1. INTRODUCTION:

Consuming milk is seen as one of the key components of a balanced and healthful diet. It is the initial nourishment mammals consume that provides necessary energy and nutrients for their growth and development. Milk's nutritional importance cannot be overlooked, as it provides essential vitamins, important minerals, and valuable proteins that are crucial for immunity^1-4.

In animal husbandry, antibiotics are used for treatment and preventive purposes or as feed additives. Numerous investigations have demonstrated that significant quantities (30–70%) of antibiotics pass into the environment unchanged, i.e., with potential antibacterial action.

Because antibiotics were used carelessly, most of their residues can be found in milk. Impacts of antibiotic residues on the public's health and the dairy industry could include antibiotic resistance, allergic reactions, carcinogenicity, mutagenicity, teratogenicity, disturbances in the normal intestinal environment, and these residues have the potential to interfere with the starter cultures during the fermentation process, which is necessary for the manufacture of cheese and yogurt^5-8.

Residues are defined as "pharmacologically active substances and their metabolites which remain in foodstuffs obtained from animals to which the drugs in question have been administered". Many global regulatory organizations, including the Food and Drug Administration (FDA), the European Food Safety Authority (EFSA), and Codex Alimentarius, have set tolerance limits for antibiotic residues (also known as the Maximum Residual Limit, or MRL) in milk and other foods for consumer protection^9-11.

The time period that must pass between the final dose of antibiotics administered to food animals and when they are consumed, or when food from them is consumed, is referred to as the withdrawal period¹².

Enrofloxacin is a third-generation fluoroquinolone and potent antibacterial that has demonstrated effectiveness in treating numerous bacterial illnesses. It inhibits the production of DNA and RNA in bacteria by inhibiting DNA gyrase. It has a wide range of activity as a bactericidal against Gram-positive and Gram-negative bacteria^13-16.

Milk from cattle treated at the indicated dose of 5 mg/kg/day intravenously or subcutaneously should be withdrawn for 72 hours (3 days) and 96 hours (4 days), respectively. The regulation specifies a MRL of 100 μg/kg for Enrofloxacin, in milk of any species. The acceptable daily intake (ADI) of Enrofloxacin for humans is 0-2µg/kg^17-20.

Because of the direct expenses and milk production loss caused by mastitis, which can lower milk production by 30% or more and have a detrimental effect on milk quality (high somatic cell count), bovine mastitis is the most common and expensive disease in the dairy industry²¹. The failure of treatment can be partially explained by a low concentration of antibacterial drugs in the breast tissue. A potential solution to the issue of low Enrofloxacin concentrations in mammary tissue is intramammary injection of 300mg for 8 days. This raises the amount of drug in milk. Peak milk concentrations on day 1 were between 18.5 and 19.8µg/mL, and on day 8, they were between 20.2 and 22µg/mL²².

There are many High-Performance Liquid Chromatography (HPLC) methods to determine Enrofloxacin in milk^23-28, but these methods are costly, require a lot of equipment, involve multiple steps, and include many separation and extraction techniques and more than one solvent. The spectrophotometric methods have become widely used due to their widely available instruments, easy processes, speed, accuracy, and precision. In contrast to other advanced analytical techniques like chromatography, spectrophotometric techniques are significantly more cost-effective and provide simpler operations²⁹.Derivative spectrophotometry is an important analytical technique for eliminating baseline shifts and tilts and obtaining qualitative and quantitative data from spectra containing overlapping bands³⁰.

2. MATERIALS AND METHODS:

2.1. Instruments:

The T80+ UV/Vis Spectrophotometer Instrument Ltd (UK) is the ultraviolet spectrophotometric device. It has a computer connection. Quartz cells with a width of one centimeter are used. A Sartorius analytical balance from Germany, model 2474, is employed for the weighing process. Further instruments and devices needed to complete the task include an ultrasonic bath made by Power Sonic, specifically the model 405, from Korea, a vortex shaker (IKA, model Vortex 3, Germany), a piece of equipment known as a centrifuge device, specifically the 90-1 Centrifuge made by the Shanghai Surgical Instruments Factory in China, volumetric flasks, glass tubes with caps, and various sizes of glass pipettes.

