Spectrophotometric Methods As Solutions To
7Spectrophotometric Methods as Solutions toPharmaceutical Analysis of β-Lactam AntibioticsJudyta Cielecka-Piontek1, Przemysław Zalewski1,Anna Krause2 and Marek Milewski21Poznan2PozLabUniversity of Medical Sciences, Department of Pharmaceutical ChemistryContract Research Organization at Centre of Transfer of Medical TechnologiesPoland1. IntroductionFollowing the discovery of the first analog of penicillin by A. Fleming (1929), the -lactamantibiotics are still a developing group of chemotherapeutics and are used in treatment ofmajority of diseases with bacterial etiology. -lactam antibiotics have a broad spectrum ofantibacterial activity, favourable pharmacokinetic parameters and low side effects. In lactam therapy two main problems are still current. The increasing resistance of somebacterial strains which implicates necessity to combine the therapy with inhibitors of lactamases and other chemotherapeutics. The second problem of therapy of -lactamantibiotics is their significant instability [1-3]. The analogs from that group are easilydegraded in aqueous solutions and in solid state. They are a special group of drugs becauseparallel to losing the antibacterial efficiency, the strong allergic properties can also appear asa results of their degradation. Therefore in terms of quality control, the stability of -lactamantibiotics in solutions was widely studied. The evaluation of stability concerned also thestudies of their metabolites and intravenous solutions after preparations of pharmaceuticaldosage forms. Moreover, the evaluation of concentration changes during storage ofsubstance in solid state was also conducted. As problem of the instability of some -lactamanalogs has been solved their oral administration is possible. An intake of oral formulationsis connected with appearance of excipients, which can influence rate of degradation andcause formation of different degradation products.The common element of chemical structure of all -lactam antibiotics is five-membered lactam ring. Currently, higher significance in treatment have derivatives in which the lactam ring is fused to: thiazolidine ring in penam analogs,2,3-dihydro-2H-1,3-tiazine ring in cephem analogs,2,3-dihydro-1H-pyrrole in carbapenem analogs,2,3-dihydrotiazole in penem analogs (Fig .1).These connections implicate the different intra-ring stress. The presence of sulphur atomand/or double bonds influence on length of bond and intra-molecular angle in molecule of-lactam analog. Finally for some derivatives, the differences in stability are noticeable.Additionally, the factor distinguishing a stability of derivatives of -lactam analogs arewww.intechopen.com
110Macro to Nano SpectroscopyCOORCOORONCH3SROCH3RNPenam analogSRCephem analogCOOHCOOHOONNRRRRCarbapenem analogRSThiopenem analogFig. 1. Chemical structure of penam, cephem, carbapenem and thiopenem nuclei.chemical structures of substituents at C2, C3, C5, C6, and C7. The amount and type ofdegradation products of -lactam antibiotics often depend on affecting factors (solvents,concentration of substance and hydrogen ions, temperature). Moreover, most of the lactam antibiotics obtained by chemical synthesis or fermentation contain impurities beingremnants of the process. In the development of analytical methods for the determination of-lactam antibiotics, selectivity is a fundamental validation parameter. A reliable, selectivemethod is expected to allow separation and determination of parental substance in thepresence of related ones. Current International Conference on Harmonization (ICH)guidelines require the development of analytical methods permitting analysis in thepresence of related products (Q1A–R2) [4]. These requirements are restrictions but alsochallenges during the development of analytical methods for the determination of -lactamantibiotics. The problem of the overlapping of the “background” originating from relatedproducts (impurities, degradation products and metabolites) and/or the presence of otheractive substances in a sample (inhibitors of -lactamases, other drugs) was solved during thedetermination of -lactam antibiotics by using chromatographic techniques (highperformance liquid chromatography, thin layer chromatography). On the other hand, searchof new solutions and analytical methods, especially being in accordance with the “greenchemistry” concept, is very important and up-to-date. Analytical methods based ondetermination of spectrophotometric properties of -lactam analogs are a developing tools intheir analysis. Non-destructive investigations of -lactam analogs, did not producing residues,were reported in fields of many spectrophotometric methods. A few methods of determinationof -lactam analogs were developed by using infrared spectrophotometry enriched bychemometric procedures [5-6]. Most of all analytical methods for the determination of -lactamanalogs were developed in range of visible and ultraviolet radiations. Desired, selective signalswere possible to obtain by application of following techniques: as spectrophotometric methods direct spectrophotometry direct spectrophotometry enriched by chemometric procedures derivative spetrophotometry derivative spectrotometry enriched by chemometric proceduresas visible spectrophotometric methods measurement of absorption of species being a result of reaction between analyteand derivatizating reagent measurement of absorption of species being a result of reaction betweendegradation products of analyte and derivatizating reagent (Table 1).www.intechopen.com
111Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of -Lactam AntibioticsDerivativeAnalog of penamUltraviolet regionVisible region1231*2* Analog of cephemI generation II generationIII generation Analog of penam IV generationAnalog of carbapenem 1. direct spectrophotometry enriched by chemometric procedures2. derivative spetrophotometry3. derivative spectrotometry enriched by chemometric procedures1.* measurement of absorption of species being a result of reaction between analyte andderivatizating reagent2.* measurement of absorption of species being a result of reaction between degradationproducts of analyte and derivatizating reagentTable 1. Possibilities of application of visible and ultraviolet spectrophotometricdeterminations for analysis of -lactam antibiotics in the period of time 1994–2011.2. Spectrophotometric methods for determination of β-lactam antibiotics2.1 Direct spectrophotometrySpectra of -lactam antibiotics recorded by using direct spectrophotometry do not havedesired selectivity due to the presence of related products. A comparison of sharp zeroorder spectra and/or value of absorption maxima for some -lactam analogs with onesobtained for CRS (chemical reference substance) is recommended by pharmacopeias for antheir identification [7] . Lack of desired absorbing species in chemical structure of penamanalog often do not allow to apply direct spectrophotometry even for qualitative studies ofsubstance of high purity.Paradoxically, the significant instability of analogs can sometimes solve this problem due toformation of degradation products that can absorb ultraviolet radiation permittingdetermination of parental substance.Significant susceptibility of -lactam analogs to degradation in basic medium was reportedduring analysis of cephem analogs. It was confirmed that formation of piperazine-2,5-dionewww.intechopen.com
112Macro to Nano Spectroscopyderivative, peak at 340 nm, was possible via intra-molecular nucleophilic attack of theprimary amine from the side chain on -lactam ring (pH 11 was required) (Fig. 2) [8].ONHSHNHNOClCOO-products‘The degradation products of cefactor - piperazine- The spectrum of piperazine-2,5-dione derivative of2,5-dione derivative.cefaclor.Fig. 2. Chemical structures of degradation products of cefaclor (1.0 mmol/l) formed at pH11.0 and its spectrum [8].The degradation of penam analogs in acidic conditions was also a base forspectrophotometric determination. As it is shown in Fig. 3, different pathways ofdegradation (including enzymatic one) can lead to obtaining absorbing species in the rangeof ultraviolet radiation. As a results of chemical degradation of penam analog in acidicconditions, the penicilloic acid, penillic acid and penicillenic acid are formed and absorb theultraviolet radiation in the range 320–360 nm, respectively [9]. While during the enzymaticdegradation under the influence of penicillin acylase, D-4-hydroxyphenylglycine (D-HPhG)and 6-aminopenicillanic acid are formed. Then the D-HPhG was catalyzed by Dphenylglycine aminotransferase to form L-glutamate and hydroxybenzoylformate whichstrongly absorb UV light at 335 nm [10].AmoxicillinHHNRHHSO-H CH3CH3NHHOHNOHHHCOOHHNRCH3OHSHNOPenicillenic enylglycineOHHHHS6-aminopenicillinic acidOOHOHOOCPenicilloic acidSCH3CHROOH3OHOHOHOHPenillic acidChemical degradation of penicillin in acidicconditions2-oxoglutaric Penamaldic acidNN CH3COOHONH2OHHSHNOCH3HNHNRCH3CH3CH3OHO HNSNOCOOHHH2N CH3NOOHSNRO NH2OOHOHOHO4-hydroxybenzoylformic acidL-glutamic acidEnzymatic degradation of amoxicillin usingbienzymatic factorsFig. 3. The pathways of obtaining of absorbing degradation products of penam analog [9-10].www.intechopen.com
Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of -Lactam Antibiotics1132.2 Direct spectrophotometry enriched by chemometric proceduresThe other way of improving the selectivity of direct spectrophotometry for thedetermination of -lactam antibiotics is the enrichment of data analysis by chemometricprocedures. A literature review revealed the application of the following determinations of-lactam antibiotics enriched by chemometric procedures that solved the problem ofspectral overlap without additional separation techniques at the stage of samplepreparation, were used: a separation of analog of cephem in the presence of impurities originating from synthesis(e.g., cephalexin in the presence of 7-aminocephalosporanic acid and acid-induceddegradation