Green validated chromatographic methods for simultaneous determination of co-formulated oxytetracycline HCl and flunixin meglumine in the presence of their impurities in different matrices

Green analytical chemistry is an important area of chemical sciences. So, developing competent eco-friendly analytical tools is a big challenge. This study devotes two chromatographic techniques for the simultaneous determination of a binary formulated mixture of oxytetracycline HCl (OXY) and flunixin meglumine (FLU), and their impurities tetracycline (TRC) and 2-chloronicotinic acid (CNA), respectively. Primarily, a TLC densitometric method is proposed and validated using TLC plates sprayed with 5% EDTA of pH 9 as a stationary phase and (acetonitrile/ distilled water/ ethanol 7:2:1, by volume) as a developing system. Well-separated spots are detected at 267 nm where linear relations have been achieved at 0.05–2 μg/band and 0.01–2 μg/band for OXY and FLU, respectively. The second developed and validated method is HPLC which is fulfilled on Hypersil BDS column-C18. A mobile phase of distilled water with 0.1%TFA/ acetonitrile in a ratio of 82: 18, v/v firstly then 70: 30, v/v after 9 min is used in a sequential isocratic elution at 210 nm for FLU and 267 nm for OXY, TRC, and CNA. Sensitive and wide linearity ranges are achieved at 0.05–200 μg/mL for both drugs. The two methods are applied successfully in cattle meat and milk for cited drugs determination. According to ICH guidelines, a validation study has been accomplished for the proposed methods. Statistical comparison has been carried out with official and reported methods. Eventually, the greenness of both procedures is evaluated using Eco-Scale which gives eco-friendly results.


Introduction
Green analytical chemistry (GAC) is a part of the whole green chemistry idea which is devoted to development and analysis. The challenge is to meet the analysis and industry needs while reducing risk impacts on humanity and the environment [1]. GAC aims to use routine analytical practices that are less hazardous and more benign to the environment [2]. A model drug used for this study is containing oxytetracycline HCl (OXY) and flunixin meglumine (FLU), marketed as FLOXON injection® indicated for the treatment of infectious diseases where concurrent analgesic, anti-inflammatory therapy is desired [3].
Three methods [31][32][33] were reported for the simultaneous determination of OXY and FLU in their pharmaceutical preparation. But these methods lack the identification of impurities in active pharmaceutical ingredients (API) which is one of the most highly regarded topics. The purity of pharmaceuticals is reflected in their safety and effectiveness, and thus the international guidelines demand highly precise, sensitive, and specific analytical methods to perform impurity profiling studies [34]. Therefore, the study of the impurities in the presence of API now is mandatory.
Detection of the veterinary drug residues in foods of animal origin like meat, egg, and milk has a great concern in food safety as it may cause different levels of toxicity in consumers through their exposure to the residues, transfer of antibiotic resistance, or allergy risk [35]. The European Medicines Agency's (EMA) Committee for Medicinal Products for veterinary use (CVMP) is responsible for recommending the Maximum Residue Limit (MRL) which is the maximum allowed concentration of residue in a food product obtained from an animal that has received a veterinary medicine [36].
In light of the previous introduction, it is obvious that the aim of this work is successfully applying eco-friendly chromatographic methods for simultaneous determination of the two active drugs in the presence of their impurities and application of the proposed methods for detection and determination of drug residues in cattle meat and milk for food safety assessment. Finally, the method's greenness profile will be assessed with the analytical Eco-Scale.
The mobile phase was filtered using a 0.45-µm nylon membrane filter (Chrom Tech, USA) and degassed using J.P-Selecta ultrasonic bath sonicator (Barcelona, Spain).

Pure samples
OXY standard was kindly supplied by Farmachem SA, Mendrisio, Switzerland, and its purity was found to be 99.16 ± 0.896 according to the reported method where it was determined via the spectrophotometric method in 0.1 N HCl at 268 nm [37]. FLU standard was kindly supplied by Norbrook Lab. Ltd., Northern Ireland, UK, and its purity was found to be 99.84 ± 0.891 according to the United States Pharmacopoeia potentiometrically in anhydrous acetic anhydride and titrated using 0.1 M perchloric acid [38]. TRC standard was kindly supplied by Pharmachem S.A., Mendrisio, Switzerland, and its purity is 99.9% as referred by the supplier. CNA standard was kindly supplied by Thermofischer (Kandel), Germany, and its purity is 99% purity as referred by the supplier.

