Application of High-Performance Thin-Layer Chromatography and Bioautography Techniques for Determination of Antibiotic Residues in Mastitic Cow Milk Following Treatment

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International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36
Vol 9 (02) Feb ’19
Hosted@www.ijlr.org DOI 10.5455/ijlr.20180907015346
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Original Research
Application of High-Performance Thin-Layer Chromatography and
Bioautography Techniques for Determination of Antibiotic Residues in
Mastitic Cow Milk Following Treatment
Priyanka Das, Kautuk Kumar Sardar*, Gyanaranjan Sahoo and Subas Chandra Parija
Department of Pharmacology and Toxicology, College of Veterinary Science and A.H., Orissa
University of Agriculture and Technology, Bhubaneswar - 751 003, Odisha, INDIA
*Corresponding author: kksardar@gmail.com
Rec. Date:
Sep 07, 2018 13:53
Accept Date:
Nov 20, 2018 00:54
DOI
10.5455/ijlr.20180907015346
Abstract
A method to identify and quantify multiple antibiotic residues like ceftriaxone and enrofloxacin in mastitic
cow’s milk by high-performance thin-layer chromatography coupled with bioautography was studied. The
antibiotic residues were extracted from antibiotic treated cow’s milk suffering from mastitis with the help
of acetonitrile by eliminating fat using petroleum ether and finally isolated with dichloromethane. The
chromatogram of peaks of antibiotic residue confirmed the presence of antibiotics in the milk samples with
presence of 0.108±0.011mg/ml (ceftriaxone) and 0.173±0.015mg/ml (enrofloxacin) in the milk sample,
collected 10 days post-administration of drug with initial dose of 15g of ceftriaxone and 9g of enrofloxacin
respectively. In the present study, the presence of antibiotic residue beyond 10 days is contradictory from
the withdrawal period given by the pharmaceutical company for 5 days. Bioautography confirmed the
presence of active antibiotic residue in milk sample-1, 2, 3, 4, 5 and 6 with zone of inhibition of
6.5±0.407mm, 6.0±0.495mm, 5.0±0.666mm, 5.2±0.4mm, 5.1±0.432mm and 6.0±0.401mm, respectively.
The test microorganism used for bioautography was Streptococcus spp.
Key words: Antibiotic Residue, Bioautography, Ceftriaxone, Enrofloxacin, HPTLC, Milk
How to cite: Das, P., Sardar, K. K., Sahoo, G., & Parija, S. C. (2019). Application of High-Performance
Thin-Layer Chromatography and Bioautography Techniques for Determination of Antibiotic Residues in
Mastitic Cow Milk Following Treatment. International Journal of Livestock Research, 9(2), 289-301. doi:
10.5455/ijlr.20180907015346
Introduction
Milk is one of the most nutritionally complete foods available and its consumption has been promoted
worldwide. Milk containing proteins, saturated fat, calcium, vitamins, etc. shares a significant proportion
of the daily essential nutrients required for human growth and development of all age groups (Connie,
2010). Milk recommended for children and elderly women should be free from antibiotic residues

International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36
Vol 9 (02) Feb ’19
Hosted@www.ijlr.org DOI 10.5455/ijlr.20180907015346
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(Goulding et al., 2004). Mastitis is the most prevalent disease in dairy cows making milk unfit for human
consumption vis a vis serves a source of spread of diseases such as streptococcal intoxication, streptococcal
sore throat, colibacillosis, tuberculosis and brucellosis. Mastitis inflicts heavy economic loss to the dairy
farmers in India mainly due to loss in milk production and quality (Radostitis et al., 2007; Das et al., 2016).
Owing to involvement of heavy financial implication and the inescapable existence of latent infection,
mastitis is obviously a principal factor that limits dairy production and warrants immediate antibiotic
therapy. Antibiotics are employed widely in veterinary practice for both prophylaxis and treatment of
mastitis in dairy cows and are added directly to milk to prolong their freshness.
