HPLC ANALYSIS OF CEPHALOSPORINS AND STUDY OF DIFFERENT ANALYTICAL PARAMETERS

HPLC ANALYSIS OF CEPHALOSPORINS AND STUDY OF DIFFERENT ANALYTICAL PARAMETERS

P. N. Patil*¹ and S. Jacob²

Department of Chemistry, Bharati Vidyapeeth’s College of Engineering, Near Chitranagari, Kolhapur, Maharashtra, India

Department of Pharmaceutics, College of Pharmacy, Gulf Medical University, Ajman, UAE

 ABSTRACTCephalosporins are the most important type of antibiotics used vary widely. Analysis of these antibiotics is challenge because of their sensitivity and instability to different conditions. The present review is extended to find out different HPLC methods used for analysis for cephalosporins in formulations and biological fluids. The chromatographic conditions used for the analysis as well as analytical parameters study carried out with different experimental conditions and different combinations. During the study analytical parameters studied such as range, linearity, precision, accuracy, LOD, LOQ etc. The focus of study of analysis of cephalosporin in formulation and biological fluid are useful for the determination of various cephalosporin for budding researchers

Chromatographic methods

INTRODUCTION: Although there are several classification schemes for antibiotics, based on bacterial spectrum (broad, narrow) or route of administration (injectable, oral, topical), or type of activity (bactericidal, bacteriostatic), the most useful is based on chemical structure. Antibiotics within a structural class will generally have similar patterns of effectiveness, toxicity, and allergic potential.

Most commonly used type of antibiotics are Pencillins, Fluoroquinolones, Cephalosphorins, Macrolides and Tetracyclines. While each class is composed of multiple drugs, each drug is unique in some way. Antibiotics are obtained from three major sources such as, microorganisms, synthesis and semi synthesis.

1. Microorganisms: For example, bacitracin and polymyxin are obtained from some Bacillus species streptomycin tetracycline etc. from streptomyces species; gentamycin from Micromonospora purpura; griseofulvin and some penicillin and cephalosporin from certain genera (penicillium, acremoni) of the family Aspergillacea; and monobactam from Pseudomonas acidophila and Gluconobacter species. Most antibiotics in current use have been produced from streptomyces species.
2. Synthetic: Chloramphenicol is now produced by a synthetic process.
3. Semi-synthesis:- This mean that part of the molecule is produced by a fermentation process using the appropriate microorganism and the products is then further modified by a chemical process. Many penicillin and cephalosporin are produced in this way.

 Cephalosporins:

History: Antibiotics are the most important bioactive and chemotherapeutic compounds made by microbiological synthesis. They also include antimicrobial compounds present in higher plants and animals. They have proven their significance in varied fields like medicinal chemistry, agriculture and food industry.

Up to now about 40000 antibiotics have been found and about 80 of them are in therapeutic use. They are isolated primarily from metabolic products of living cells. Various penicillins, cephalosporins and several other antibiotics are semi-synthetic ones, which mean one part of the molecule, i.e. 6-amino penicillanic acid is prepared from say penicillin G or penicillin V, followed by synthetic introduction of an appropriate side chain ¹.

Cephalosporin compounds were first isolated from cultures of Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist Giuseppe Brotzu. He noticed that these cultures produced substances that were effective against Salmonella typhi, the cause of typhoid fever, which had beta-lactamase. Guy Newton and Edward Abraham at the Sir William Dunn School of Pathology at the University of Oxford isolated cephalosporin C.

Cephalosporins are structurally and pharmacologically related to the penicillins. Like the penicillins, cephalosporins have a β-lactam ring structure that interferes with the synthesis of the bacterial cell wall. They are used for the treatment of infections caused by Gram (+) and Gram (−) bacteria. They are among the safest and the most effective broad-spectrum bactericidal antimicrobial agents and therefore, they are the most frequently prescribed class of antibiotics ².

