Antibiotics are prescribed to treat or prevent Bacterial Infections. It also helps to prevent spreading of infections. Antibiotics are available with different types, but most of them can be classified into 6 groups, such as: Penicillins, Cephalosporin; Aminoglycosides; Tetracyclines; Macrolides; Fluoroquinolone etc. There are series of drugs available with each groups and every antibiotic has its own criteria, thus, Chemical structure could be a great option to classify the Antibiotics 1, 2. Cephalosporins antibiotic have unique structure (Figure 2) with a common β-lactam ring that act to hinder Cell wall synthesis of the bacteria 3.

Cephalosporin Antibiotics are essentially most effective and most prescribed broad-spectrum antibiotics which are active against both Gram (+) and Gram (−) bacteria 4. Although these antibiotics are helpful to protect human and animal heath from different bacterial infections but at the same time, presence of these antibiotics as residue level into the aquatic environment might initiate an unexpected issue like bacterial resistance to Antibiotics. Several scientists found resistant bacteria into the environment due to unconscious uses of Bacteria 5

Cephalosporins Antibiotic resistant bacteria have already been detected into the aquatic environment or into different sewage treatment plants as well. These antibiotics are recognized as one of the main source of Antibiotic resistance from all pharmaceutical active components present into the environment 6, 7, 8. The water quality in perspective of Cephalosporin Antibiotic residue is very censorious in developing countries where self-medication and untreated sewage into the environment is very common 9, 10, 11. There are several other reasons also involved for the presence of mass of antibiotic contaminants into the environment, like, plant agriculture with antibiotics and also waste disposal from pharmaceutical manufacturing plants. Earlier pharmaceutical manufacturing plant was not in attention but recently it is identified as one of the main source for discharging effluent with Antibiotics in some Asian countries of up to several L mg L^-1 (ppm) level for single compound 12, 13. Presently, Cephalosporins have five generations and most of them are used to treat as antibiotics to the patients. But cephalosporin 3^rd Generations was mostly prescribed drugs (Table 1) than other Generations (1^st or 2^nd) in Bangladesh 14.

According to use of Antibiotics, the Pharmaceutical manufacturing company also manufacture the products to supply into the market to meet the demand of patients, thus, it is obvious that Cephalosporin Antibiotics are mostly manufactured antibiotics from Pharmaceutical Companies. So, Cephalosporin antibiotics contaminants are also in high risk to be available in Pharmaceutical waste water during discharge to outside surface water. Pharmaceutical manufacturing site especially Cephalosporin manufacturing site is one of the main source of Antibiotic Resistance. As we already discussed that among all antibiotics, β-Lactams and Florfenicol are mostly stable into the soil mostly than other Antibiotics, thus, these groups have affinity to show activity of Antibiotic resistance 15. There are several ways by which a Pharmaceutical company can be involved to contaminate the Environment with Antibiotic Pharmaceuticals. Waste water from Cephalosporin manufacturing plant is one of the source of multiple antibiotics from Cephalosporin groups (Cephems). Increasing concentration of these multiple antibiotics can lead to multi-resistant bacteria in the environment 16. Bacteria strains can be resistant by frequently contact with minor concentrations of antibiotics 17. Due to long term presence of multiple Antibiotics into the waste water system and also pressures selection at sub-inhibitory concentrations of various antibiotics, ARG and ARB can be developed 16.

Pre-treatment of Cephalosporin waste water from the WWPTP before transferring might be an option to degrade the Cephalosporin drugs. Identification and quantification of Cephalosporin residues in waste water is required for the quality control department of a pharmaceutical company to detect the Cephalosporin residues in waste water. Measurement of Cephalosporin residues in the aquatic environment is very much important to ensure the quality of the environment, especially in the surrounding of the pharmaceutical production plants. So, an analytical method is in high demand for analyzing all the Cephalosporin drugs molecule in waste water. Literature survey reveals that the available analytical methods in the literature are either developed for formulation development/clinical/biological samples 18, 19, 20, 21, 2, 22, 23, 24, 25. Analytical HPLC method for Environmental water samples is available with a single Cephalosporin group 5, 26 or a multiple Cephalosporin group like3, 27 which have determined mostly 06 (six) Cephalosporin drugs. By evaluating all relevant literature it is found that very recently in 2021, maximum 08 (Eight) Cephalosporin molecules has been detected and quantified in Pharmaceutical formulation through a HPLC method 1, 22.