2.2. Solvents and chemicals:

Standard active veterinary pharmaceutical ingredient is Enrofloxacin ≥ 98% and of Chloroform analytical grade (Merck, Germany).

2.3. Preparation of standard solutions of Enrofloxacin:

First, 10mg of Enrofloxacin was weighed. Then it was transferred into a 10mL flask and diluted with chloroform to the mark to obtain a standard mother solution with a concentration of 1000µg/mL. Next, 1 mL was transferred to 10mL flask and mixed with chloroform to create a standard stock solution at a concentration of 100µg/mL. In conclusion, six amounts were transferred using a pipette into 10mL flasks and then mixed with chloroform to create a range of standard Enrofloxacin solutions with concentrations of 0.5, 1, 2, 4, 6, and 8µg/mL.

2.4. Preparation of milk samples:

Eight cow milk with different kind (raw, powdered, and ultra-high temperature (UHT) milk) were bring from the local market. Then, 5g of milk was weighed into a glass tube, 5mL of chloroform was put into the same tube. The tube was shaken well manually for extraction for 10 minutes, and then the tube was put into a vortex shaker for 20 minutes. Finally, the tube was moved to a centrifuge and was centrifuged at 5000RPM for 30 minutes. The top layer was eliminated. The lower chloroform layer was transferred to a volumetric flask with a capacity of 5mL, The volume was completed to 5 mL using chloroform.

2.5. Preparation of milk-containing Enrofloxacin samples:

Six milk samples with different kind (raw, powdered, and ultra-high temperature (UHT) milk) were used.

First, 10mg of Enrofloxacin was weighed in a beaker. The weight was completed to 10g using liquid milk, meaning we reached a concentration of 1 mg of Enrofloxacin in each 1g of milk. Then, the beaker was moved to an ultrasonic bath, and it was still in the bath for 30 minutes. Then, the contents of the beaker were moved to a plastic tube, and the plastic tube was put into the vortex for 20 minutes. In the end, a specific amount of the prepared samples was taken and diluted with milk to achieve various concentrations ranging from (0.1-6) µg/g.

The same protocol related to milk extraction using chloroform was applied.

3. RESULTS AND DISCUSSION:

3.1. Optimal wavelength for second-order derivative spectrophotometry:

UV spectrogram scan of pure Enrofloxacin solution; using chloroform as solvent appears in Figure (1). UV spectrogram scan of 6 milk samples; using chloroform as solvent appears in Figure (2).

We may notice a problem that there is a wide range of absorption region with no zero point (zero absorption point) in the milk spectrogram that allows us to determine the drug without interference. In other words, the Enrofloxacin absorption area is overlapped by milk. To solve this problem, we resorted to using the first and second-order derivative method, and we found that with the second-order derivative (amplified 100 times [coefficient × 100]), we obtained a zero-crossing point for all milk samples at a wavelength of 289nm as shown in Figure (3), while the drug had absorption at this wavelength as shown in Figure (4); allowing us to determine it without interference.

3.2. Method validation:

3.2.1. Linearity:

A series of Enrofloxacin solutions were prepared at concentrations ranging from 0.5 to 8µg/mL, and linearity was studied at a wavelength of 289nm; Figure (5) shows the second-order derivative of this series. As shown in Figure (6), it illustrates the relationship between concentration and absorbance, correlation coefficient (R²), in addition to the regression equation. Table (1) displays the results.

The results show approved linearity range and correlation coefficient. The data collection and management followed the guidelines set by The International Council for Harmonization (ICH), except for the robustness and stability tests³¹.

Figure 1: The zero-order UV absorption spectrogram of Enrofloxacin 6µg/mL in chloroform.