Ceftriaxone is used widely in the treatment of mastitis in cows as well as both human and veterinary diseases
because of their broad spectrum of antibacterial activity and good pharmacokinetic properties (Sar et al.,
2014). Enrofloxacin having bactericidal nature dominates the veterinary practice because of its high
therapeutic value (Lopez et al., 2015). Ceftriaxone and enrofloxacin, two widely used veterinary antibiotics,
may be present as residues in milk following treatment and hence there is an urgent need for their detection
and determination. The presence of drug residues in milk will have public health implications, and hence
knowledge about the penetration of ceftriaxone from blood to milk is worth consideration (Goudah et al.,
2006).
In many countries, Government authorities have established monitoring programs for antibiotic
determination in food (Ministry of Agriculture, HMSO, London, 1992). The monitoring of the veterinary
drug residues is an important component of the food safety control in various raw materials and foods of
animal origin, and maximum residue limits (MRL) of veterinary drugs recruited with the food animals have
been set to ensure the safety of foods of animal origin for consumers (Commission Regulation 37/2010;
Elizabeta et al., 2011; Navratilova et al., 2011; Langiano et al., 2012). Several techniques like thin-layer
chromatography (TLC), gas–liquid chromatography (GLC), radioimmunoassay, electrophoresis, high-
performance liquid chromatography (HPLC), high performance thin layer liquid chromatography (HPTLC)
as well as microbiological and immunological assays are heavily relied to determine antibiotic residues in
milk. The coupling of TLC with microbiological detection (bioautography), a simple, cheap and quite
sensitive method, has been used for the identification and quantification of several antibiotics (Petz et al.,
1987; Choma and Grzelak, 2011).
To the best of our knowledge, no currently available studies have identified the presence of ceftriaxone and
enrofloxacin residue in milk collected from mastitic cows following treatment at dairy farmers’ door step.
Therefore, the present study was undertaken to determine the antibiotic residues in mastitic cow milk
following treatment.

International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36
Vol 9 (02) Feb ’19
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Materials and Methods
Standard Antibiotic Solutions and Solvent System
Stock solutions: Ceftriaxone (M/s Intas Pharmaceuticals Ltd., Gujarat, India) and enrofloxacin (M/s Intervet
India Pvt. Ltd., Pune, India) were dissolved individually in methanol at a concentration of 10 mg/ml and
stored at 4 °C. The standards were prepared from the antibiotics used in treatment of mastitis in cows in
field conditions. Each stock solution was diluted with 1ml of methanol to make a final concentration of
1mg/ml. The developing solvent used for HPTLC was a mixture of dichloromethane:acetone:
methanol:glycerin (64:20:15:1 v/v) (Ramirez et al., 2003).
Selection of Animal and Collection of Milk Sample
The study was undertaken in crossbred Jersey dairy cows (n=67) suffering from mastitis which were treated
with ceftriaxone and enrofloxacin by the field veterinarians. Of them, 30 cows treated with ceftriaxone and
enrofloxacin maintained by the livestock farmers were selected in the present study, and were divided into
six groups. Milk samples (20 ml) were collected from each antibiotic treated mastitic cow that got cured
completely after treatment. Group-1 were treated with ceftriaxone @ 3g/day for 5 days, and milk samples
were collected after 7days post-treatment. Group-2 cows were administered with enrofloxacin @ 1.5 g /day
for 6 days and milk samples were collected 10 days after treatment. Similarly, the Group-3 cows were
treated with ceftriaxone @ 2g/day for 5days and milk samples were collected after 10 days of treatment.
The Group-4 cows were injected with enrofloxacin @ 1.5 g/day for 5 days and milk samples were collected
after 10 days of treatment. Likewise, Group-5 and Group-6 cows were treated with ceftriaxone @ 2g/day
for 6 days and 3g/day for 4 days, respectively, and milk samples were collected after 10 days post-treatment.