To date, more than 20 cephalosporins have been approved by the U.S. Food and Drug Administration, and new ones continue to appear. Cephalosporins have an excellent safety profile and a basic structure that lends itself to numerous alternations, resulting in compounds that have widely different spectra of antimicrobial activity. The continuing emergence of resistant strains of bacteria necessitates the development of new antibiotics.

Structure: Cephalosporins like penicillins having a b-lactam ring antibiotic containing a dihydrothiazine ring with D-a-aminoadipic acid. Cephalosporins are less toxic and broad-spectrum antibiotics comparable in action to ampicillin. Organisms known to produce cephalosporin C include Acremonium chrysogenum and Acremonium chrysogenum ATCC 36225 ^{3, 4}.

The cephalosporin ring structure is derived from 7-aminocephalosporanic acid (7-ACA) while the penicillins are derived from 6-aminopenicillanic acid (6-APA). The first cephalosporin, known as cephalosporin c, was isolated from the fermentation products of a fungus, Cephalosporium acremonium from a sewer in Sardinia in 1948 by Italian scientist Giuseppe Brotzu. He noticed that these cultures produced substances that were effective against Salmonella typhi, the cause of typhoid fever Hydrolysis of this compound produced aminocephalosporanic acid, which was modified with different side chains to create the whole family of cephalosporin antibiotics.

The cephalosporin nucleus, 7-aminocephalosporanic acid (7-ACA), was derived from cephalosporin C and proved to be analogous to the penicillin nucleus 6-aminopenicillanic acid, but it was not sufficiently potent for clinical use. Modification of the 7-ACA side-chains resulted in the development of useful antibiotic agents, and the first agent cephalothin (cefalotin) was launched by Eli Lilly in 1964.

The cephalosporin molecule is composed of a nucleus and two side chains. The nucleus 7-aminocephalosporinic acid consists of a four member’s β-lactam ring fused to a six-member dihydrothiazine ring. By comparison, the penicillin nucleus, 7-aminopenicillanic acid has β-lactam ring fused to a five-member thiazolidine ring.

The cephalosporin nucleus has two advantages over the penicillin nucleus. First, it is inherently more stable to   β-lactamases; bacteria    that produce penicillinase are usually susceptible to cephalosporin. The second advantage is the presence of R2 substituent. The penicillin molecule can be modified at only one site (R1), while the cephalosporin molecule has two sites (R1 and R2). Thus, compared with penicillins the potential number of cephalosporins is considerably greater.

The presence of an intact β-lactam ring is essential for the antimicrobial activity of the cephalosporin. Modifications of the R1 substituent, which is in close proximity to the β-lactam ring, affect antibacterial activity by altering β-lactamase stability and/or binding affinity for the penicillin-binding proteins.

Changes in the R2 which is attached to the dihydrothiazine ring, affect the pharmacokinetic property of the drug. Substitution of an acetoxy group ([-CH.sub.2]-O-CO [-CH.sub.3]) for R2 is associated with significant metabolism to desacetyl derivatives. The introduction of a methoxy group (-O[CH.sub.3]) on the β-lactam ring at position 7 is associated with a marked increase  in both Β-lactamase stability and activity against anaerobes.

CORE STRUCTURE OF CEPHALOSPORIN

STRUCTURE OF CEPHALOSPORIN

Classification of Cephalosporins:

Analysis of Cephalosporin: There are various methods used for the analysis of cephalosporin in the various forms like chromatographic, UV, electrophoresis etc. The applications of HPLC to the analysis of antibiotics introduce a powerful tool for therapeutic drug monitoring as well as clinical research ⁵.

GC methods are fast but it requires elevated temperature, it may cause thermal degradation of drugs. To avoid that it requires derivatization to improve volatility and to improve chromatographic behavior. So these methods are not applicable for antibiotics.

While other chromatographic methods having high limit of detection value so they are also not preferred.  HPLC technique can provide valuable tool which generating high pure compound and HPLC has ability to analyze both volatile and nonvolatile compounds with ultra trace level may be employed in clinical research. Many antibiotics contain ionizable group can be analyzed by ion exchange chromatographic methods. High resolving power of HPLC serves as a particularly important method for isolation and purification of antibiotics ⁵.

