Solid-state stability study of meropenem – solutions based on spectrophotometric analysis
© Cielecka-Piontek et al.; licensee Chemistry Central Ltd. 2013
Received: 13 February 2013
Accepted: 27 May 2013
Published: 8 June 2013
B-Lactam antibiotics are still the most common group of chemotherapeutic drugs that are used in the treatment of bacterial infections. However, due to their chemical instability the potential to apply them as oral pharmacotherapeutics is often limited and so it is vital to employ suitable non-destructive analytical methods. Hence, in order to analyze such labile drugs as β-lactam analogs, the application of rapid and reliable analytical techniques which do not require transferring to solutions or using organic solvents, following the current green approach to pharmaceutical analysis, is necessary. The main objective of the present research was to develop analytical methods for the evaluation of changes in meropenem in the solid state during a stability study.
The UV, FT-IR and Raman spectra of meropenem were recorded during a solid-state stability study. The optimum molecular geometry, harmonic vibrational frequencies, infrared intensities and Raman scattering activities were calculated according to the density-functional theory (DFT/B3LYP method) with a 6-31G(d,p) basis set. As the differences between the observed and scaled wavenumber values were small, a detailed interpretation of the FT-IR and Raman spectra was possible for non-degraded and degraded samples of meropenem. The problem of the overlapping spectra of meropenem and ring-containing degradation products was solved by measuring changes in the values of the first-derivative amplitudes of the zero-order spectra of aqueous solutions of meropenem. Also, molecular electrostatic potential (MEP), front molecular orbitals (FMOs) and the gap potential between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were determined.
Based on the findings of this work, it appears possible to use time-saving and reliable spectrophotometric analytical methods, supported by quantum-chemical calculations, for solid-state stability investigations of meropenem. The methods developed for this study may be considered a novel, green solution to pharmaceutical analysis of labile drugs – an alternative for the recommended chromatographic procedures.
The chemical instability of drugs may reduce pharmacotherapeutic possibilities and affect the selection criteria when deciding on analytical methods for the quality control of labile medicines. In that group of drugs, the problem of the susceptibility of β-lactam antibiotics to degradation in solutions and in the solid state has often been reported [1–3]. Since oral administration of antibiotics is not possible in the case of some β-lactam analogs, the remaining solution is the parenteral route, which involves the risk of unwanted side effects [4, 5]. In view of the significant lability of β-lactam analogs, the application of suitable analytical methods is of utmost importance. The quality control of drugs in the solid state is expected to rely on methods ensuring minimization of sample transformation, for example elimination of sample derivatization or limitation of reactions with chemical reagents. As it has been established, organic solvents affect the rate of degradation of β-lactam antibiotics as well as the formation of degradation products [6–8]. Therefore, the methodology of studying β-lactam antibiotics in solutions is required to eliminate such reagents that provide inconsistent environments.
Therefore, in light of the therapeutic potential of meropenem, including its bactericidal activity against resistant strains, it is vital to develop methods of quality control able to take into account its significant susceptibility to degradation. Meropenem is known to be very unstable in aqueous solutions and in the solid state . Depending on affecting factors and the form of meropenem, different degradants are formed. For example, during the degradation of meropenem in acidic solutions degradation products with an open β-lactam ring are formed whereas thermal degradation of meropenem leads to the formation of 4-methyl-3-(1H-pyrrol-3-ylsulfanyl)-5H-pyrrole-2-carboxylic acid. Mendez et al. suggested that the modification of the side chain of meropenem in the direction of a pyrrolic-ring degradation product is a result of decarboxylation and aromatization of the pyrrolidine ring and hydrolysis of the β-lactam ring, considered an intermediate stage of degradation [19, 20]. Also, it was observed that during solid-state stability studies of carbapenem analogs different degradants were formed during thermolysis as a consequence of the highly strained fused ring system [21, 22].