Extraction of Sample
The whole procedure was conducted in the Central Instrumentation facility, Orissa University of
Agriculture and Technology, Bhubaneswar. The Malisch multiresidue method for the determination of
residues of chemotherapeutics was used to extract the analytes (Malisch, 1986). Milk sample (20 ml) from
mastitic cows were extracted by thoroughly mixing in a commercial blender at a high speed with acetonitrile
(40ml) for 1 min. The liquid phase was decanted into 250 ml separating funnel. Petroleum ether (20ml) was
added and shaken for 1 min, after which the upper phase was discarded. Then sodium chloride (2g) was
poured carefully into the separating funnel and shaken gently to dissolve as much as possible. After that
30ml dichloromethane was added to it and shaken again for 1 min, then the lower phase was drained into a
round bottom flask and evaporated to dryness at 40 oC in a rotary vacuum evaporator. Then the residue was
reconstituted with 1ml methanol and put in the eppendorf tube to be used for spotting on HPTLC plate.

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Method for HPTLC
Chromatography was done on a silica gel HPTLC plate (10 × 10 cm, silica gel 60F254, 200 µm thickness;
M/s E Merck, Germany). Each sample group (5µl) was applied 10 mm above the base of the plate using
application device ‘LINOMAT- 5’, ( M/s CAMAG, Switzerland with all the accessories) fitted with a
syringe of 100 µl capacity using nitrogen gas at 6 psi followed by air drying of the HPTLC plate at room
temperature. The plates were then developed in a twin trough chamber using the mobile phase
dichloromethane:acetone:methanol:glycerin (64:20:15:1 v/v). The plates were developed in a pre-
equilibrated closed chamber till the solvent reached to 90% height of the plates. The plates were removed
and the solvent fronts were marked and dried on a plate dryer. The developed plates were visualized at 254
nm and 366 nm under a visualizer and photographed by the documentation system. Then the plates were
scanned under CAMAG TLC SCANNER-3 at 254 nm and 366 nm. The chromatographic conditions were
optimized previously to achieve the best resolution and peak shape. The concentration in the unknown
sample peak corresponding to the standard peak was calculated by comparing the peak area (AU) of the
peak to that of the peak of the standard and the respective concentration of the standard solution, by using
the formula: AU (sample peak) / AU standard peak x concentration of standard.
Bioautography
Milk samples (n=30) collected from mastitic cows in and around Bhubaneswar including other areas of
Odisha State were cultured on Edward medium agar for isolation of the organism. The Gram positive
bacteria were isolated and identified up to genus level as streptococci based on the morphology, cultural
and biochemical reactions employing CAMP test (Bhagat et al., 2015). In bioautography, Molten Mueller–
Hinton agar was taken in a petridish and spread all over the petridish and left for drying. The broth
containing culture of Streptococcus agalactiae was applied on the surface of the petridish. The HPTLC
plates were cut into uniform pieces containing antibiotic residue and put on the petridish in inverted manner,
and the petridish were then incubated for 48 h. Inhibition zone diameters were measured with Vernier
calipers and compared with antibiotic standards inhibition zones (Ramirez et al., 2003).
Statistical Analysis
All the experimental results were expressed as mean ± standard error (SE) in replicates (n=5).
Result and Discussion
Detection of Antibiotic Residue
The antibiotic residue in milk sample collected from six groups were shown in Table 1. The standard-1
showed a single peak that started at 0.87 Rf and ended at 0.94 Rf having area of 7092.8 AU (arbitrary units)

International Journal of Livestock Research eISSN : 2277-1964 NAAS Score -5.36
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containing ceftriaxone. The standard -2 gave six peaks of which one peak was from 0.43 Rf to 0.77 Rf of
area 108812.6 AU showing presence of enrofloxacin. It also showed another peak which corresponded to
peak of standard -1 (ceftriaxone) with start position 0.82 Rf and end position 0.99 Rf having area of 19152.1
AU.
