Development of TLC Chromatographic-Densitometric Procedure for Qualitative and Quantitative Analysis of Ceftobiprole
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processes Article Development of TLC Chromatographic-Densitometric Procedure for Qualitative and Quantitative Analysis of Ceftobiprole Zaneta˙ Binert-Kusztal, Małgorzata Starek , Joanna Zandarek˙ and Monika D ˛abrowska* Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Medical College, Jagiellonian University, 9 Medyczna St., 30-688 Kraków, Poland; [email protected] (Z.B.-K.);˙ [email protected] (M.S.); [email protected] (J.Z.)˙ * Correspondence: [email protected] Abstract: Currently, there is still a need for broad-spectrum antibiotics. The new cephalosporin antibiotics include, among others, ceftobiprole, a fifth-generation gram-positive cephalosporin, active against Staphylococcus aureus methicillin agonist (MRSA). The main focus of the work was to optimize the conditions of ceftobiprole qualitative determination and to validate the developed procedure according to ICH guidelines. As a result of the optimization process, HPTLC Cellulose chromatographic plates as a stationary phase and a mixture consisting of ethanol:2-propanol: glacial acetic acid: water (4:4:1:3, v/v/v/v) as a mobile phase were chosen. The densitometric detection was carried out at maximum absorbance of ceftobiprole (λ = 232 nm). Next, the validation process of the developed procedure was carried out. The relative standard deviation (RSD) for precision was less than 1.65%, which proves the high compatibility of the results, as well as the LOD = 0.0257 µg/spot and LOQ = 0.0779 µg/spot values, which also confirm the high sensitivity of the procedure. The Citation: Binert-Kusztal, Z.;˙ Starek, usefulness of the developed method for the stability studies of ceftobiprole was analyzed. Study M.; Zandarek,˙ J.; D ˛abrowska, M. was carried out under stress conditions, i.e., acid and alkaline environments, exposure to radiation Development of TLC ◦ Chromatographic-Densitometric imitating sunlight and high temperature (40–60 C). It was found that cefotbiprole is unstable in Procedure for Qualitative and an alkaline environment and during exposure to UV-VIS radiation. Moreover, the lipophilicity Quantitative Analysis of Ceftobiprole. parameter, as a main physicochemical property of the biologically active compound, was determined Processes 2021, 9, 708. https://doi. using experimental and computational methods. org/10.3390/pr9040708 Keywords: ceftobiprole; TLC method; validation of the method; stability testing; lipophilicity Academic Editor: Ofelia Anjos Received: 24 March 2021 Accepted: 14 April 2021 1. Introduction Published: 16 April 2021 Cephalosporins, which belong to the group of semi-synthetic β-lactam antibiotics, have their origins as early as in the middle of the 20th century. The first research work Publisher’s Note: MDPI stays neutral on their use in the therapeutics was carried out at the turn of the 19th and 20th century. with regard to jurisdictional claims in The discoverer of cephalosporins is considered to be the Italian scientist Giuseppe Brotzu published maps and institutional affil- iations. (1895–1976), professor and microbiologist of the Medical Faculty in Cagliari (Sardinia). In 1945 the scientist became interested in people who swam in rivers polluted with faeces or consumed shellfish fished from them. These people did not get sick, while in other parts of Italy, there was a typhoid pandemic—at the time known as abdominal typhus. On this basis, Brotzu hypothesised that these sewage systems probably contain microorganisms Copyright: © 2021 by the authors. that inhibit the growth of pathogenic intestinal bacteria—Salmonella typhi—that can prevent Licensee MDPI, Basel, Switzerland. the spread of typhus [1]. In 1948, together with Howard Florey, they discovered that a This article is an open access article strain of the fungus can produce seven antibiotics—five varieties of cephalosporin P, N distributed under the terms and conditions of the Creative Commons and C. The latter is considered to be the most important, as it is on its basis that all the Attribution (CC BY) license (https:// cephalosporin antibiotics used today are created. Despite its considerable advantages, creativecommons.org/licenses/by/ such as high resistance to staphylococcal penicillinase enzyme and hydrolysis in an acidic 4.0/). environment, like the rest of the natural penicillin, it has unfortunately not been used in Processes 2021, 9, 708. https://doi.org/10.3390/pr9040708 https://www.mdpi.com/journal/processes Processes 2021, 9, 708 2 of 18 Processes 2021, 9, 708 2 of 18 the therapeutic field due to its low activity. For this reason, we are currently using its semi-syntheticsemi-synthetic derivatives derivatives obtained obtained from from 7- 7-aminocephalosporicaminocephalosporic acid, whichwhich waswas formedformed as asa a product product of of the the hydrolytic hydrolytic decomposition decomposit ofion cephalosporin of cephalosporin C. The C. basis Thefor basis the structurefor the structureof cephalosporins of cephalosporins is the aforementioned is the aforem 7-aminocephalosporicentioned 7-aminocephalosporic acid, which acid, contains which the containscharacteristic the characteristicD2-cephem Δ system2-cephem (Figure system1)[ 2(Figure]. 