Official Methods of Analysis: The United States Pharmacopeia XXX ⁶ prescribes a polarographic method for the assay of cefamandole naftate and HPLC methods for the assay of the other cited cephalosporins while the European Pharmacopeia 2002 ⁷ prescribes HPLC methods for their assay. The analysis of cephalosporins in biological materials from human origin and in food-producing animals ⁸ foods, waters and pharmaceuticals was performed with liquid chromatographic, capillary electrophoretic, spectro- scopic and electrochemical methods. None of them, however, was analyzed the seven cephalosporins simultaneously ^{9, 10}.

Analysis in Bulk Drug and Dosage Forms: There are various methods available for the analysis of antibiotics in different formulations as well as in biological fluids. Some methods found in literature survey are for single cephalosporin while some methods are available with different combination. Various methods found are on different instrumental methods such as, HPLC, HPTLC, GC, CE, TLC, UV spectrophotometric, AAS, Electrochemical methods.

Chromatographic methods:

High-Performance Liquid Chromatographic Methods (HPLC): The most common methods for the analysis of cephalosporins in formulation and in biological fluids are HPLC method for analysis. As reported in the literature, several analytical procedures have been described for analysis of cefotaxime, ceftazidime and ceftriaxone in pharmaceutical formulations and biological fluids. These include high-performance thin layer chromatography ¹¹, high-performance liquid chromatography ^12-14, differential pulse adsorptive stripping voltammetry ⁹, NMR spectrometry ¹⁵, polarography ¹⁶ and UV derivative spectrophotometry ¹⁷.

There are various HPLC methods are reported for the analysis of a single cephalosporin in biological fluid ^18-25. All these methods present a unique preparatory and chromatographic protocol for single cephalosporin or its metabolite or both of them. Several methods have been used for the analysis of Ceftazidime the third generation cephalosporin for estimation of ceftazidime alone which includes High Performance Liquid Chromatography ^26-28 and in combination with pyridine ²⁹ Vancomycin ³⁰ and cefepime ³¹.

The impressive increase in the use of high-performance liquid chromatography in the past thirty years did not pass the b-lactam antibiotics. HPLC has been used frequently in all fields of B-lactam research, not only as an assay method but also as a tool for purification of the antibiotics. Due to the insolubility of these compounds in organic solvents, normal phase LC was sparingly used. Most methods employ reversed-phase or ion-pair reversed-phase LC and chemically bonded packing materials.

A review on the evidence of chemical structure and stability profiles of ceftazidime ¹⁷ and cefotaxime ³² has been reported. Recently, full spectrum quantitation (FSQ) was used for rapid multi-component analysis of complex biological and pharmaceutical mixtures ³³. The technique was successfully applied for simultaneous analysis of binary mixtures of cortisonecortisol ³⁴, cephalexinxephradine, phenobarbiton ephenytoin sodium and ternary mixture of aspirin-caffeine salicylic acid ³⁵.

Moreover, FSQ has been used to evaluate the stability of chloramphenicol succinate in injections ³⁶ and to determine famotidine ³⁷ in the presence of its acid induced degradation products. The present work reports on the use of FSQ and HPLC to quantify cefotaxime, ceftazidime and ceftriaxone in the presence of their alkali-induced degradation products and in commercial injections. Furthermore, the application of HPLC as a stability-indicating assay to study the kinetics of degradation of the investigated antibiotics in aqueous solutions of varying pH (2-10) is demonstrated.

Spectroscopic methods:

Ultraviolet Spectrophotometric Methods:Cefotaxime, ceftriaxone and ceftazidime were determined in the presence of their alkali-induced degradation products through spectrophotometric full spectrum quantitation over the range of 265-230 nm ³⁷. Various UV spectrophotometric methods are reported for the analysis of Ceftazidime alone in presence of other drugs ^38-42.

