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Food Safety and Toxicology

Investigation of the persistence of penicillin G and dihydrostreptomycin residues in milk of lactating buffaloes (Bubalus bubalis) using ultra high performance liquid chromatography tandem mass spectrometry Claudia Chirollo, Antonio Vassallo, Fabrizio Dal Piaz, Barbara Lamagna, Gennaro Tortora, Gianluca Neglia, Nunziatina De Tommasi, and Lorella Severino J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00229 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Journal of Agricultural and Food Chemistry

Investigation of the persistence of penicillin G and dihydrostreptomycin residues in milk of lactating buffaloes (Bubalus bubalis) using ultra high performance liquid chromatography tandem mass spectrometry

Claudia Chirollo a, Antonio Vassallo b,*, Fabrizio Dal Piaz c, Barbara Lamagna a, Gennaro Tortora a, Gianluca Neglia a, Nunziatina De Tommasi d and Lorella Severino a,

*.

a

Department of Veterinary Medicine and Animal Production, University of Napoli

‘‘Federico II’’, Via Delpino 1, 80137 Napoli, Italy; b

Department of Science, University of Basilicata, Viale dell’Ateneo Lucano 10, 85100

Potenza, Italy c

Department of Medicine, University of Salerno, Via Giovanni Paolo II 132, 84084

Fisciano, Italy d

Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084

Fisciano, Italy

*

Corresponding authors :

Antonio Vassallo (Tel: +39 0971 205624;

Fax: +39 0971 205678; E-mail:

[email protected]); Lorella Severino (Tel: +39 081 2536272; Fax: +39 081 2536274; E-mail: [email protected])

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ABSTRACT

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The purpose of this research was to evaluate the persistence of penicillin G and

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dihydrostreptomycin in milk of lactating buffaloes following intramuscular injection of

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procaine penicillin G (200,000 IU/mL) and dihydrostreptomycin sulfate (250 mg/mL)

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every 24 h for 3 days. Milk samples were collected twice daily up to 13th milking post-

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treatment and analyzed by ultra-high-performance liquid chromatography coupled to

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tandem mass spectrometry. The analytical method has been validated according to

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Commission Decision 2002/657/EC. The highest concentrations of penicillin G (275 µg

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kg-1) and dihydrostreptomycin (220.5 µg kg-1) were detected in the milk of the first

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milkings post-treatment and levels were below the maximum residue limit of 4 µg kg-1

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and 200 µg kg-1 in all treated buffaloes at milkings 12 and 2 respectively. The results of

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this study demonstrate that 9 milking withdrawal time set for bovine milk could be not

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adequate for depletion of penicillin G in lactating buffaloes.

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Keywords: buffaloes, residues, milk, penicillin G, dihydrostreptomycin, HPLC-

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MS/MS.

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INTRODUCTION

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Water buffalo (Bubalus bubalis) is recognized as the world second most important milk

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producing species and plays an important role in the economy of developing countries

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like India, Nepal and Pakistan (80% of the world’s production of buffalo milk). Among

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Europe, Italy is the only country where Mediterranean buffalo breeding has a

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remarkable zootechnical importance for the production of “mozzarella di bufala

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campana” cheese with a protected designation of origin (PDO) according to EU

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legislation. 1

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Therapeutics, such as antimicrobial, are used in lactating buffaloes for the treatment of

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mastitis and post-partum diseases.

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requires that pharmacologically active substances authorized in cattle (Bos taurus) may

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also be targeted to buffaloes (Bubalus bubalis) with the same withdrawal periods,

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although no data on depletion of antibiotics in buffalo milk are available in literature.

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Synergistic effects of combination compounds are frequently preferred in livestock, to

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increase the effectiveness and to improve treatment outcome as the combined effect of

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both antibiotics is better than their individual effect. In this regard, penicillin plus

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aminoglycoside is considered a first-line antibiotic choice in dairy animals with clinical

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metritis and mastitis.

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Penicillin G (penG) belongs to the oldest group but still one of the most used among the

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classes of antimicrobials.

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been described,

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molecules belonging to the β-lactams group are scarcely tolerated in the European

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Union among antimicrobials .7 In order to adequately protect the consumer and secure

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dairy production, the Committee for Veterinary Medicinal Products recommends the

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maximum residue level (MRL) of 4 µg kg-1 for penG in milk of all producing animals.