Pharmaceutical formulation
Pharmaceutical formulation FLOXON injectable solution was labeled to contain 108 mg/mL of OXY and 33 mg/mL of FLU, batch number 170362. It was manufactured by Pharma Swede-Egypt Company and was purchased from the local market.

Standard solutions
Stock solutions of 1.00 mg/mL of OXY, FLU, TRC, and CNA were prepared in methanol. Working solutions of 100.00 µg/mL of OXY, FLU, TRC, and CNA were prepared in 0.1 N methanolic HCl for TLC densitometric method while working solutions of 300.00 µg/mL of OXY, FLU, TRC, and CNA were prepared in the first ratio of the mobile phase (distilled water with 0.1%TFA /acetonitrile 82: 18) for HPLC method.

TLC densitometric method
The separation was done on TLC plates precoated with silica gel aluminum plates 60 F254 (20 × 10 cm) with 0.25 mm 1 3 thickness. The bands were spaced 10 mm apart from each other and 10 mm from the bottom edge of the plate with a band length of 6 mm. The plates were previously impregnated with 5% EDTA of pH 9 (adjusted using 2.0 M NaOH) and left to dry horizontally at room temperature, and then they were developed in a chromatographic jar pre-saturated for 30 min with a developing system composed of (acetonitrile/ distilled water/ ethanol 7:2:1, by volume), by ascending chromatography to a distance of 8 cm from the spotting line. The plates were allowed to dry at room temperature, and then the separated bands were visualized under a UV lamp (254 nm) and the plates were scanned under the following conditions: source of radiation was deuterium lamp, scan mode was absorption mode, and scanning speed was 20 mm/s and at wavelength 267 nm.

HPLC method
This separation was performed on Agilent 1260 series HPLC apparatus using Hypersil BDS C18 column (150 × 4.6 mm I.D.), particle size (5 μm). The mobile phase was composed of distilled water with 0.1%TFA/ acetonitrile 82: 18, v/v for 9 min, and then the ratio changed to 70: 30, v/v. It was filtered and degassed before use and mixed in a sequential isocratic elution at a 1 mL/min flow rate. All determinations were performed at ambient temperature (25 ºC). Detection was achieved at wavelength 210 nm for FLU and 267 nm for OXY, TRC, and CNA. The injection volume was 20 µL.

TLC densitometric method
Accurately measured aliquots from OXY and FLU standard working solutions (100.00 µg/mL) were separately spotted on TLC plates using the apparatus autosampler to get bands of the final concentration range (0.05-2.00 µg/band) for OXY and (0.01-2.00 µg/band) for FLU and then were chromatographed as under the previously mentioned chromatographic conditions. Calibration curves relating the integrated peak area versus the corresponding concentration of each OXY and FLU were constructed, and regression equations were computed.

HPLC method
Accurately measured aliquots from OXY and FLU standard working solutions (300.00 µg/mL) were transferred into a series of 10-mL volumetric flasks, and the volume was completed to the mark with the first ratio of the mobile phase. The final concentration ranges (0.05-200 µg/mL) for OXY and FLU were chromatographed as under the previously mentioned HPLC chromatographic conditions. Calibration curves were obtained for each OXY and FLU by plotting the peak area versus the corresponding concentrations, and the regression equations were computed.

Application to Pharmaceutical Formulation (FLOXON®)
A stock solution of Floxon was prepared by accurately transferring 1 mL of dosage form into a 100-mL measuring flask, and then the volume was completed to the mark with methanol.

TLC densitometric method
Further dilution of Floxon stock solution in 0.1 N methanolic HCl was done to obtain the final concentration (54.00 and 16.50 µg/mL) for OXY and FLU, respectively. After that, the drugs were spotted on the plate using the auto-sampler to get bands of 0.648 and 0.198 µg/band of OXY and FLU, respectively. Then the procedure was completed as described for the standard chromatographic condition.

HPLC method
Suitable dilution of Floxon stock solution was done using the first ratio of the mobile phase to prepare a solution with a final concentration equal to 27 and 8.25 µg/mL of OXY and FLU, respectively. The procedure under the chromatographic condition was followed.