Table 1: Rf value, peak position and peak area of scanned HPTLC plate
Track
Peak
Max. Position
Max. Height
Max. %
Area
Area %
Assigned Substance
1
1
0.03Rf
11.7AU
7.03%
115.2AU
1.60%
Unknown
1
2
0.94Rf
1545 AU
92.97%
7098.8 AU
98.40%
Ceftriaxone
2
1
0.02 Rf
42.7 AU
3.50%
290.4 AU
0.22%
Unknown
2
2
0.05 Rf
62.3 AU
5.12%
1242.4 AU
0.93%
Unknown
2
3
0.10 Rf
19.5 AU
1.60%
329.0 AU
0.25%
Unknown
2
4
0.29 Rf
54.6 AU
4.48%
3275.7 AU
2.46%
Unknown
2
5
0.68 Rf
708.1 AU
58.11%
108812.6 AU
81.75%
Enrofloxacin
2
6
0.93 Rf
331.4 AU
27.19%
19152.1 AU
14.39%
Unknown
3
1
0.01 Rf
63.3 AU
13.72%
424.8 AU
2.25%
Unknown
3
2
0.14 Rf
20.6 AU
4.47%
626.5 AU
3.32%
Unknown
3
3
0.73 Rf
300.5 AU
65.20%
14152.1 AU
75.00%
Unknown
3
4
0.94 Rf
76.5 AU
16.61%
3666.6 AU
19.43%
Unknown
4
1
0.02 Rf
11.8 AU
7.50%
69.5 AU
1.13%
Unknown
4
2
0.14 Rf
18.8 AU
11.95%
708.1 AU
11.51%
Unknown
4
3
0.61 Rf
21.6 AU
13.72%
491.6 AU
7.99%
Unknown
4
4
0.69 Rf
53.5 AU
33.97%
2368.0 AU
38.49%
Unknown
4
5
0.95 Rf
51.7 AU
32.86%
2515.8 AU
40.89%
Unknown
5
1
0.02 Rf
12.9 AU
11.87%
117.1 AU
2.90%
Unknown
5
2
0.14 Rf
20.9 AU
19.33%
535.9 AU
13.27%
Unknown
5
3
0.64 Rf
19.4 AU
17.93%
711.1 AU
17.61%
Unknown
5
4
0.95 Rf
55.1 AU
50.86%
2674.2 AU
66.22%
Unknown
6
1
0.01 Rf
120.4 AU
62.73%
901.7 AU
22.41%
Unknown
6
2
0.14 Rf
13.2 AU
6.89%
275.8 AU
6.85%
Unknown
6
3
0.94 Rf
58.3 AU
30.38%
2847.0 AU
70.74%
Unknown
7
1
0.01 Rf
82.3 AU
48.45%
658.3 AU
14.78%
Unknown
7
2
0.14 Rf
15.4 AU
9.04%
363.9 AU
8.17%
Unknown
7
3
0.94 Rf
72.3 AU
42.52%
3432.3 AU
77.05%
Unknown
8
1
0.14 Rf
10.4 AU
4.98%
99.8 AU
1.26%
Unknown
8
2
0.65 Rf
33.7 AU
16.22%
1231.7 AU
15.50%
Unknown
8
3
0.66 Rf
36.1 AU
17.38%
1116.1 AU
14.05%
Unknown
8
4
0.87 Rf
30.7 AU
14.79%
1079.0 AU
13.58%
Unknown
8
5
0.94 Rf
96.9 AU
46.63%
4419.8 AU
55.62%
Unknown
Milk sample (Group -1) showed the area 14152.1 AU starting from 0.61 Rf to 0.77 Rf containing
ceftriaxone. Milk sample (Group-2) demonstrated peak starting from 0.90 Rf to 1.00 Rf with area 2515.8
AU containing enrofloxacin. It also gave another peak corresponding to ceftriaxone starting from 0.64 Rf
to 0.72 Rf with an area of 2368.0 AU. Similarly, milk sample (Group-3) and (Group-6) showed peak
corresponding to both ceftriaxone and enrofloxacin. Milk sample (Group-4) contained only enrofloxacin
residue with a peak area of 2847.0 AU that started from 0.89 Rf to 0.99 Rf.

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Fig. 1: Chromatogram (254 nm) showing antibiotic residue in the milk of mastitic cows having eight
peaks i) Ceftriaxone, ii) Enrofloxacin, iii) Group -1, iv) Group -2, v) Group -3, vi) Group -4, vii) Group -5
and viii) Group -6
The chromatogram peaks of antibiotic residues at 254 nm were shown in Fig.1 and the photographs of the
scanned HPTLC plate at 366 nm and 254 nm (Fig. 2a and 2b), confirming the presence of antibiotics in the
milk samples. The standard-1 contained the ceftriaxone (1mg/ml) while standard-2 contained both
enrofloxacin (1mg/ml) and ceftriaxone (0.34 mg/ml) (Table 2).