The most common techniques for the determination of meropenem during stability studies are based on chromatography. Due to the insolubility of carbapenems in organic solvents, only reversed-phase (RP) liquid chromatography is used in the analysis of meropenem . Since the degradation of β-lactam analogs is known to be additionally affected by solvolysis, it appears justified to eliminate the use of organic solvents . Therefore, in order to ensure the reliability of determination, it is vital to use such analytical methods that do not require the application of organic solvents or the placement of samples in media other than target intravenous solutions during studies of meropenem in the solid state. The analytical procedures that overcome those problems are spectral methods. They allow a rapid analysis of meropenem in the solid state (FT-IR, Raman spectroscopy) and in aqueous solutions, relying exclusively on dissolution in water samples (UV). For the determination of carbapenems, including meropenem, only first-derivative, first-derivative spectrum ratio and third-derivative UV spectroscopies were used, which permitted eliminating interferences originating from degradation products with an open β-lactam ring and dimers [20, 25, 26] formed in aqueous solutions. A literature review has not demonstrated the use of spectroscopic methods as an alternative mode of quality control of meropenem in the solid state.
The main objective of the present study was to develop and apply spectrophotometric methods for the quality control of meropenem in the solid state, which involved investigating vibrations within the bonds in a meropenem molecule and their changes during degradation by using spectral methods (FT-IR, Raman spectroscopy) on the basis of chemical-quantum calculations and developing a selective analytical method for the determination of changes in meropenem concentrations in the solid state with the use of water instead of organic solvents.
Substances and spectroscopic measurements
The meropenem reference standards (purity > 98%) were supplied by Pharmachem International Co., (China). The commercial preparation of meropenem, Meronem®, contained meropenem 500 mg and sodium carbonate 140 mg.
The first derivatives of ultraviolet spectra of meropenem were recorded by using a UV–VIS Lambda 20 (PerkinElmer) spectrophotometer equipped with 1.0 cm-in-width quartz cells and controlled via the UV WinLab software. Water was used as solvent. The vibrational infrared spectra of meropenem were recorded between 400 and 7000 cm-1 in powder, at room temperature, with an FT-IR Bruker Equinox 55 spectrometer equipped with a Bruker Hyperion 1000 microscope. The Raman scattering spectra were obtained with a LabRAM HR800 spectrometer (Horiba Jobin Yvon) with laser excitation λexc = 633 nm (He-Ne laser). In each case the power of the laser beam at the sample was less than 1mW to avoid damaging the sample. High quality pure water was prepared using an Exil SA 67120 purification system (Millipore).
For stability studies, 5 mg samples of meropenem were weighed into 5 ml vials. To evaluate their stability at increased air humidity, they were placed in heat chambers at 313 K in desiccators containing saturated solutions of sodium chloride (relative humidity (RH) ≈ 76.4%). The changes of meropenem concentration were studied after 15; 30; 45; 60; 75; 90; 105 and 120 minutes of degradation. To evaluate the stability of meropenem in dry air, the vials were immersed in a sand bath placed in heat chambers at 323 K. The sand was first dried, and then kept at 323 K with preventing the absorption of water vapor from the environment. The changes of meropenem concentration were studied after 2.5; 5; 7.5; 10; 12.5; 15; 17.5; and 20 hours of degradation.
The UV method was validated according to the International Conference on Harmonization Guidelines  in terms of linearity, precision, accuracy, and limits of detection and quantitation for meropenem were established.
The derivative spectrophotometric method was based on the transformation of the zero-order spectrum of meropenem into its first derivative (∆A/∆λ) by using the UV WinLab software. In order to interpret the experimental results of IR absorption and Raman scattering, quantum-chemical calculations were performed by using the Gaussian 03 package . The GaussView software was utilized to propose the initial geometry of the investigated molecules and to visually inspect the normal modes. The molecular geometries were optimized by means of a density functional theory (DFT) method with the B3LYP hybrid functional and a 6-31G(d,p) basis set.
Results and discussion
The FT-IR and Raman spectra of non-degraded and degraded samples of meropenem obtained in this study allowed characterization of molecular vibrations as well as changes in its structure resulting from degradation during storage in the solid state. The calculated IR and Raman scattering spectra, obtained by means of the density functional theory, were used as reference. The application of the first derivative of the zero-order UV spectra permitted determination of meropenem in the presence of 4-methyl-3-(1H-pyrrol-3-ylsulfanyl)-5H-pyrrole-2-carboxylic acid, which is a degradation product that can form under the recommended storage conditions for meropenem preparations . Additionally, the FMOs and the MEP of meropenem were determined based on quantum-chemical calculations. The following sections discuss spectral analysis and the findings of theoretical calculations.