Figure 2: The zero-order UV absorption spectrogram with zoom of 7 milk samples in chloroform.

Figure 3: The second-order derivative spectrogram shows 8 milk samples spectrums.

Figure 4: The second-order derivative spectrogram shows Enrofloxacin 6 µg/mL.

Figure 5: The second-order derivative spectrogram of Enrofloxacin series.

Table 1: Linearity data.

Concentration (µg/mL)	Absorbance at 289 nm			Average (µg/mL)	SD	RSD%
Concentration (µg/mL)	Found concentration (µg/mL)			Average (µg/mL)	SD	RSD%
0.500	0.512	0.497	0.512	0.507	0.008367	1.651
1.000	0.946	0.975	0.9 46	0.956	0.016735	1.750
2.000	1.975	1.975	1.975	1.975	0.000000	0.000
4.000	4.048	4.062	4.062	4.057	0.008367	0.206
6.000	5.946	6.193	6.149	6.096	0.131504	2.157
8.000	7.888	7.975	7.888	7.917	0.050204	0.634

3.2.2 Limit of quantification (LOQ) and limit of detection (LOD):

The limit of quantification (LOQ) and limit of detection (LOD) were calculated from the equations below. σ represents the standard deviation of y-intercepts in regression lines while S denotes the slope of the calibration curve. LOQ and LOD were 0.09μg/mL and 0.03μg/mL respectively. The results show low LOD—LOQ.

3.3x σ 10x σ

LOD= -------------------- LOQ= -----------------

S S

Figure 6: The relationship between concentration and absorbance.

3.2.3. Stability:

The stability was studied at refrigerator temperature using chloroform as solvent. According to our research, Enrofloxacin remains stable for a duration of 1 week. The findings from the stability investigation are presented in table (2). This study was done for 3 concentrations of 4, 6, and 8µg/mL each day.

3.2.4. Accuracy:

Accuracy study was achieved at 289nm of second-order derivative. Three sets of concentrations were examined, each with three repetitions. Table (3) shows the recovery, the mean recovery, and the relative standard deviation (RSD%) values for all 9 samples in total of Enrofloxacin.

3.2.5 Precision

The precision study was achieved at 289nm of second-order derivative. In order to assess the intraday repeatability precision, three different concentrations were studied at each of the wavelengths tested, with three replicates for each concentration. 27 samples were assessed for intermediate inter-day precision, with equal numbers evaluated after 24hours and 48hours. Table (4) shows the recovery, the mean recovery, and the relative standard deviation (RSD%) values of the total 27 samples of Enrofloxacin.

3.2.6. Robustness:

A study on robustness was conducted by making three different modifications to instrumental parameters, with each parameter being changed independently in three repeats for a modest alteration. Initially, the scanning speed was adjusted to both fast and slow from medium. Next, the scanning range was adjusted from 200–400nm to 201–399nm. Finally, Next, the interval was adjusted from 1nm to 0.5nm. It has been proven that our method is robust for Enrofloxacin with the scanning speed and the scanning range, but not for the interval setting. Enrofloxacin had recovery ranges of: 98.638– 101.826% for scanning speed parameter, and 98.058– 101.246% for scanning range parameter.

Table 2: Data of the stability study.


Concentrations (µg/mL)	Found concentration (µg/mL)				Average (µg/mL)	SD	RSD%
Concentrations (µg/mL)	1^st Day	2^nd Day	3^rd Day	7^th Day	Average (µg/mL)	SD	RSD%
4.000	4.004	4.048	4.062	4.077	4.048	0.031307926	0.773
6.000	5.946	6.019	6.062	6.106	6.033	0.06797704	1.127
8.000	7.975	8.033	8.062	8.135	8.051	0.066282235	0.823

Table 3: Data of the accuracy study.