Fig. 2: (a) Photograph of HPTLC plate at 366 nm
Fig. 2(b): Photographed at 254 nm showing eight
different lanes i) Ceftriaxone, ii) Enrofloxacin, iii)
Group -1, iv) Group -2, v) Group -3, vi) Group -4,
vii) Group -5 and viii) Group -6

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Table 2: Determination of antibiotic residue in milk sample (n=30) using HPTLC
Sample
Total dose of
Administration
Name of the
Antibiotic Given
Total
Antibiotic
Given
Amount of
Ceftriaxone (mg/ml)
Amount of
Enrofloxacin
(mg/ml)
Group
No.(n=30)
(dose x days)
S1
1mg/ml
_
S2
0.34mg/ml
1mg/ml
1
3gx5
Ceftriaxone
15g
0.418±0.023mg/1ml
_
2
15mlx6
Enrofloxacin
9g
0.108±0.011mg/1ml
0.173±0.015mg/ml
3
2gx5
Ceftriaxone
10g
0.220±0.009mg/1ml
0.039±0.012mg/ml
4
15mlx5
Enrofloxacin
7.5g
0.156±0.015mg/ml
5
2gx6
Ceftriaxone
12g
0.282±0.012 mg/1ml
_
6
3gx4
Ceftriaxone
12g
0.370±0.016mg/1ml
0.061±0.018mg/ml
As we took enrofloxacin injected to the animals as standards, the chromatogram showed two peaks
containing both enrofloxacin of 1mg/ml and trace amount of ceftrixaone of 0.34 mg/ml. Milk sample
(Group-1) contained only ceftriaxone (0.418± 0.023 mg/ml) with total administered dose (15g) of injection
of ceftriaxone intramuscularly and the milk sample was collected after 7days of treatment. There was
presence of 0.108±0.011 mg/ml (ceftriaxone) and 0.173±0.015mg/ml (enrofloxacin) with total dose (9g) of
enrofloxacin (Group-2), and the milk sample was collected after 10 days of post-administration. Similarly,
the cows treated with ceftriaxone (10g) in Group-3 displayed ceftriaxone (0.220±0.009 mg/ml) and
enrofloxacin (0.039±0.012 mg/ml) after 10 days of drug administration. The Group-4 cows administered
with enrofloxacin (7.5g) and the Group-5 cows injected with ceftriaxone (12g) contained enrofloxacin and
ceftriaxone with concentration of 0.156±0.015 mg/ml and 0.282±0.012 mg/ml, respectively, 10 days post-
treatment (Table 2). The Group-6 cows treated with ceftriaxone (12g) recorded 0.370±0.016 mg/ml
(ceftriaxone) and 0.061±0.018 mg/ml (enrofloxacin) after 10 days of post-administration (Table 2).
The experiment showed that milk sample (Group-1) and sample (Group -5) contained ceftriaxone residue
of 0.418±0.023 mg/ml and 0.282±0.012 mg/ml, collected after 7 days and 10 days of post-treatment,
respectively. But in case of standard -2, both ceftriaxone and enrofloxacin were detected. The milk sample
of Group - 2, 3 and 6 contained both the antibiotic residues and the milk sample of Group-4 had only
enrofloxacin. In our study, enrofloxacin was estimated to be 0.173±0.015 mg/ml, 0.039±0.012mg/ml,
0.156±0.015 mg/ml and 0.061±0.018mg/ml in milk sample of Group-2, -3, -4 and -6, respectively, after 10
days of treatment which showed peak value of standards and samples (Fig. 1). Presence of veterinary
antibiotic residue on chloramphenicol, sulfonamides, tetracyclines, gentamicin, streptomycin,
dihydrostreptomycin, flumequine and enrofloxacin in milk was studied by Bilandzic et al. (2011) who
detected 4.11 μg/L of enrofloxacin in three months duration contrast to the present study. However,

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consistent to our finding, European Commission regulation (EU) No. 37/2010 has also reported that the
MRL value of ceftriaxone (Cephalosporin) and enrofloxacin (Fluroquinolone) in milk is 100 μg/kg each
and recommended use of thin-layer chromatography-direct bioautography for the screening of
ciprofloxacin and enrofloxacin at the maximum residue level stipulated for milk (Commission Regulation
(EU) No. 37/2010; Navratilova et al., 2011).