Comparison of observed and calculated vibrational modes of meropenem before degradation
Wavenumber (cm -1 )
O-H b in carboxylic group
O-H b in carboxylic group
def. pyrrolidine ring + C-O-H b in carboxyl group
def. pyrrolidine ring + C-O-H b in carboxyl group
breathing β-lactam ring + C=O b in dimethylcarbomoyl group
carbonyl group b in carboxylic group
breathing β-lactam ring + C-N s between N and methyl group in dimethylcarbomoyl group
C-C between β-lactam and dimethylcarbomoyl group + C-N in β-lactam
C-C s in basis ring + CH3 w at pyrrolidine ring
C-C in β-lactam ring + CH t/w in trans-hydroxyethyl group
CH t/w in trans-hydroxyethyl group
CH t/w + C-N s in pyrrolidine ring
CH2t and methyl t in dimethylcarbomoyl group
CH t in CH2 and methyl group in dimethylcarbomoyl group
C-N s in pyrrolidine ring + C-O-H b in carboxyl group
C-H w at β-lactam ring
C-N s at 4 CH3 + C-H w
C-C s between pyrrolidine ring and C at COH group
C-H w in CH in trans-hydroxyethyl group + CH w in pyrrolidine ring
C-C s between carboxyl group and pyrrolidine ring + C-N s in basis ring + C-H sc in CH3 in trans-hydroxyethyl group and methyl group
C-N s between CO and CH3 in dimethylcarbomoyl group
C-H sc in dimethylcarbomoyl group
C-H w in dimethylcarbomoyl group
CH3 w/sc in dimethylcarbomoyl group
CH3 w/sc in dimethylcarbomoyl group
C=C s in pyrrolidine ring
C=O s in trans-hydroxyethyl group
C=O s in carboxyl group
C=O s in pyrrolidine ring
C-H s in dimethylcarbomoyl group
C-H s in dimethylcarbomoyl group
C-H s in trans-hydroxyethyl group group
C-H s in methyl group
By analyzing changes in the FT-IR spectra of meropenem exposed to increased relative air humidity (RH = 76.4%, T = 40°C) it was possible to determine a cleavage of the β-lactam ring. The observed changes were not characteristic and were expressed by the appearance of bands at 1747 and 1748 cm-1 at increased temperature and RH = 0% and at increased relative air humidity, respectively.
The identification and evaluation of qualitative changes in meropenem that may occur during storage in commercial packaging based on analyzing the FT-IR spectra of that carbapenem analog may be considered a time-efficient, cost-effective and reliable analytical tool.
UV spectral studies
By achieving the desired selectivity of determination of meropenem after applying first-derivative spectrophotometry, it was possible to analyze it quantitatively. The calibration curve was described by the equation y=(111,30 ±2,27) × (λ = 320 nm). The b values calculated from the equation y = ax + b were not significant. A good intra-day repeatability was determined (1.64%–2.29%) for the three levels (80%, 100% and 120%) of nominal concentration of meropenem in the linearity range 25–131 μg/mL. Inter-day repeatability also had acceptable values. The percentage recovery of meropenem ranged from 99.9% to 101.3% in a pharmaceutical dosage form. The limits of detection and quantitation, describing the smallest concentration of an analyte that can be reliably measured by an analytical procedure, were 3.58 μg/mL and 11.0 μg/mL, respectively.
The application of first-derivative spectrophotometry for the determination of meropenem may be a solution to the problem of overlapping bands. The achievement of required validation parameters and the advantages of spectrophotometric determination of meropenem make this method suitable for a routine analysis of that carbapenem analog, allowing its determination in the presence of solid-state degradation products.