But in this study, we aimed to develop an analytical method for determination of most frequently prescribed and mostly manufactured eleven Cephalosporin drugs, which will be applicable for the analysis of waste water transferred from Cephalosporin manufacturing unit to the outside surface water. It will be used as an important tool for regular environmental monitoring program of factory quality control to ensure one health also. Our developed method will be a cost-effective and time-saving method as eleven different Cephalosporin molecules (Figure 3) will be identified and quantified in a single HPLC run. The method was developed and validated with well separated peaks as per ICH Q2(R1) guidelines.

All 11 Cephalosporin molecules were identified and quantified into a minimum level by the developed method with a Gradient mobile phase consisting of a TBAH buffer, pH 6.8 and Acetonitrile at the ratio 85:15 & 25:75 V/V as Solution A & B for the Gradient program with a 250 cm x 4.6 mm, 5 µ column as stationary phase at 35°C COT and 1.2 FR. The developed method demonstrated a unique chromatogram with separated individual specific and selective peaks for all 11 Cephalosporin compounds such as Cefepime, Cefadroxil, Ceftazidime, Cephradine, Cefaclor, Cefotaxime, Ceftibuten, Ceftriaxone, Cefixime, Cefuroxime and Cefpodoxime and placebo interference has also check by injecting Purified water as placebo, where the final sample is waste water. The stability of analytical solution was determined and got the stability up to 24 hours for the solutions in refrigerator (2-8°C) condition. The system was suitable during every analysis and the method was found precise during method precision and intermediate precision with different analyst and different instrument. The Method is linear for individual Cephalosporin compound by giving a correlation coefficient (r) value ≥0.999 at the concentrations level 0.5 – 7.5 µg mL^-1 for each Cephalosporin compound. A precise LOQ level was found with the concentration value as 0.10 ppm (100 ppb) and Lo=OD level was 0.05 ppm (50 ppb) for each Cephalosporin compound. The % Recovery for individual Cephalosporin compound was determined for LOQ, 50%, 100% & 150% level and found the % recovery between 95.0% to 105.0% that were within limit.

Waste water from any pharmaceutical plant is transferred to Central Effluent treatment plant to surrounding outside water like Ground water, surface water etc. Waste water from antibiotic plant like Cephalosporin unit must be pre-treated with chemical before transferring to Central ETP or surface water to conform the absence of Cephalosporin compounds in the waste water. There should have a specific method which can determine the Cephalosporin compounds. In this method, 11 most commonly manufactured Cephalosporin compounds in Bangladesh has been considered for the development of a HPLC method to quantify all these Cephalosporins into the residue level in the waste water. The developed and validated method for the analysis of waste water was is very simple method to be executed. HPLC method is modernized in respect of cost, and time by changing the Mobile phase, stationary phase and chromatographic conditions. The validation results indicated that the developed HPLC method for determination of 11 Cephalosporin antibiotics from different generations such as Cefepime, Cefadroxil, Ceftazidime, Cephradine, Cefaclor, Cefotaxime, Ceftibuten, Ceftriaxone, Cefixime, Cefuroxime Axetil and Cefpodoxime proxetil in a single run is precise, accurate, linear over the test concentration range, rugged and specific.

The gradient solvent system to determine all these Cephalosporins in a single run will not only save the cost and time but also it will be a unique method to control all type of Cephalosporin wastes discharged from the manufacturing unit to the environment and it will be a better option for the analytical & quality control labs to control Cephalosporin residues. Therefore, this HPLC method by this study will be helpful and distinctive from the previous methods published into different journals. This method will be more efficient and simple to apply where, each drug from different Cephalosporin compounds need a specific method and a separate solvent system.

HPLC : High Performance liquid chromatography; DAD : Photodiode-array Detection; TSP : Total survey prescriptions; TAP : Tital antibiotic suggested prescriptions; FA-TAP : Frequency of Antibiotics out of TAP; FA-TSP : Frequency of antibiotics out of the TSP; Max : Maximally prescribed antibiotics in their respective group; ICH : International Council for Harmonisation; ARG : Antibiotic-resistant Gene; ARB: Antibiotic-resistant bacteria; RSD : Relative standard Deviation; WWPTP : Waste water pre-treatment plant; LOD : Limit of detection; LOQ : Limit of quantification; COT : Column oven temperature; FR : Flow rate; TBAH : Tetra-butyl Ammonium Hydroxide

The authors are very much thankful to the management of The ACME Laboratories Ltd., Dhaka, Bangladesh for providing support to carry out this study.