In the present study, in Group -1, the ceftriaxone residual amount was found to be 0.418±0.023 mg/ml after
7days of treatment, whereas, Johal and Srivastava (1998) reported ceftriaxone concentration in plasma
measured at 8h in crossbred calves to be 0.32 ± 0.03 mg/ml with initial dose of 10mg/kg.. Following single
intramuscular dosing, Sar et al. (2013) studied that ceftriaxone exhibited absorption-reabsorption pattern in
plasma. Kumar et al. (2010) detected MIC90 of 0.5 μg.ml−1 for ceftriaxone in milk 36 h post-administration.
Ceftizoxime was also available from 72-360 h post-dosing in milk in presence of fibrosin following
intramammary administration of ceftriaxone suggesting that the polyherbal drug played a key role in the
penetration of ceftriaxone from milk to systemic circulation (Sar et al., 2011, 2014). Similar study was done
by Baynes et al. (2016) confirmed that the U.S. milk withdrawal time for cattle treated with tetracycline
was 96 h. Study done by Buket et al. (2013), revealed that the mean levels of quinolones were found to be
30.81 ± 0.45 µg/kg and 6.64 ± 1.11 µg/kg in chicken and beef samples, respectively, by ELISA method
where the samples were collected randomly from local market within one month. According to study of Do
et al. (2016), the sulfamethazine residues were figured out at levels ranging from 11-1600 mg/kg in pork
meat by LC-MS/MS method where the samples were collected from local market at a random basis. In the
study of Kawalek et al. (2016), the highest concentrations of florfenicol was 1.6 ± 2.2 μg /ml of milk at 22
h of administration of drug with initial dose of 2.5 mg /kg body weight following intramammary infusion
(Shanoy et al., 2016). Liquid chromatography tandem-mass spectrometry demonstrated that florfenicol
residues concentration ranged from 0.4 - 0.6μg/g in liver of white-tailed deer after 10days of drug
administration with initial dose of 20 mg/kg body weight, intramuscularly (Anderson et al., 2016). For
qualitative and quantitative determination of florfenicol in white-tailed deer tissues, the study was done by
utilising solid phase extraction and liquid chromatographic separation followed by mass-spectroscopy
product and parent ion pattern.
Detection of sulfathiazole in milk was 30 ng/ml using magnetic solid phase extraction-HPLC-UV method
(Osboo et al., 2015). Chowdhury et al. (2015) reported that the average concentrations of amoxicillin
residue in local milk and local egg were 9.84 µg/ml and 10.46 µg/g, respectively, where the samples were
collected randomly from local household farms at Raozan Upazila, Bangladesh, whereas, amoxicillin
residue was found to be 56.16 µg/ml and 48.82 µg/g for commercial milk and commercial egg, respectively,
collected from commercial farms in the region of Chittagong Metropolitan area in Bangladesh. The study
established that milk collected from commercial dairy farm and eggs from commercial layer farms had

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higher concentration of antibiotic residues than milk and egg from local farms which might be attributed to
indiscriminate use and improper monitoring system for antibiotic withdrawal period of specific antibiotics
in the commercial dairy and layer farms (Chowdhury et al., 2015). Drug residues in milk have a potential
hazard for the consumer and may cause several adverse reactions, interfere the intestinal flora and develop
resistant populations of bacteria, thereby rendering antibiotic treatment ineffective (Dewdney et al., 1991).
An efficient control of the residues in milk is very important to ensure the safety of milk and milk products
(Navratilova et al., 2011).
In the present study, the amount of antibiotic residue present in milk sample 10 days post administration
revealed that enrofloxacin was better than ceftriaxone from public health point of view as lower residue
was present in milk compared to ceftriaxone and its half-life is also less than ceftriaxone.