1) [2]. (a) (b) FigureFigure 1. 1.ChemicalChemical structure structure of of7-am 7-aminocephalosporicinocephalosporic acid acid (a) ( aand) and Δ2-cephemD2-cephem (b ().b ). CephalosporinsCephalosporins belong belong to tothe the well-tolerated well-tolerated group group ofof antibiotics, antibiotics, which which is isextremely extremely diversediverse and and can can be be grouped grouped into into five five classes classes in in terms terms of of their their activity. activity. The The classification classification intointo a aparticular particular generation generation is isbased based on on the the differences differences in in action: action: bactericidal bactericidal effect effect on on Gram-negativeGram-negative bacteria bacteria (including (including BacteroidesBacteroides andand Gram-positive Gram-positive anaerobics), anaerobics), ProteuProteus, s, β PseudomonasPseudomonas, reaching, reaching the the cerebrospinal cerebrospinal fluid fluid and and sensitivity sensitivity to to β-lactamases.-lactamases. It Itcan can be be assumedassumed that that a lower a lower generation generation has has greater greater activity activity against against Gram-positive Gram-positive bacteria, bacteria, and and a highera higher generation generation has has greater greater activity activity ag againstainst Gram-negative Gram-negative bacteria. bacteria. There There are are some some exceptions, such as cefepime, which is included in the fourth generation, and its activity exceptions, such as cefepime, which is included in the fourth generation, and its activity against Gram-positive bacteria is comparable to that of the first generation, and against against Gram-positive bacteria is comparable to that of the first generation, and against Gram-negative bacteria is equal to the third generation [3]. Currently, in a hospital and Gram-negative bacteria is equal to the third generation [3]. Currently, in a hospital and out-of-hospital treatment, there are infections caused by resistant strains of bacteria that are out-of-hospital treatment, there are infections caused by resistant strains of bacteria that insensitive to methicillin. These are mainly Staphylococcus aureus, Streptococcus pneumoniae, are insensitive to methicillin. These are mainly Staphylococcus aureus, Streptococcus Enterococcus spp. The use of similar chemotherapy regimens varies according to the type of pneumoniae, Enterococcus spp. The use of similar chemotherapy regimens varies according hospital or out-of-hospital infection, as using the same regimens is not equally effective. to the type of hospital or out-of-hospital infection, as using the same regimens is not Therefore, the need to look for new antibacterial compounds that will be able to overcome equally effective. Therefore, the need to look for new antibacterial compounds that will the existing resistance mechanisms is growing [4]. The new group of cephalosporin be able to overcome the existing resistance mechanisms is growing [4]. The new group of antibiotics includes the fifth generation. Unlike the fourth generation, these compounds cephalosporin antibiotics includes the fifth generation. Unlike the fourth generation, these have a broad spectrum of effect on Gram-positive bacteria and an extended spectrum of compoundsresistance tohave methicillin, a broad i.e.,spectrum MRSA of and effect other on resistant Gram-positive enterococci, bacteria mainly and VRSA an extended (S. aureus spectrumresistant of to resistance vancomycin) to methicillin, [5]. i.e., MRSA and other resistant enterococci, mainly VRSA (AnS. aureus example resistant of generation to vancomycin) V cephalosporin [5]. is ceftaroline. It exhibits a wide spectrum of activityAn example against of generation Gram-positive V cephalosporin and Gram-negative is ceftaroline. pathogens It exhibits and most a wide importantly spectrum it ofshows activity activity against against Gram-positive VISA (S. aureusand Gram-negativestrain moderately pathogens resistant and to most vancomycin), importantly hVISA it shows(precursor activity of against VISA), VISA MRSA (S. andaureus VRSA. strain It moderately has been provenresistant effective to vancomycin), against bacterialhVISA (precursorpathogens of of VISA), the respiratory MRSA and system, VRSA. e.g., It S.has pneumoniae been proven[6]. Ceftarolineeffective against