aqueous layer with a Pasteur pipette and place in a 2ml vial. Note that the aqueous layer will be homogeneous; therefore it is not necessary to remove 100% of layer; 50-75% of the aqueous layer is adequate for two injections). Using a 3ml syringe, filter the aqueous layer through nylon filter 0.45µm, 13mm. Store in sealed vials.
Apparatus
Equipment. Determinations were made in a HPLC system from Merck Hitachi, with variable UV detector, a 250×4.6mm reversed-phase C18 column, 5µm particle size (LiChroCART 250-4, Merck 1.50838), and a 2cm guard column.

Operating conditions. Flow rate of 1.0ml/min (sulfonamides) or 1.2ml/min (nitrofurans), standard injection volume of 5µl and sample injection volume of 20µl. Running time of 45min for sulfonamides and chloramphenicol, and of 15min for nitrofurans. Chart speed of 0.5cm/min. Detector settings at 275nm for sulfonamides and chloramphenicol, and at 370nm for nitrofurans.

Chromatographic Systems. Two chromatographic systems were used:

1) For the 6 sulfonamides and chloramphenicol, a gradient system was used employing the two mobile phases indicated above as 1) and 2), and the following time schedule:

Time (min) 0 5 20  30  35 45

% B 0 0 100 100 0  0 


2) For the nitrofurans determination an isocratic system was used, employing mobile phase B.

Samples were first analyzed using the isocratic system and then with the gradient system, after an equilibration time of 1h.


Quality assurance of HPLC methodology
The HPLC analysis of the 10 studied residues in milk has been described previously (Pérez et al., 2001) and it is based on the AOAC official method 993.32 for multiple sulfonamide residues (AOAC, 1995), with some modifications in the chromatographic system. Method validation (Pérez et al., 2001) showed that the detector response was linear over the range of 25 to 100ng/ml for all drugs under study, with correlation coefficients in excess of 0.99. Method detection limits ranged from 4µg/kg (nitrofurazone) to 16µg/kg (sulfamethazine). Average recoveries ranged from 65.5% (sulfathiazole) to 104.2% (chlora­mphenicol) and precision (coefficients of variation, CV<13%) was acceptable according to the Codex Committee on Veterinary Drug Residues, which considers as acceptable the coefficients of variation below 20% (CODEX, 1989).
Results and Discussion
Residues of sulfonamides were detected in all four milk brands analyzed. In milks A, B and D, all 6 sulfonamides tested were found. However in Milk C, sulfamonomethoxine and sulfachloropyridazine were not detected (Figure 1).

The percentage of positive samples to any sulfonamide residues were: 47.2% (Milk A), 58.3% (Milk B), 44.7% (Milk C) and 50% (Milk D). These values are above those reported in milk from other countries such as Costa Rica (2%; Monge et al., 1993) and Italy (37%; Ferrini et al., 1994).

According to Honkanen and Reybroeck (1997), during the last decades the occurrence of antimicrobial residues in milk has decreased considerably in those countries where residue testing is regularly performed, and the number of inhibitor positive ex-farm milk is in general 1-5‰. In Mexico there is not an antimicrobial milk residue program established which could reflect the high values of positive samples found in this study.

The sulfonamide residues with higher frequencies were sulfathiazole (33% Milk A, 20.8% Milk B, 16.7% Milk D) and sulfamerazine (37.5% Milk B, 20.8% Milk C, 16.7% Milk D).

Figure 1 presents the average residue levels for each sulfonamide tested and found in samples in the four milk brands under study. Mean values and ranges (µg/kg) were 38.2, 4.1-92.6 (Milk A); 55.4, 4.3-180 (Milk B); 21.9, 1.9-90.68 (Milk C) and 27.5, 8.7-74.4 (Milk D). The sulfonamide residues with higher mean levels were sulfa­methoxazole (57.4µg/kg, Milk A, and 42.15µg/kg, Milk C), sulfamerazine (49.53µg/kg, Milk B) and sulfathiazole (37.24µg/kg, Milk B).

In Mexico no maximum residue limits (MRL) have been established for sulfonamide residues in milk. Thus, MRL values from the Codex Alimentarius, EC regulation and FDA regulations (Honka­nen and Reybroeck, 1997) were considered. Only three samples (Milk B) exceeded 1.1, 1.4 and 1.8 times the MRL of 100µg/kg; the first sample presented multiple sulfonamide residues (sulfathiazole, sulfamerazine and sulfamethoxazole) with a total amount of 110.5µg/kg. The second and third ones presented sulfamerazine residues in concentrations of 141.2 and 180µg/kg, respectively.