Mixtures of ceftazidime, cefuroxime sodium, cefotaxime sodium and their degradation products were analyzed by first-derivative spectrophotometry at 268.6, 306, 228.6 nm, respectively. Cefotaxime and cefuroxime were determined through the reaction with 1 chlorobenzotriazole and the absorbance was measured at 298 nm.

UV, first-derivative, second-derivative and H-point standard addition methods were applied for the determination of cefalexin in pharmaceutical preparations. Derivative spectrophotometry was also applied for the determination of some cephalosporins in binary mixtures ⁴³. A spectrophotometric method was reported for the determination of cefalexin bulk drug and its acid-induced degradation products ⁴⁴. UV spectrophotometry and difference UV spectrophotometry were applied to determine cefalexin in tablets. In addition, derivative spectrophotometry was used to determine the triethylammonium salt of cefotaxime in the presence of related compounds resulting from the synthesis.

Dissociation constants of cefepime and cefpirome were determined by UV spectrometry. Cefuroxime axetil and probenecid were simultaneously determined in solid dosage forms by UV spectrophotometric method. Derivative spectrophotometry was reported for the determination of cefprozil in pharmaceutical dosage forms in the presence of its alkali induced degradation products. Binary mixtures of cefalotin and cefoxitin were determined by first-derivative spectrophotometry. A spectrophotometric method has been developed for determination of cefadroxil in bulk powder and its pharmaceutical dosage forms based on the reaction of primary amine group with acetylacetone-formaldehyde reagent, which gives a yellow coloured chromogen. Another method was reported for the determination of cefadroxil in pharmaceutical dosage forms through mixing with sulfanilic acid; the absorbance was measured at 440 nm.

Thin-layer Chromatographic Methods: Qureshi et al., has determined Cefradine and cefalotin ­ by spectrodensitometric method after contact with iodine vapors. Cephalosporins were applied to TLC plates coated with a mixture (2:1) of layered double hydroxide of aluminum(III) and magnesium(II) and silica gel G and developed with a range of mobile phases, the spots were detected with iodine vapours and the cephalosporin content was determined. Degradation products of ceftazidime, cefuroxime sodium and cefotaxime sodium were prepared by acid hydrolysis, mixtures of these drugs and their degradation products were analyzed by quantitative densitometric TLC­. Some cephalosporins in phosphate buffer of pH 3.6 were spotted on TLC plates coated with silica gel with a fluorescent indicator or silica gel RP 18; the spots were visualized at 254 nm or with a color-forming agent.

Ceftriaxone, cefixme, cefotaxime, cefaclor and cefalexin were determined in their pharmaceutical dosage forms using HPTLC and the measurement of each spot was carried out at specified wavelengths using a scanner in absorbance/reflectance mode ^45,46 reported a method for simultaneous determination of cefadroxil and cefalexin in pharmaceutical preparations using quantitative TLC. Cefalexin was analysed by HPTLC on silica gel F254 plates. The plates were scanned in reflectance mode at 263 nm ⁴⁷. Few methods have been reported for the quantification of Cephalexin single drug and in Combination by HPTLC ^46-48.

The use of RP-HPLC procedures for determination in plasma, serum and urine has been reported ^49-51. Few HPLC methods also have been reported for quantification of Cephalexin ^{44, 52}. Some spectrophotometric and colorimetric methods also have been reported ^{53, 54}.

However till now, no stability indicating method for estimation of Cephalexin has been reported Cefuroxime axetil and cefuroxime were determined using TLC densitometry after separation on silica gel using chloroform/ ethyl acetate/glacial acetic acid/water (4:4:4:1) as a mobile phase.

Dhanesar proposed a densitometric method for quantitation of some cephalosporins on a hydrocarbon impregnated silica gel HPTLC plate. Simultaneous determination of cefalexin and probencid in pharmaceutical preparation was performed by HPTLC using silica Gel 60 F254 HPTLC plate and they were detected at 254 nm. Also HPTLC method was used for the determination of ceftriaxone in injection solutions using butanol/acetonitrile/water (3:1:1) as developing solvent and detection at 254 nm ¹¹.