Antibiotic Sensitivity Test for Antibiotic Residue in HPTLC Plate (Bioautography)
Antibiotic sensitivity test of the HPTLC plate containing antibiotic residue showed the zone of inhibition
(Table 3 and Fig. 3).
Table 3: Antibiotic sensitivity test for antibiotic residue in HPTLC plate (Bioautography)
Sample Group No. (n=30)
Zone of Inhibition (in mm)
Assigned Substance
S1
8
Ceftriaxone
S2
7.1
Enrofloxacin
1
6.5±0.407
Sample gr-1
2
6.0±0.495
Sample gr-2
3
5.0±0.666
Sample gr-3
4
5.2±0.405
Sample gr-4
5
5.1±0.432
Sample gr-5
6
6.0±0.401
Sample gr-6
Fig. 3: Antibiogram showing presence of active antibiotic residue in the HPTLC plate

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Ceftriaxone and enrofloxacin demonstrated the zone of inhibition of 8.0 mm and 7.1 mm, respectively. The
detected antibiotic residue present in milk sample (Group - 1) elicited the zone of inhibition of 6.5±0.407
mm. Similarly, other milk sample (Group -2 to Group - 6) showed the zone of inhibition of 6.0±0.495 mm,
5.0±0.666 mm, 5.2±0.4 mm, 5.1±0.432 mm and 6.0±0.401mm, respectively. Choma (2006) screened
enrofloxacin and ciprofloxacin residues in milk by HPLC followed by TLC with direct bioautography.
Moreover, in TLC-direct bioautography technique, semi-quantitative determination of flumequine and
cefacetril in milk was also determined by TLC-DB which was further compared with quantitative HPLC
analysis (Piech et al., 2016). The zone of inhibition showed by enrofloxacin was less than that of ceftriaxone
showing less residual amount of antibiotic in milk (Table 3).
Ceftriaxone and erofloxacin as standards showed the zone of inhibition of 8.0 mm and 7.1 mm, respectively.
Enrofloxacin showed the zone of inhibition of 5.2±0.4 mm in Group- 4 and ceftriaxone with 6.5±0.407 mm
zone of inhibition in Group-1 whereas Ramirez et al. (2003) recorded zone of inhibition of 8.5 mm, 6.5 mm
and 5.5 mm for ampicillin, chloramphenicol and dicloxacillin, respectively, where the milk was fortified
with each antibiotic working standard solution to a level of 0.1 mg/ ml. The maximum diameter of zone of
inhibition observed from muscle of chicken containing chloramphenicol residue was 3.8 ± 0.4 mm for
Staphylococcus aureus (Tajik et al., 2010), collected arbitrarily from five different poultry meat center in
northwest of Iran. According to study of Patil et al. (2013), the zone of inhibition using 2.0 mg/ml phenyl
tetrazolium chloride against Bacillus cereus and Sclerotium rolfsii were 30mm and 20mm, respectively, in
biautography method. The maximum zone of inhibition of Staphylococcus aureus was recorded by using
dimethyl sulphoxide extracts of green tea variety, qimen was 10.00±0.0 mm by bioautography method
which represents the antibacterial property of qimen (Bashir et al., 2014).
Conclusion
One goal of total quality management is to prevent the occurrence of antibiotic residue in raw milk shipped
from the dairy farmers to consumers. The potential of HPTLC and bioautography as a rapid analytical tool
for the detection and determination of antibiotic residue in milk were demonstrated. The presence of
ceftriaxone and enrofloxacin residue in milk sample collected from mastitic cows was detected through
HPTLC that brought to a close confirmation about the milk withdrawal period to be maintained by the
consumers from public health concern. The finding from this study will serve as a basis in developing a
database related to presence of antibiotic residues in milk for wellbeing and safe human consumption.
Acknowledgments
The authors acknowledge the cooperation extended by the dairy owners in and around Bhubaneswar
including other areas of Odisha State during collection of samples and also to the Hon’ble Vice-Chancellor,

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Orissa University of Agriculture and Technology, Odisha, India for providing necessary facilities to
undertake the study.
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