Meat sample
Two and a half grams of cattle meat was accurately weighed and spiked with 50 mg of OXY and FLU, and then 25 mL of methanol was added. The prepared sample was homogenized for 5 min followed by sonication for 15 min and then centrifuged at 3000 rpm for 10 min. Extraction was done two times with 10 mL methanol for each. All extractions were collected and filtered with a 0.45 µm syringe filter. The filtrate was transferred into a 50-mL volumetric flask and completed to the mark with the same solvent to have a stock solution of a final concentration of 1 mg/mL of OXY and FLU in methanol.
Further dilution was performed to prepare a solution of 100 µg/mL in 0.1 N methanolic HCl and continue under TLC densitometric method with a concentration range (0.05-2.00 µg/band).
For HPLC measurements, serial dilutions were performed from the working standard solution (300 µg/mL) with a concentration range (0.05-200 µg/mL) in the first ratio of mobile phase solution and then were chromatographed under the same stated HPLC chromatographic method.
Calibration curves were constructed for each of OXY and FLU by plotting the peak areas versus the corresponding concentrations and the regression equations were computed.

Milk sample
A mixture of 5 mL milk and 25 mg of OXY and FLU was mixed well and then add 5 mL of 1 M HCl for protein precipitation. Filtration was done using Whatman filter paper no.44 (12.5 cm diameter). Then the filtrate was transferred into a 25-mL volumetric flask, and the volume was completed with methanol to obtain a concentration of 1 mg/mL of OXY and FLU in methanol. The solution was diluted to prepare 100 µg/mL in 0.1 N methanolic HCl, and the TLC densitometric method was followed over a concentration range (0.05-2 µg/band). For HPLC measurements, further dilution was performed to prepare a working solution (300 µg/mL) using the first ratio of the mobile phase as a diluent and then chromatographed under the same stated HPLC chromatographic method with a concentration range (0.05-200 µg/mL). Calibration curves were constructed, and the regression equations were computed.

Results and discussion
Green analytical chemistry (GAC) focuses on lessening or removal of harmful chemicals, reducing energy consumption, and minimizing generated waste without affecting the performance of a method [1]. Green analytical chemistry is a laboratory attitude that deals with the procedure as an environmental problem [39]. The pharmaceutical industry is a worldwide business aiming to improve healthcare and human life quality through manufacturing high-quality pharmaceuticals that meet both patient safety and biological efficacy but drug impurities threaten this purpose [34].
Chromatography plays an important role in many pharmaceutical industries and also in the chemical and food industry. It is an important technique that enables the separation, identification, and purification of multi-components for qualitative and quantitative analysis [40]. OXY impurity is tetracycline, a commonly used antibiotic due to its low toxicity and the broad spectrum of action. However, as a result of the growing number of tetracycline-resistant isolates of clinically significant bacteria, its clinical utility has dwindled [41]. 2-Chloronicotinic acid is FLU's official impurity, a key intermediate in the production of antibiotics, anti-cardiovascular drugs, insecticides, and herbicides [42].
According to our knowledge, two spectrophotometric and one chromatographic methods were reported for simultaneous determination of OXY and FLU in their pharmaceutical preparation [31][32][33]. The presented chromatographic methods were more straightforward and rapid than spectrophotometric ones because of a direct separation of the binary mixture with fewer mathematical steps. Moreover, the proposed chromatographic methods are more advantageous than the three reported spectrophotometric and chromatographic methods in their higher sensitivity of 0.05 µg/mL rather than the spectrophotometric one which was 5 µg/mL [31], while in the other spectrophotometric methods were 2.5 and 4 µg/ mL for FLU and OXY, respectively [32] and also mentioned to be 32 and 160 µg/mL for FLU and OXY, respectively in the reported chromatographic method [33] that allow the application of the suggested methods for minor traces determination in biological matrices. Furthermore, the proposed methods were more selective than published ones owing to awarding simultaneous separation of both drugs and their impurities in active pharmaceutical ingredients (API). Therefore, our scientific motivation is to develop and validate two new, simple, accurate, precise, and green chromatographic methods for the simultaneous determination of both drugs without any preliminary separation and to be easily applied in quality control laboratories. In addition, the application of these methods in the qualitative and quantitative determination of both drug residues in cattle meat and milk has a great benefit in food safety testing for human health.
Several trials were carried out to obtain good and optimum separation of OXY, FLU, TRC, and CNA without using harmful solvents. The search for alternatives to classical procedures which use chloroform, dichloromethane, dioxan, and other undesirable solvents for analysis has been one of the major challenges that have faced. Guide for solvent selection and to what degree it is considered green is very important as there is no universal green solvent; it is according to many parameters such as environment, health, safety, waste, and others [43]. Finally, the solvents with lower issues for health and environment were selected and applied.

