The success of a pharmaceutical product relies on its qualitative analysis with a thorough validation process [1]. With the advancements in medicinal chemistry, pharmacology, and thereby the launch of new formulations, the demand for suitable testing methods has increased a lot in recent times. The acceptability of the method of analysis lies in the convenience of application and less costly approach [2]. When no conclusive approaches are available, new methodologies for evaluating novel pharmaceutical combinations are needed. Novel procedures have been developed to analyze pharmaceutical formulations with greater precision and strength. Preliminary runs are employed to optimize and validate these approaches [3]. The analytical method is an important aspect of regulatory filing in Europe and the United States, where enormous technological breakthroughs in analytical instrumentation are achieved with an emphasis on reducing analysis time without sacrificing precision and accuracy [4]. All of the new drug molecules take a too long time to have their inclusion in standard pharmacopeia books. Standards and analytical procedures for these substances and their different combinations may not be available in pharmacopeias under these circumstances [5]. As a result, new analytical procedures for these medications must be developed to assure the identification, purity, potency, and performance of drug items [6].

The validation data to support analytical procedures is collected in a planned and systematic manner by the applicant as part of the method validation process for analytical procedures. Validation is required for all analytical procedures intended for use in the analysis of clinical samples. Analytical methods are validated in accordance with International Conference on Harmonization (ICH) guidelines [7].

Oxytetracycline (OXY) has been engaged as an antimicrobial agent in the management of mastitis in dairy cows. As a consequence, (1) OXY is found in fairly large amounts in pasteurized milk along with other antibiotic residues. Similarly, honey is observed with considerable contents of OXY. OXY is used in the treatment of European foulbrood disease and American foulbrood disease due to Paenibacilus larvae and Streptococcus pluton bacteria, respectively. (2) The honey containing these antibiotic residues is not meant for sale in advanced countries in particular [8]. (3) Further, OXY has got a wide application in dealing with fish bacterial diseases induced by Aeromonas salmonicida, Aeromonas hydrophila, and Aeromonas sobia. (4) Polymixin B (PMB) is also used in the treatment of various bacterial diseases associated with aquatic life. The use of antibiotics becomes unavoidable in certain circumstances, but the setting up of limits on the food contents may allow these items to be consumed safely by humans. Thus, there is an immense need for accurate and precise estimation of these antibiotics in different food samples. The capacity of an analytical process to produce test results that are directly proportional to the concentration (quantity) of analyte in the sample (within a specific range) is known as linearity. The robustness of an analytical procedure is a measure of its ability to remain unaffected by modest but deliberate changes in method parameters, and it indicates its dependability in everyday use [9]. Combination products, commonly known as fixed-dose drug combinations (FDCs), are single dose pharmaceutical formulations that combine two or more active medications. A new investigational drug in conjunction with an existing drug or two or more novel entities could also be included in the FDC formulation. FDC drugs are frequently utilized to treat single or numerous diseases. Patients are benefited from FDC products because they simplify dosing and lower prescription costs [9].

OXY is a wide-spectrum antibiotic that belongs to the tetracycline family [10, 11]. However, as these medications leave residues in milk or meat that might be directly poisonous or induce allergic reactions in some hypersensitive individuals, their use has become a severe problem in terms of infectious illnesses. Even more importantly, low-dose antibiotics in foods taken over long periods can cause problems with the proliferation of drug-resistant bacteria [12]. Different techniques are commonly used as screening methods to identify tetracycline residues in food [13], but these approaches lack specificity, give only semi-quantitative assessments of residues identified and can produce false positives. High-performance liquid chromatography (HPLC) with diode array detector (DAD) [14] and fluorimetric [15] detection has been employed for quantitative tetracycline determinations, but some chromatographic techniques are suited for multi-residue analysis [16, 17]. OXY and PMB are available as a fixed-dose topical gel formulation (0.5% strength) for cutaneous infections. The simultaneous presence of OXY and PMB in different food items and FDCs comprising these antibiotics insist on the requirement of a novel method of analysis for the efficient detection of these two drugs.

OXY and PMB have not been analyzed simultaneously till date. To reduce the analytical error and offer readers a good approach for determining the two antibiotics in a single injection, it hypothesized that these two drugs were estimated simultaneously. In this work, the authors have developed a simple, rapid, and precise method for the quantitative and qualitative measurement of both OXY and PMB in different samples.

The liquid chromatographic method was designed and fine-tuned to ensure reproducibility and accuracy. Both the hit-and-trial approach and the polarity of the mobile phase were used as selection criteria in the process. In the early stages of the measurement of OXY and PMB, a number of solvents in varying proportions were utilized [17, 20]. Although several mixtures of organic solvents, either used in 100% concentration or in conjunction with water in varying proportions, were tried, however, the separation did not meet the required mark. Following system suitability parameters were considered for mobile phase selection which is written below:

(1) A sharp peak was observed

(2) Tailing and fronting were not observed

(3) Appropriate elution time

(4) Suitable number of theoretical plates and home-exercise therapy program (HETP)

As the longitudinal broadening is inversely proportional to the flow rate of the mobile phase system, maintaining the uniformity of the flow rate is highly important [21]. When the flow rate is too high or too low, it has an effect on the Gaussian peak and can lead to symmetry problems in the overall peak. Hence, a low mobile phase composition is able to reduce peak broadening and tailing and able to improve theoretical plates. During the final analysis, ACN: water (pH 3.5 adjusted with orthophosphoric acid) in the ratio of 80:20 (v/v) was the selected and optimal mobile phase, as it gives excellent separation of molecules and detection [22].

For a particular range of analyte concentration, a procedure’s linearity refers to its capacity to produce test findings that are directly proportional to the sample’s concentration [23]. According to the least squares method of analysis, the developed method was shown to be linear. LOD is the ability of the method to detect the lowest amount of drug and LOQ is the ability of the method to quantify the lowest amount of drug [24]. According to guidelines, LOD and LOQ are observed to be within uniform limits. Recovery experiments were conducted at 80%, 100%, and 120% of the test concentrations of both drugs to verify the method’s accuracy. It was necessary to produce the samples in triplicate and to compute the percent recovery for each sample [25]. Moreover, the robustness and ruggedness define the toughness and high sensitivity of the developed method as it doesn’t show any fluctuations through minuscule fluctuations [25, 26]. Analysis revealed RSD (%) values < 2% for the performed studies which define the corrected validation of the developed method as per ICH guidelines. For the assay determination, the active content of the marketed formulation showed an acceptable range of 90–110% in the ointment which successfully tested the acceptability of the developed method for routine analysis.

In conclusion, the RP-HPLC method has been used for the estimation of OXY and PMB in a bulk material. The method was finally optimized as: mobile Phase; ACN:water (80:20) with pH 3.5 using 0.5% (v/v) orthophosphoric acid, and flow rate: 0.9 mL/min. For both OXY and PMB, validation studies were done and RSD (%) was found to be < 2%. For the marketed product, an assay was performed to check the purity of OXY and PMB in the marketed product and the percentage purity of both drugs was calculated. Percentage estimation for OXY was found to be 98.64%, and for PMB it was found to be 90.84%. The developed HPLC method for determining OXY and PMB was simple, specific, accurate, precise, rapid, and economical, indicating its adequacy for routine pharmaceutical analysis of OXY and PMB.