The analysis of the optimized geometry of meropenem indicated that the intraring stress is a result of the fusion of the heterocyclic moieties with the substituents, which can be spatial obstacles to each other. Such molecular configuration of meropenem ensures its binding with PBP (penicillin-binding protein) enzymes that is responsible for antibacterial activity. At the same time that configuration determines the significant susceptibility to degradation of meropenem under the conditions of acid–base hydrolysis and in the presence of oxidizing factors as well as during thermolysis.
The proposed methods (UV, FT-IR and Raman spectroscopy) for the quality assessment of meropenem and its determination in the presence of degradation products formed during solid-state storage may be considered superior to commonly applied chromatographic techniques. The spectral and theoretical studies conducted in this work permitted evaluation of molecular changes in meropenem and characterization of the inter- and intra-molecular interactions observed during its storage in the solid state. The analysis of the frontier molecular orbitals and the molecular electrostatic potential revealed the sites prone to electrophilic and nucleophilic attacks in meropenem.
This study was supported by a grant from the Foundation for Polish Science (no. VENTURES/2011-8/7).
- Nicolau DP: Carbapenems: a potent class of antibiotics. Expert Opin Pharmacother. 2008, 9: 23-37. 10.1517/146565188.8.131.52.View ArticleGoogle Scholar
- Hurt M, Lamb H: Meropenem: a review of its use in patients in intensive care. Drugs. 2000, 59: 653-680. 10.2165/00003495-200059030-00016.View ArticleGoogle Scholar
- Zhanel G, Wiebe R, Dialy L, Thomsaon K, Rubistein E, Hoban D, Noreddin A, Karlowsky J: Comparative review of the carbapenems. Drugs. 2007, 67: 1027-1052. 10.2165/00003495-200767070-00006.View ArticleGoogle Scholar
- Edwards SJ, Emmas CE, Campbell HE: Systematic review comparing meropenem with imipenem pluscilastatin in the treatment of severe infections. Current Medical Research and Opinion. 2005, 21: 785-794. 10.1185/030079905X46223.View ArticleGoogle Scholar
- Deshpande AD, Baheti KG, Chatterjee NR: Degradation of β-lactam antibiotics. Curr Scien. 2004, 12: 1684-1695.Google Scholar
- Sajonz P, Wu Y, Natishan TK, McGachy NT, Tora DD: Challenges in the analytical method development and validation for an unstable active pharmaceutical ingredient. J Chrom Scien. 2006, 44: 132-140. 10.1093/chromsci/44.3.132.View ArticleGoogle Scholar
- Markovic BD, Dobricic VD, Vladimirov SM, Cudina OS, Savic VM, Karlikkovic-Rajic KD: Investigation of solvolysis kinetics of New synthesized fluocinolone acetonide C-21 esters—an In vitro model for prodrug activation. Molecules. 2011, 16: 2658-2671. 10.3390/molecules16032658.View ArticleGoogle Scholar
- Llinas A, Page MI: Intramolecular general acid catalysis in the aminolysis of beta-lactam antibiotics. Org Biomol Chem. 2004, 5: 651-654.View ArticleGoogle Scholar
- Cielecka-Piontek J, Michalska K, Zalewski P, Jelińska A: Recent advances in stability studies of carbapenems. Cur Pharm Anal. 2011, 7: 213-227. 10.2174/157341211797457989.View ArticleGoogle Scholar
- Kataoka H: New trends in samples preparation for clinical and pharmaceutical analysis. Tr Anal Chem. 2003, 22: 232-244. 10.1016/S0165-9936(03)00402-3.View ArticleGoogle Scholar
- Görög S: The changing face of pharmaceutical analysis. Tr Anal Chem. 2007, 27: 12-17.View ArticleGoogle Scholar
- Papp-Wallace KM, Endimiani A, Taracila MA, Bonomo RA: Carbapenems: past, present and future. Antimicrob Agents Chemother. 2011, 55: 4943-10.1128/AAC.00296-11.View ArticleGoogle Scholar
- Mohr JF: Update on the efficacy and tolerability of meropenem in the treatment of serious bacterial infections. Clin Infect Dis. 2008, 47: S41-S51. 10.1086/590065.View ArticleGoogle Scholar