Concentrations (µg/mL)	Found concentration (µg/mL)			Mean concentration (µg/mL)	Recovery %			Mean Recovery %	SD	RSD %
Concentrations (µg/mL)	1	2	3	Mean concentration (µg/mL)	1	2	3	Mean Recovery %	SD	RSD %
3	3.033	3.033	3.048	3.038	101.111	101.111	101.594	101.272	0.008367395	0.275
5	4.917	5.019	5.091	5.009	98.348	100.377	101.826	100.184	0.087358171	1.453
6	6.004	5.932	6.106	6.014	100.072	98.865	101.763	100.233	0.087358171	1.744

Table 4: Data of the precision study.

	Concentration (µg/mL)	Found concentration (µg/mL)			Mean concentration (µg/mL)	Recovery %			Mean recovery %	SD	RSD%
	Concentration (µg/mL)	1	2	3	Mean concentration (µg/mL)	1	2	3	Mean recovery %	SD	RSD%
Intraday	4	4.019	4.004	4.062	4.029	100.471	100.109	101.558	100.713	0.030169072	0.749
	6	6.106	5.932	6.091	6.043	101.763	98.865	101.522	100.717	0.096497509	1.597
	8	7.888	7.932	7.975	7.932	98.605	99.149	99.692	99.149	0.043478261	0.548
Inter-day 24 h	4	3.975	4.077	4.048	4.033	99.384	101.920	101.196	100.833	0.052254366	1.296
	6	6.062	5.946	5.932	5.980	101.039	99.106	98.865	99.670	0.071491056	1.195
	8	7.903	7.932	7.932	7.922	98.786	99.149	99.149	99.028	0.016734790	0.211
Inter-day 48 h	4	3.932	3.961	3.946	3.946	98.297	99.022	98.659	98.659	0.014492754	0.367
	6	6.004	5.903	6.033	5.980	100.072	98.382	100.556	99.670	0.068490082	1.145
	8	8.048	7.961	8.033	8.014	100.598	99.511	100.417	100.175	0.046587685	0.581

4. Application of the proposed method for milk:

As mentioned earlier in the "Preparation of milk-containing Enrofloxacin samples" section, the drug was added to drug-free milk, which gave us zero absorption at the wavelength of 289nm. Table (5) shows the amounts of drug added to the milk samples and the recovery. The recovery values are within the acceptable range according to Veterinary International Conference on Harmonization (VICH) guidance, which states that the acceptable range for analyte concentrations of a veterinary drug should range from -20 to +10 percent³². Figure (7) shows the spectra of the second-order derivative of a some of milk samples before and after the addition of the drug.

Table 5: Data of the analysis of Enrofloxacin in milk with the proposed method.

Milk type	Added concentration (µg/mL)	Found concentration (µg/mL)	Recovery %
Powdered	0.5	0.468	93.62
	1	0.946	94.64
	2	1.857	92.85
	6	5.653	94.22
Range	(0.5-6)	(0.468-5.653)	(92.85-94.64)
UHT	0.1	0.091	91.30
	1	0.917	91.74
	2	1.830	91.52
	6	5.526	92.10
Range	(0.1-6)	(0.091-5.526)	(91.30-92.10)
Raw	0.5	0.454	90.73
	1	0.903	90.29
	2	1.816	90.81
	6	5.494	91.57
Range	(0.5-6)	(0.454-5.494)	(90.29-91.57)

Figure 7: The spectra of the second-order derivative of a some of milk samples.

5. CONCLUSION:

This first unique method can be considered a fast, simple, and cost-effective way (as it does not require complex and numerous separation and extraction techniques), The method was characterized by being precision, accuracy, stability, and robustness, ultimately ensuring the preservation of public health by monitoring the levels of this veterinary drug in milk before its marketing and use by humans. Food laboratories and health regulatory authorities can use this method as an initial and immediate way to monitor residues.

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Received on 04.08.2024 Revised on 28.10.2024

Accepted on 07.12.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):699-705.

DOI: 10.52711/0974-360X.2025.00103

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