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Dihydrostreptomycin (DHS) is an aminoglycoside antibiotic that acts directly on

5, 6

4

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Existing legislation on veterinary medicines

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However, allergic reactions in sensitive population have

due to β-lactams group residues in milk. For this reason, the



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bacterial protein synthesis.

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infections induced by Gram-negative and Gram-positive bacteria. Residues of this

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compound in the milk of all ruminants must be below the MRL of 200 µg kg-1 set by

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European Commission. In veterinary medicine, DHS is commonly combined with penG

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to increase the single drug potential in severe post- partum infections. 9

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If these antibiotics are not properly managed, drugs residues can remain in animal

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tissues and carry over into the milk. This condition can represent a risk for consumer

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safety, owing to toxicity, allergic reactions and increase in antimicrobial resistance.

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The screening methods largely used to detect these compounds are microbial assays.

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However, confirmatory methods are based on high performance liquid chromatographic

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(LC) methods coupled to different detectors. The β-lactam antibiotics can be detected

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by LC using diode array,

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using different

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methods for sample preparation.

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Many procedures have been evaluated for the analyses of aminoglycosides using very

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different approaches,

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MS/MS) with electrospray ionization (ESI) has been chosen as the best option as

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confirmatory method.

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the presence of amino and hydroxyl groups in the structure; moreover, they do not have

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chromophore and fluorophore groups and try to create connections with matrix proteins.

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20, 21

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preceded by organic solvents and/or an acid deproteinization of the matrix

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techniques are time consuming and may increase the costs for daily laboratory analyses.

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Detailed penG e DHS pharmacokinetic studies have been conducted for cattle 22,

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information concerning antibiotic depletion are lacking for buffalo species. In this

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paper, a very simple in-house LC-MS/MS method has been carried out and validated for

11, 12

17, 18

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It is available in many formulations for the treatment of

fluorescence, 13, 14 and mass spectrometry detectors,

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15, 16

but liquid chromatography tandem mass spectrometry (LC-

This class of antibiotics have nevertheless a high polarity for

In literature there are many studies for aminoglycosides using SPE extraction,


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.These

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but

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the simultaneous determination of dihydrostreptomycin and penicillin G residues in

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buffalo milk, modifying the extraction procedure described by Jank et al.

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detection of β-lactams in bovine milk. This method has several advantages, such as a

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very simple sample preparation procedure (centrifugation steps at low temperature),

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high extraction capacity, good reproducibility, and environmental friendly.

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for the

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MATERIALS AND METHODS

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Animal studies

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Eight lactating water buffaloes with post-partum disease were selected for this study

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from a farm located in Campania Region. Each animal received three intramuscular

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injections of 30 mL of Repen® (Fatro, Italia) containing a suspension of procaine

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penicillin G (200,000 IU/mL) and dihydrostreptomycin sulfate (250 mg/mL) at 24 h

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intervals. Milk samples were collected twice daily up to milking 13 post-treatment (4

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milkings more compared to bovine withdrawal period) and stored at -25 °C until

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analysis. The selected buffaloes were not treated with any antimicrobial drug during the

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previous 2 months. Animals were under veterinary supervision during the first three

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days of the study (every 12 h) for possible adverse reactions, then daily until the end of

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the trial.

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Materials and reagents

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Dihydrostreptomycin (DHS), as its sulfate salt, and penicillin G (penG) used as

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analytical standards (AS), as its procaine salt, were purchased from Sigma Aldrich

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(Bornem, Belgium). HPLC grade water, acetonitrile, and C18 bulk were from Baker

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Mallinckrodt (Phillisburg, New Jersey). Formic acid was obtained from Merck

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(Darmstadt, Germany).

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Sample preparation

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Milk samples were thawed at room temperature prior to analyses to avoid separation of

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aqueous and lipidic phases. Milk samples (2 g ± 0.1 g) were transferred into 50 mL

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centrifuge tubes. Extraction process was based on a procedure described by Jank et al. 24

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using only 4 mL of acetonitrile (ACN) and 100 mg of C18 bulk to each sample. Three

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centrifugation steps (10 min 4000 rpm) at low temperature (the first two at 4 °C, the last

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at 2 °C) were necessary to complete the purification. The final phase was characterized

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by the evaporation of the supernatant in a water bath (≤45 °C) under N2 stream. The

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milk extracts (250 µL), after an additional ultracentrifugation step (10 min at 13000

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rpm) in tubes of 1.5 mL, were put into an HPLC vials and 1µL injected to LC-ESI-

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QTrap-MS system.