Analysis of Cephalosporin in Biological Fluids:

High- Performance Liquid Chromatographic Methods: This technique is the most frequently applied technique for the determination of cephalosporins in biological fluids (blood, plasma, urine, cerebrospinal fluid, etc.), animal tissues, food, etc. summarises the recent HPLC reported methods for the analysis of cephalosporins in biological fluids, animal tissues, food, etc.

Capillary Electrophoretic Methods: Nine cephalosporins were determined using capillary zone electrophoresis after hydrodynamic injection on a fused-silica capillary and detection was performed at 210 nm Mrestani et al., proposed a CZE (Capillary Zone Electrophoresis) method for the determination of four cephalosporins after injection on a fused-silica capillary with detection at 270 nm and 30 kV separations potential. Cefixime and five of its metabolites were determined in human digestive tissues by high performance capillary electrophoresis on a fused-silica capillary tube with detection at 280 nm. CZE was used to determine β-lactam antibiotics, aminoglycosides, quinolones and tetracyclines in biological samples.

Cefotaxime and its deacetyl metabolite were determined by CZE using a fused-silica capillary with borate buffer pH 9.2 as electrolyte .CZE was used for the determination of four cephalosporins in clinical samples.

Micellar electrokinetic capillary chromatography (MEKC) Cefuroxime was determined in human serum by MEKC using a fused-silica capillary with 150mM sodium dodecyl sulfate in 20mM sodium phosphate and borate (pH 9.0) as electrolyte, with applied potential of 15 kV and detection at 274 nm.

Yeh et al., proposed a MEKC method for determination of ceftazidime in plasma and cerebrospinal fluid using Tris buffer with sodium dodecyl sulfate as background electrolyte and detection was performed at 254 nm. MEKC was used for determination of cefotaxime and it’s deacetyl metabolite using a fused-silica capillary with phosphate buffer pH 8.0 containing 165mM sodium dedecylsulphate as separation electrolyte. Cefpirome was estimated in human microdialysis and plasma samples by MEKC.

The effect of various parameters such as pH, pre-concentration time, deposition potential, supporting electrolytes, possible interferences and other variables were investigated for the examined drugs. Statistical analysis was carried out for the obtained results. Good linearity's were obtained. The proposed method was successfully applied to the analysis of the studied drugs in their available pharmaceutical formulations ⁵⁵ and in biological samples (serum and urine). The interference of some amino acids urea, ascorbic acid and some metal ions was investigated.

The developed Liquid chromatographic method with UV-Visible detection offers, sensitivity, precision and accuracy. It produces symmetric peak shape, good resolution and reasonable retention time for Ceftazidime and sulbactam sodium.

Moreover there is no pretreatment of the sample which makes the method simple and easy to perform. It can be used for the simultaneous determination of Ceftazidime and sulbactam sodium in the pharmaceutical companies and research laboratories for routine analysis and in plasma samples ⁵⁶.

TABLE 1: DIFFERENT HPLC METHODS FOR THE ANALYSIS OF CEPHALOSPORIN WITH ANALYTICAL PARAMETERS

TABLE 2: DIFFERENT CHROMATOGRAPHIC CONDITIONS

SPECTRA1: A TYPICAL LC CHROMATOGRAM OF A MIXTURE OF CEFTAZIDIME PENTAHYDRATE 500 ppm (PEAK NO. 1) AND SULBACTAM SODIUM, 250 ppm (PEAK NO. 2) ACHIEVED USING THE PROPOSED METHOD ⁵⁶

SPECTRA 2:- CHROMATOGRAM OF CEFTAZIDIME PENTAHYDRATE AND SULBACTAM SODIUM AFTER SUBJECTED TO ACID DEGRADATION⁵⁶

SPECTRA 3: CHROMATOGRAM OF CEFTAZIDIME PENTAHYDRATE AND SULBACTAM SODIUM AFTER SUBJECTED TO THERMAL DEGRADATION ⁵⁶

SPECTRA 4: A TYPICAL HPTLC DENSITOGRAM OF CEFPROZIL STANDARD ⁶¹.