Sulfamethazine residues, which are of high concern because of its possible carcinogenic properties (Paulson et al., 1992), were found in some of the four groups of milk samples: 8.3% (Milks A and B), 16.7% (Milk C) and 4.2% (Milk D). These values were higher than those reported in milks from Germany (2%; Grether et al., 1994) and Sweden (1.8%; Mellgren et al., 1996). The residue levels of this antimicrobial in positive samples were under the MRL established by the Codex (25µg/kg); however, if the MRL of the FDA (10µg/kg) is considered, two samples exceeded the limit, with residue levels of 18.0µg/kg (Milk A) and 23.6µg/kg (Milk C).

The residue values that violate established regulations may occur when withdrawal periods are neglected or if higher than the recommended doses are used. The presence of these unacceptable residue levels in commercial milk, which is pooled milk from different producers and, therefore, residue levels are diluted, should be taken into account. It is well known that high volumes of good quality milk can be contaminated with the milk of only one careless producer.

The high frequency of positive samples and the appearance of some cases exceeding MRL, reveals the need to establish regular residue testing programs in Mexico.

No cases of nitrofuran residues were found in milks tested. In Mexico these drugs are recommended for treatment of mastitis in dry cows (DMVNZ, 1997). Our results may reflect that these antimicrobials are used correctly only in non lactating cows. Also it would be possible that nitrofuran residues, if present, were under the detection limits of the method used, of 4, 8 and 13µg/kg for nitrofurazone, furazolidone and furaltadone, respectively. No reports of nitrofuran residues in milk were found in the literature, probably because the use of these substances is banned in animal husbandry in several countries (Rugraff and Coulon, 1999).

Chloramphenicol residues were detected in one sample (Milk A) at levels of 27.2µg/kg. This drug is not allowed for use in dairy cattle and its presence in milk means that farmers may still use it in a non authorized form. The presence of chloramphenicol residues in milk has also been reported in Slovenian milk (Gacnick et al., 2000), one in 217 milk samples analyzed, in levels of 4.6µg/kg.

Mexican authorities should pay more attention to the presence of antimicrobial drug residues in milk and should establish periodical residue testing programs. In this way, the occurrence of positive samples would decrease considerably.
REFERENCES
AOAC (1995) Method 993.32 (Multiple sulfonamide residues in raw bovine milk). In Handbook of Official Analytical Methods of Analysis. 16th ed. Association of Official Analytical Chemists. Arlington, VA, USA. Vol. 1, p. 13.

CODEX (1989) Codex Alimentarius. ALINORM 89/31., Appendix VI. Geneve, Switzerland. pp. 1-9.

DMVNZ (1997) Directorio de Medicina Veterinaria, Nutrición y Zootecnia. Mercadeo Estadístico SC, Mexico.

Ferrini AM, Filesi C, Mannovi V, Sanzini E, Aureli P, Bellomonte G (1994) Indagine sulla presenza di residui di sulfamidici in latti dell´ Agro Pontino. La industria del latte 30: 31-39.

Gacnick KS, Kirbis A, Cerkvenik V (2000) Residues of veterinary drugs in Slovenian milk 1995-1998. Mljekarstvo 50: 1991-1998.

Grether A, Hammer P, Heeschen W (1994) Zum immunochemischen Nachweis ausgewählter sulfonamide in milch. Kieler Milchwirstchaftliche Forschungsber. 46: 101-126.

Honkanen BT, Reybroeck W (1997) Antimicrobials. In Residues and Contaminants in Milk and Milk Products. IDF. Brussels, Belgium. pp. 26-33.

Mellgren C, Sternesjo A, Hammer P, Suhren G, Bjorck L, Heeschen W (1996) Comparison of biosensor, microbiological, immunochemical and physical methods for detection of sulfamethazine residues in raw milk. J. Food Prot. 59: 223-1226.

Monge R, Arias ML, Ellner R (1993) Contamination of bovine milk with residues of inhibitory substances in Costa Rica. Revista de Biología Tropical. 41: 855-856.

Paulson GD, Fiel VJ, Giddings JM, Lamoreux CH (1992) Lactose conjugation of sul­pho­­namide drugs in the lactating dairy cow. Xeno­biotica 22: 925-939.

Pérez N, Gutiérrez R, Noa M, Díaz G, Luna H, Escobar I, Munive Z (2001) Liquid chromatographic determination of multiple sulfonami­des, nitrofurans and chlo­ram­phenicol residues in pasteurized milk. J. AOAC Int. 85: 20-24.

Rugraff Y, Coulon S (1999) Residues in pig meat: results of surveys. Techni-Porc. 22: 21-23.



Thomson CA, Sporns P (1995) Direct ELISAs for sulfathiazole in milk and honey with special emphasis on enzyme conjugate preparation. J. Food Sci. 60: 409-414.

Zomer E, Saul S, Charm SE (1992) HPLC receptogram: a method for confirmation and identification of antimicrobial drugs by using liquid chromatography with microbial receptor assay. I Sulfonamides in milk. J. AOAC Int. 75: 987-993.


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