products) using H-point standard additions method (HPSAM) [11]determination of analog of penam in the presence of other drugs (e.g., amoxicillin in thepresence of diclofenac) using partial least squares (PLS) regression analysis [12]determination of analog of cephem in the presence of alkali-induced degradationproducts using full spectrum quantitation (FSQ) (e.g., cefotaxime, ceftazidine,ceftiaxome, in the presence of degradation products) [13].Each chemometric method relies on different tools of regression analysis of multicomponentsystem permitting simultaneous determination of two or more components.The determination of -lactam analyte in the presence of known and unknown inferenceswas possible by the application of HPSAM procedure, where analyte concentration iscalculated from the following equation:A0 -b0 (A, -b)M λ1 - M(λ2 ), -CX (A -b)(1)M λ1 - M(λ2 )where b0 and A0 are the absorbance values for -lactam analyte, b and A, ones for theinterferent, at λ1 and λ2 and M λ , M λ are slopes of plots at selected wavelengths.In PLS technique, analytical sensibility was defined as SENk‖σr ‖where SENk ‖bk‖, σr is avalue estimated from standard deviation of blank samples, bk value is a vector of theregression coefficient for the k analytes and k is a number of components in a mixture.The FSQ technique during a determination of -lactam antibiotics applies Fourier preprocessing of the entire absorption spectra of the individual -lactam analogs with theirdegradation products at variable concentration to calculate matrix calibration coefficients.2.3 Derivative spectrophotometryA derivative spectrophotometry using derivatives of absorbance with respect to wavelength f λ)’, second f λ ; third f λ ; respectively) is a suitable tool for(firstovercoming the overlapping spectra problem in analysis of many -lactam analogs.Possibility of application of derivative spectrophotometry with zero-crossing point is widelyused in analysis of all -lactam analogs. The direct correlation between order of usedderivative spectrophotometry and similarities of chemical structures of nuclei of -lactamanalogs has not been observed, e.g., both second-derivative and first-derivative weredeveloped for cephem analogs including the same nuclei (Fig. 4).www.intechopen.com
114Macro to Nano SpectroscopyFirst-derivative spectrum of cefprozil (1 mg%) in0.1 M HCl.Second-derivative spectrum of cefepime (20µg/ml) in water.Fig. 4. The application of derivative spectrophotometry for analysis of cephem analogs [15-16].The application of derivative spectrophotometry for determination of -lactam antibioticswas used in the following areas: a separation and determination of penam/cephem analogs and inhibitors of lactamase in aqueous solution (e.g., determination of ampicillin sodium in the presenceof sulbactam sodium; determination of cefsulodin in the presence of clavulanic acid)[14-15]a separation and determination of cephem/carbapenem analog and excipients used inparenteral pharmaceutical dosage forms (e.g., determination of cefepime in thepresence of L-argininie) [16]a separation and determination of cephem analog and its degradation products (e.g.,determination of cefprozil in the presence of its degradation products) [17]a separation and determination of cephem analog and related compounds from thesynthesis (e.g., determination of triethylammonium salt of cefotaxime in the presence of2-mercaptobenzothiazole) [18]a separation and determination of penam/cephem/carbapenem analogs in biologicalmatrix (e.g., determination of amoxicillin, cefuroxime, imipenem in urine) [19].The separation of often structurally very similar species (e.g., two analogs of cephem,cephem analog and its impurities from synthesis or carbapenem analog and its degradationproducts) was possible by using derivative spectrophotometry (Fig. 5).It was proved that the derivative spectrophotometry can be recommended as a method forroutine control analysis of pharmaceutical preparation of -lactam antibiotics. Derivativespectrophotometry ensured the rapid analysis of parenteral dosage forms and also removeda “background” excipients in oral pharmaceutical dosage forms.The special potency of derivative spectrophotometry was possibility of its usage indetermination of -lactam analogs in biological matrix. In this case, to meet therequirements of analytical methods, the selectivity had to be extended in regard withinterference of biological endogenous components. It was noticed that the selectivedetermination of penam/cephem/carbapenem in the presence of metabolites (open-ringdegradation product) and endogenous substance of urine was possible to achieve.www.intechopen.com
Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of -Lactam AntibioticsChemical structures of components of mixture115Derivative spectraThe separation of -lactam analog with the same rst-derivative of spectrum of cephalothinCL (20.0 µg/ml) and cefoxitin CX (20.0µg/ml) and mixtures of each componentM)NH2HNSSHH3COSOHOThe separation of -lactam analog and impurties from synthesisFirst derivative spectrum oftriethylammonium salt of cefotaxime A (402µg/ml) , 7-aminocephalosporanic acid B (40mercaptobenzothiazoleµg/ml), S-(2-benzothazolyl)2-amino- (methoxyimino-4-thiazoleethanethioate) C(0.03 µg/ml).Triethylammoniumsalt of cefotaximeOCOONaCH3ONONNCH3SHHNNSH2NSOSThe separation of -lactam analog and degradation productsOpen-ring hydrolysisproducts of biapenemBiapenemFirst-derivative spectra of biapenem duringdegradation at 313 K: in HClOHHOHH HCH 3H 3CSNO NNHNNOHOHOOCSNNNOOOFig. 5. Separation of some -lactam analogs using derivative spectra [18, 20-21].www.intechopen.com
116Macro to Nano Spectroscopy2.4 Derivative spectrophotometry enriched by chemometric proceduresThe application of chemometric procedures coupled with derivative spectroscopy permitsachievement of higher selectivity in determination of -lactam antibiotics. Currently,chemometric procedures based on the estimated ratio of spectra derivative for the selectivedetermination of -lactam analogs are the most common. It was proved that the applicationof the ratio of different-order spectra derivatives permitted the separation of binary andtertiary mixtures of -lactam antibiotics [22]. During the determination of concentrations ofthree components (e.g., penicillin-G sodium, penicillin-G procain and dihydrostreptomycinsulphate salts) in a mixture the equation describing the ratio spectra derivativespetrophotometry is as follows:/,, Cb,/,/, Cc,(2)iswhere, is the absorbance of the ternary mixture of a, b and c at wavelength λ,,the absorbance of pure component at wavelength λ, Cb and Cc – are the concentrations of band c, kb,λ and kc,λ are the products of the molar absorption coefficient of b at wavelength λand the thickness of the absorption cell. Equation 2 is divided by Cb while divisor can be anycomponent of ternary mixture (Fig. 6):Equation 3 is drawn:,,//,, J Cc d d Ccd,,//,,,,///dλ,,(3)(4)Finally, after the next derivation J (as the left side of equation 3), is proportional to the Ccvalue and can be used to determine concentration of component in the ternary mixture(when , and , are fixed) [23].Depending on chemometric procedure, the selective determination of following analogs waspossible: a separation and determination of carbapenem and degradant (e.g., the determinationof ertapenem and its degradant) when the substraction technique was used [24-25]a separation and determination of penam and cephem analogs (e.g., the determinationof penicillin-G, penicillin-G procain in the presence of dihydrostreptomycin sulphatesalts or the determination of cefotaxime and cefadroxil), cephem analog and inhibitor of-lactamases (e.g., the determination of cephradine and clavulanic acid) andcarbapenem analog and degradation products (e.g., the determination of meropenemand its degradant) when ratio spectra of derivative with all orders were used [26-29]a separation and determination of carbapenem and degradation products (e.g., thedetermination of ertapenem) when the substraction technique was used [30]a separation and determination of carbapenem and degradation products (e.g., thedetermination of ertapenem) when the Kraise’s method was used [31]a separation and determination of penam analogs (e.g., the determination of ampicillinand flucloxacillin) when multivariate methods (classical least squares and principlecomponent regression) were used.www.intechopen.com
Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of -Lactam Antibiotics117First order derivative ratio spectra of dicloxacillin sodium 50.0–400.0mg/l using of 68.47 µg/ml ampicillin sodium as a divisor [26]First order derivative ratio spectra of meropenem 4.0–60.0 µg/l usingof 32 µg/ml degradate as a divisor [29]Fig. 6. The application of ratio spectra of derivative spectrophotometry in analysis of lactam antibiotics.3. Visible spectrophotometric methods for determination of β-lactamantibioticsThe -lactam analogs itselfs do not absorb in visible region of radiation. However, manyvisible spectrophotometric methods were developed for the determination of -lactamantibiotics using the effect of formation of “species” giving signals in visible region as theresult of chemical derivatization (Fig. 7).Absorption spectra of amoxicillin trihydrate(a) 30 µg/ml; (b) after reaction with Fe (III) [31]Fig. 7. The application of derivatization for detetmination of penam analog.Formation of “species” absorbing visible radiation can be a result of reactions of chemicalreagents with: -lactam analogwww.intechopen.com
118 Macro to Nano Spectroscopydegradation product of some -lactam analog.Above-mentioned methods were described
Spectrophotometric Methods as Solutions to Pharmaceutical Analysis of à -Lactam Antibiotics 113 2.2 Direct spectrophotometry enrich ed by chemometric procedures The other way of improving the selectivity of direct spectrophotometry for the determination of Ã-lactam antibiotics is the enrichment of
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