SPECTRA 5: DETERMINATION OF MAX OF CEFUROXIME AXETIL BY UV SCANNING

SPECTRA 6:- TYPICAL ELECTROPHEROGRAM OBTAINED FROM A MIXTURE OF THE CEPHALOSPORINS. CE CONDITIONS: APPLIED POTENTIAL 20 KV; BUFFER 50 MM SODIUM TETRABORATE, pH 9; DETECTION WAVELENGTH 214 NM.

1, CEFRADINE; 2, CEFTIZOXIME; 3, CEFOTOXIME; 4, CEFUROXIME; 5, CEFACLOR; 6, CEFADROXIL; 7, CEFTRIAXONE, AND 8, CEFIXIME ⁶⁶

SPECTRA 7:- TYPICAL ELECTROPHEROGRAM OBTAINED FROM A MIXTURE OF THE CEPHALOSPORINS. CE CONDITIONS: APPLIED POTENTIAL 30 KV; BUFFER 50 MM SODIUM TETRABORATE, pH 9; DETECTION WAVELENGTH 214 NM.

1, CEFRADINE; 2, CEFTIZOXIME; 3, CEFOTOXIME; 4, CEFUROXIME; 5, CEFACLOR; 6, CEFADRO-XIL; 7, CEFTRIAXONE AND 8, CEFIXIME ⁶⁶.

DISCUSSION & CONCLUSION: This review contains different analysis methods includes some chromatographic methods especially HPLC, HPTLC and some capillary electrophoresis methods of analysis.

The different methods of analysis are specifically studied about the analytical parameters which include linearity, range, accuracy, precision, LOD, LOQ and robustness of the method along with combination of more than one antibiotic from formulation and biological fluids.

Analysis of antibiotics present in formulated and unformulated samples demands for a highly specific and rapid method as many antibiotics (e.g. β-lactams) have serious stability problems. This has made HPLC preferred over other traditional microbiological methods. Also chromatographic techniques are usually sensitive enough for most antibiotics as they achieve a limit of quantification (LOQ) of 0.3-0.5ug/ml. Sensitivity can be further enhanced by coupling it with fluorimetric, electrochemical or mass-spectrometric detection methods. By using different types of columns and varying combinations of solvent systems, scope of HPLC method can be further expanded to a wide range of samples, although reversed phase columns were used more frequently.

The purpose of this study is to find out the methods which are giving good results like precision. Some of these methods are used at very low concentration range of linearity.  HPLC, HPTLC, UV spectrophotometric, capillary zone electrophoresis methods are commonly used for determining drugs in pre and final pharmaceutical products. Some reported methods are suitable for the identification and quantification of the cephalosporins at many ranges of analysis.

In conclusion, the different HPLC method has been successfully used on a routine basis and allows the quantification of the drug in pharmaceutical formulations and body fluids in a short analytical time.

These methods are sensitive, simple, fast, ease extraction procedure and possess excellent linearity and precision characteristics. These observations made it possible to anticipate the use of this method as an official procedure.

The data of validation also shows that the proposed methods are selective, linearity, precise and accurate with low LOD, LOQ values and adequate to determine the content of drug substance in formulated, non formulated products and in body fluids.

The HPTLC method by V. Jagapathi Raju et al., has shows a method for analysis of cephalosporin in tablets with nano gram level and high precision value. H. Fujitomo et al., proposed two methods for the determination of cephalosporins in oral suspension with little change in analysis range and variation in method.

The HPLC is more sensitive, linear, precision and high accurate method. Lisoni et al., showed that the analysis of cefatanet proxitil in drug substance and powder form with very low precision and high % of recovery. The LOD and LOQ value of the said method is also low concentration.

Masoom Raza et al., and Ravi Shankar et al., develop a method which can work on ppm level of concentration with high accuracy and precision with low LOD, LOQ value. Some HPLC methods can be used for the multi-component analysis with 7 - 10 cephalosporins at a time.

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