- Jones RN, Sader HS, Fritsche TR: Comparative activity of doripenem and three other carbapenems tested against Gram-negative bacilli with various β-lactamase resistance mechanisms. Diagn Microbiol Infect Dis. 2005, 52: 71-74. 10.1016/j.diagmicrobio.2004.12.008.View ArticleGoogle Scholar
- Rhomberg PR, Jones RN: Contemporary activity of meropenem and comparator broad-spectrum agents: MYSTIC program report from the United States component (2005). Diagn Microbiol Infect Dis. 2007, 57: 207-215. 10.1016/j.diagmicrobio.2006.07.009.View ArticleGoogle Scholar
- Payen MC, Wit D, Martin C, Sergysels R, Muylle I, Laethem Y, Clumeck N: Clinical use of the meropenem-clavulanate combination for extensively drug-resistant tuberculosis. Inter J Tuber Lung Dis. 2012, 16: 558-560. 10.5588/ijtld.11.0414.View ArticleGoogle Scholar
- Hugonnet JE, Tremblay LW, Boshoff HI, Barry CL, Blanchard JS: Meropenem-clavulanate is effective against extensively drug-resistant mycobacterium tuberculosis. Science. 2009, 27: 1215-1218.View ArticleGoogle Scholar
- Cielecka-Piontek J, Zając M, Jelińska A: A comparison of the stability of ertapenem and meropenem in pharmaceutical preparations in solid state. J Phamraceutical Biomedical Analysis. 2008, 46: 52-57. 10.1016/j.jpba.2007.08.024.View ArticleGoogle Scholar
- Mendez A, Chagastelles P, Palma E, Nardi N, Schapoval E: Thermal and alkaline stability of meropenem: degradation products and cytotoxicity. Int J Pharm. 2008, 350: 95-102. 10.1016/j.ijpharm.2007.08.023.View ArticleGoogle Scholar
- Elragehy NA, Abdel-Moety EM, Hassan NY, Rezk MR: Stability-indicating determination of meropenem in presence of its degradation product. Talanta. 2008, 77: 28-32. 10.1016/j.talanta.2008.06.045.View ArticleGoogle Scholar
- Cielecka-Piontek J, Jelińska A, Dołhań A, Zalewski P: Kinetic and thermodynamic analysis of degradation of doripenem in solid state. Inter J Chem Kinet. 2012, 44: 722-728. 10.1002/kin.20722.View ArticleGoogle Scholar
- Sajonz P, Vailayay A, Sudah O, McPherson L, Capodanno V, Natishan TK, Helmy R, Antia FD: Development of a gradient eluation preparative high performance liquid chromatography method for the recovery of the antibiotics ertapenem from crystallization process streams. J Chrom A. 2008, 8: 365-372.Google Scholar
- Berthoin K, Le Duff C, Marchand-Brynaert J, Carryn S, Tulkens P: Stability of meropenem and doripenem solutions for administration by continuous infusion. J Antimicrob Chemother. 2010, 65: 1073-1075. 10.1093/jac/dkq044.View ArticleGoogle Scholar
- Cielecka-Piontek J, Michalska K, Zalewski P, Zasada S: Comparative review of analytical techniques for determination of carbapenems. Cur Anal Chem. 2012, 8: 91-115. 10.2174/157341112798472206.View ArticleGoogle Scholar
- Cielecka-Piontek J, Jelińska A: The UV-derivative spectrophotometry for the determination of doripenem in the presence of its degradation products. Acta Spectrochim A. 2010, 77: 554-557. 10.1016/j.saa.2010.06.019.View ArticleGoogle Scholar
- Cielecka-Piontek J, Lunzer A, Jelińska A: Stability-indicating derivative spectrophotometry method for the determination of biapenem in the presence of its degradation products. Cent Eur J Chem. 2011, 9: 35-40. 10.2478/s11532-010-0125-9.Google Scholar
- : Validation of analytical procedures, Proceeding of the International Conference of Harmonisation (ICH), Commission of the European Communities. 1996, Geneva, SwitzerlandGoogle Scholar
- Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JA, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, et al: Gaussian 03, Revision B.05. 2003, Pittsburgh PA: Gaussian, IncGoogle Scholar
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