TLC densitometric method
Tetracyclines tend to form complexes with trace metals in the adsorbents used. So the plates were impregnated with a 5% EDTA solution pH 9 (adjusted with 2.0 M NaOH), to overcome lower separation performance due to spots tailing or spots retention on the baseline [44,45]. Different solvents were tried for optimization of the developing system and good separation such as (1-butanol/ acetone/ ammonia 6:4:0.1, by volume), (butanone/ ethyl acetate/ glacial acetic acid 6:4:0.1, by volume) and (methanol/ acetone/ distilled water 4:4:2, by volume), but no well-defined separated spots were achieved. Using a developing system of (acetonitrile/ distilled water/ ethanol 7:2:1, by volume) minimum tailing was exhibited with a maximum resolution of the four components with R f 0.32, 0.44, 0.58, and 0.73 for OXY, TRC, CNA, and FLU, respectively. This separation allows the determination of OXY and FLU at 267 nm without any interference from each other or their impurities (Fig. 2). Although sensitivity for FLU was higher at 210 nm, the presence of EDTA makes a noise that hinders the quantitative determination of FLU.

HPLC method
HPLC was also described for the simultaneous determination of OXY, TRC, FLU, and CNA. To optimize the proposed HPLC method, all the experimental conditions were investigated. Sequential isocratic was used as an alternative to gradient elution for the separation of complex mixtures that differ widely in retention. It consists of a series of successive isocratic experiments with a different composition of the mobile phase in each run [46]. The advantage of this technique is having better detection possibilities than using gradient elution. Further, LC detectors may be operated at a high sensitivity range, as there are no problems with baseline drift or solvent impurity [46].
Several trials were carried out to obtain a good and optimum separation. Different compositions of mobile phases with different ratios were tried such as (ethanol/ acetonitrile/ distilled water with 5% σ-phosphoric acid 70:20:10, by volume) and (acetonitrile/ phosphate buffer pH = 4 20:80, v/v). The best resolution was obtained upon using (distilled water with 0.1% TFA/ acetonitrile 82:18, v/v) for 9 min and then (70:30, v/v) in a sequential isocratic elution with a flow rate of 1 mL/min at wavelength 210 nm for FLU and 267 nm for OXY, TRC, and CNA. The maximum absorbance for each drug was found to be at different wavelengths the λ max was 267 nm for OXY and 210 nm for FLU. Accordingly, to reach higher sensitivity for both drugs, each one was determined at its λ max without affecting the concern of separation quality and system suitability. Using Hypersil BDS C18 (150 × 4.6 mm I.D., 5 μm), the t R values were found to be 3.18, 5.37, 7.40, and 13.04 for CNA, OXY, TRC, and FLU, respectively. The conditions of the suggested method such as lower mobile phase components and small column length minimize the pump wearing, decrease the effect of longitudinal diffusion and result in a better peak shape and symmetry, especially for the highly retained compound (FLU) (Fig. 3).

Method validation
Validation is an important step to confirm the suitability of the developed analytical procedure for its intended use. It is an integral part of any good analytical practice. Validation parameters were done according to the ICH Q2 guideline [47].

System suitability tests
The suitability of the chromatographic system was determined according to USP [6]. All the calculated parameters are within the acceptable limits which indicate good selectivity and resolution of the method and ensure system performance as shown in Tables 1 and 2.

Linearity
By applying the suggested methods, we found that linear relationships were obtained by plotting peak areas against  Table 3.

Accuracy
Six pure samples of each OXY and FLU within their linearity ranges were analyzed to assess the accuracy of the proposed methods. The concentrations of both drugs were calculated from their corresponding regression equations, and the obtained results are shown in Table 3. The standard addition technique was also applied for further assessment of the accuracy of the proposed methods, as given in Table 4.