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LC-MS and LC-MS/MS analyses

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A NexeraX2 UHPLC apparatus (Shimadzu, Kyoto, Japan), coupled with an ABSciex

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(Foster City, CA, USA) API6500 Q-Trap instrument, working in positive Multiple

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Reaction Monitoring (MRM) system was selected for the LC-MS/MS analyses. A

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standard solution (10 µg mL-1 in water into the source at a flow rate of 0.01 mL/min)

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was diluted with acetonitrile and water (50/50) for the instrument optimization. All the

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analysis and the MS data were performed in positive ion mode.

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Samples (1 µL) were loaded on a Luna Omega Polar column (Phenomenex) (1.6 µm

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Polar C18 100 A, 50 x 2.1 mm) and compounds separation was obtained using a linear

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gradient from 2% to 25% of acetonitrile (eluent B) in H2O containing 0.1% of formic

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acid (eluent A) over 7 minutes. Flow rate was 0.4 mL min−1 and injection volume was 1

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µL for standards and milk extract samples, the total run-time was 7 min.


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All the parameters, fragmentation ions of each analytes and dwell times are showed in

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Table 1. All data were processed using Analyst® software (ABSciex, Foster City, CA,

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USA).

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Calibration

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A primary stock standard solution of 1 mg mL−1 for DHS and penG was obtained by

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adding the correct amount of standard in water. Solutions were stored frozen (-20 °C)

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and were stable for 1 month. Eight point working solutions of 1 to 1000 ng mL-1 of

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DHS and of 0.1 to 500 ng mL-1 of penG were assembled by dilution of the respective

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stock solutions with water. The working standard solutions were stored at -20 °C in

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small plastic tubes to allow the use of only one cap during each extraction day and to

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avoid degradation. Negative buffalo milk samples were fortified, to obtain calibration

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curves, with penG and DHS standard solutions to give matrix final concentrations of 1,

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10, 20, 50, 100, 200, 500, 1000 µg kg-1 for DHS and 0.1, 1, 2, 4, 10, 50, 100, 500 µg

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kg-1 for penG.

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Validation procedures

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The validation of LC-MS/MS method was carried out following the requirements of the

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Commission Decision 2002/657/EC.. 25 All calibration curves were obtained comparing

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the standard and the known concentration of each chemical plotting the areas of the

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generated curves; all the analysis were performed in triplicate. Linearity, selectivity,

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within-laboratory repeatability (WLr), within-laboratory reproducibility (WLR) and

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recovery were determined. Validation precision (accuracy and relative standard

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deviation “RSD”) was performed using buffalo bulk milk (resulted compliant for dairy

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industry during official controls)

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level of 4 µg kg-1 and 200 µg kg-1 for penG and DHS respectively.

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and fortifying samples at 0.5, 1 and 2 times MRL

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RESULTS AND DISCUSSION

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Extraction and clean up

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ACN had been selected as an extraction solvent for its capacity to precipitate protein. 27

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The centrifugations at low temperature (4 °C and 2 °C) had been used to avoid

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compounds degradation because, at refrigerator conditions, these antibiotics have a

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good stability for a short-term storage of a maximum of 3 days. 28

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Optimization of chromatographic separation

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Chromatographic separation was obtained using an HPLC technique already confirmed

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for β-lactams

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extraction procedure and change the column. The gradient composition was the same,

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while methanol was replaced by ACN as organic solvent in mobile phase to improve

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resolution, reproducibility and peak shape. The chromatographic column initially used

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(Kinetex 2.6 µm F5 100 Å, LC Column 100 x 2.1 mm) was also modified since that

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column has given insufficient results for DHS added to the method scope. DHS showed

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unsatisfactory responses (not retained) while the retention time for penG was 6.12 min.

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Thus, other columns were evaluated and Luna Omega column (1.6 µm Polar C18 100

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A, 100 x 2.1 mm) was chosen, due to the absence of stabilization time between

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subsequent injections and the runs were shorter (

1 Investigation of the persistence of penicillin G ... - ACS Publications

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