Investigation of the Persistence of Penicillin G and ... - ACS Publications

Subscriber access provided by Miami University Libraries

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

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])

1 ACS Paragon Plus Environment


1

ABSTRACT

2 3

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

4

dihydrostreptomycin in milk of lactating buffaloes following intramuscular injection of

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

14

adequate for depletion of penicillin G in lactating buffaloes.

15 16

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

17

MS/MS.

18 19 20 21 22 23 24 25 26 2 ACS Paragon Plus Environment

Page 2 of 24

Page 3 of 24


27

INTRODUCTION

28

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

29

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

30

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

31

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

32

remarkable zootechnical importance for the production of “mozzarella di bufala

33

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

34

legislation. 1

35

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

36

mastitis and post-partum diseases.

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

metritis and mastitis.

45

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

46

classes of antimicrobials.

47

been described,

48

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

49

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

50

dairy production, the Committee for Veterinary Medicinal Products recommends the

51

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

52

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

5, 6

4

2

Existing legislation on veterinary medicines

3

However, allergic reactions in sensitive population have

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



8

Page 4 of 24

53

bacterial protein synthesis.

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

However, confirmatory methods are based on high performance liquid chromatographic

63

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

64

by LC using diode array,

65

using different

66

methods for sample preparation.

67

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

68

different approaches,

69

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

70

confirmatory method.

71

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

72

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

73

20, 21

74

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

75

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

76

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

77

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

78

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

11, 12

17, 18

19

It is available in many formulations for the treatment of

fluorescence, 13, 14 and mass spectrometry detectors,

10

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,


22

.These

23

but

Page 5 of 24


79

the simultaneous determination of dihydrostreptomycin and penicillin G residues in

80

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

81

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

82

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

83

high extraction capacity, good reproducibility, and environmental friendly.

24

for the

84 85

MATERIALS AND METHODS

86

Animal studies

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

the trial.

97 98

Materials and reagents

99

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

100

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

101

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

102

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

103

(Darmstadt, Germany).

104 5 ACS Paragon Plus Environment


105

Sample preparation

106

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

107

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

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

QTrap-MS system.

116 117

LC-MS and LC-MS/MS analyses

118

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

119

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

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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


Page 6 of 24

Page 7 of 24


129

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

130

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

131

USA).

132 133

Calibration

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

kg-1 for penG.

144 145

Validation procedures

146

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

147

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

148

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

149

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

150

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

151

recovery were determined. Validation precision (accuracy and relative standard

152

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

153

industry during official controls)

154

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

26

and fortifying samples at 0.5, 1 and 2 times MRL

155 7 ACS Paragon Plus Environment


156

RESULTS AND DISCUSSION

157

Extraction and clean up

158

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

159

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

160

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

161

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

162 163

Optimization of chromatographic separation

164

Chromatographic separation was obtained using an HPLC technique already confirmed

165

for β-lactams

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

subsequent injections and the runs were shorter (

Investigation of the Persistence of Penicillin G and ... - ACS Publications

Recommend Documents