Precision
The precision of the proposed methods was determined by the analysis of three different concentrations of each of OXY and FLU in triplicate on a single day (repeatability) and on three consecutive days (intermediate precision). The obtained results are listed in Table 3 and showed satisfactory precision for the proposed methods.

Specificity
The specificity of the suggested methods was investigated by resolution. A good resolution was obtained between the corresponding four peaks as in Figs. 2, 3 and listed in Tables 1, 2. Further assessment of specificity was done by analyzing the pharmaceutical dosage form. The proposed methods were successfully applied for the determination of OXY and FLU in FLOXON injection®. The obtained satisfactory results are shown in Table 4.

The detection limit (LOD) & the quantitation limit (LOQ)
LOD and LOQ for both drugs were calculated for the proposed chromatographic methods by using the following equations: LOD = 3.3 * σ /S, LOQ = 10 * σ /S where "σ" is the standard deviation of response and "S" is the slope    Table 3.

Robustness
The capability of the method to remain unaffected by small modifications in the experimental conditions was investigated. For the TLC densitometric method, a small change in the ratio of the developing system acetonitrile: distilled water/ethanol (7: 1.5: 1.5) and a change in wavelength 267 ± 2 nm were assessed. For the HPLC method, a change in wavelength (210 ± 2 nm and 267 ± 2 nm) and a small change in the 1st ratio of mobile phase into (80:20) and the 2nd ratio to be (68:32) were tested. The robustness was  checked by measuring the resolution and asymmetry factor which were nearly unaffected with an acceptable recovery, only R f and R t were changed according to the polarity of the solvent as shown in Table 5.

Biological application
The developed methods were used to determine drug residues in both meat and milk of cattle. The accepted MRL for OXY was found to be 100 g/Kg in both meat and milk by the European Medicines Agency [48], while the MRL for FLU was found to be 20 g/Kg in meat and 40 g/Kg in milk by the same Agency [49].

TLC densitometric method
Linear calibration curves were obtained for each OXY and FLU with recoveries of 100.03 ± 1.635 and 99.58 ± 1.460 for OXY in milk and meat, respectively, while they were found to be 100.50 ± 1.603 and 100.21 ± 1.665 for FLU in milk and meat, respectively Table 6.

HPLC method
Linear calibration curves were obtained for each OXY and FLU with recoveries of 99.67 ± 1.041 and 100.62 ± 1.709 for OXY in milk and meat, respectively, while they were found to be 99.74 ± 1.862 and 99.77 ± 1.524 for FLU in milk and meat, respectively Table 7.

Statistical comparison
Statistical comparison was accomplished between the proposed methods and the reported direct spectrophotometric method for OXY [37] and the official direct spectrophotometric method for FLU [38]. The obtained results show no significant difference between them, Table 8.

Greenness assessment
It is a big issue to determine the greenness of an analytical process. Analytical Eco-Scale is one of the used green impacts which take into consideration many factors that have negative behavior on the environment, such as the amount and type of used chemicals, generated wastes, occupational hazards, and energy consumption. It is described as a semiquantitative tool that could be used to evaluate the greenness of analytical methods. The result of the calculation is ranked on a scale, where the score: > 75 represents excellent green analysis, > 50 represents acceptable green analysis, < 50 represents inadequate green analysis [50]   Penalty points and Eco-Scale were calculated for the developed methods as shown in Table 9. The TLC densitometric method shows acceptable green analysis while the HPLC method represents excellent green analysis.

Conclusion
In the present work, green, sensitive, economical chromatographic methods (TLC densitometric and HPLC) were developed for the simultaneous determination of OXY and FLU in their pharmaceutical dosage form and in the presence of their impurities. The proposed methods can be used in the qualitative and quantitative determination of both drug residues in cattle meat and milk, which has a great benefit in food safety testing for human health. Finally, the proposed methods can be easily implemented in QC laboratories. In addition, both methods can be used for API impurity profiling and detection of OXY and FLU residues in biological samples.
Author contributions All authors contributed sufficiently and equally to this work, and there have been no involvements that might raise the question of bias in the work reported or conclusions and all authors agreed to publish the work in this journal.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). No funding was received for this work.

Declarations
Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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