Stability and degradation products of imipenem applying high-resolution mass spectrometry: An analytical study focused on solutions for infusion.

Carbapenems show recognized instability in aqueous solutions; therefore some care must be taken in their handling and preparation and their use in the hospital environment. The stability and degradation products of imipenem were investigated from conditions that simulate its clinical use. For this, a simple stability-indicating method by HPLC-DAD was validated with a focus on the quantitation of drug concentration remaining from infusion solutions (sodium chloride 0.9% and glucose 5%). The degradation products formed were identified by high-resolution mass spectrometry (ESI-Q-TOF-MS/MS), with detection of the [M + H]+ ions at m/z 318 (DP-1), m/z 599 (DP-2) and m/z 658 (DP-3). The most probable elemental compositions were obtained with a high degree of confidence, where the error between the masses observed and calculated was 1.25 ppm for DP-1, -0.33 ppm for DP-2 and 1.82 ppm for DP-3. The DP-1 degradation product resulted from cleavage of the ß-lactam ring; DP-2 corresponded to the drug dimer; and DP-3 was generated from the interaction between imipenem and cilastatin. The proposed method provides a safe and reliable alternative for the quantitation of imipenem, and the stability data obtained by ESI-Q-TOF help in understanding the drug behavior under the conditions of clinical use.

Pooled population pharmacokinetic model of imipenem in plasma and the lung epithelial lining fluid.

AIMS: Several clinical trials have confirmed the therapeutic benefit of imipenem for treatment of lung infections. There is however no knowledge of the penetration of imipenem into the lung epithelial lining fluid (ELF), the site of action relevant for lung infections. Furthermore, although the plasma pharmacokinetics (PK) of imipenem has been widely studied, most studies have been based on selected patient groups. The aim of this analysis was to characterize imipenem plasma PK across populations and to quantify imipenem ELF penetration. METHODS: A population model for imipenem plasma PK was developed using data obtained from healthy volunteers, elderly subjects and subjects with renal impairment, in order to identify predictors for inter-individual variability (IIV) of imipenem PK. Subsequently, a clinical study which measured plasma and ELF concentrations of imipenem was included in order to quantify lung penetration. RESULTS: A two compartmental model best described the plasma PK of imipenem. Creatinine clearance and body weight were included as subject characteristics predictive for IIV on clearance. Typical estimates for clearance, central and peripheral volume, and inter-compartmental clearance were 11.5 l h(-1) , 9.37 l, 6.41 l, 13.7 l h(-1) , respectively (relative standard error (RSE) <8%). The distribution of imipenem into ELF was described using a time-independent penetration coefficient of 0.44 (RSE 14%). CONCLUSION: The identified lung penetration coefficient confirms the clinical relevance of imipenem for treatment of lung infections, while the population PK model provided insights into predictors of IIV for imipenem PK and may be of relevance to support dose optimization in various subject groups.

Further development on DMFC device used for analytical purpose: real applications in the pharmaceutical field and possible in biological fluids.

The analytical research devoted to the utilization of the direct methanol fuel cell (DMFC) for analytical purposes has been continued. The research reported in this paper concerns two points, one of which was the possibility of improving the features, from the analytical point of view, of a catalytic fuel cell for methanol and ethanol, by introducing an enzyme, immobilized into a dialysis membrane small bag, in the anodic area of the fuel cell. This objective has been fully achieved, particularly using the enzyme alcohol dehydrogenase, which has increased the sensitivity of the method and reduced dramatically the response time of the cell. The second point concerned the opportunity to determine two particular antibiotics having an alcohol functional group in their molecule, that is, imipenem and chloramphenicol. Also, this goal has been reached, even if the sensitivity of the method is not so high. Graphical abstract Imipenem and Chloramphenicol determination using the DMFC and Ethanol determination using the enzymatic DMFC.

Transcriptome profiling in imipenem-selected Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistance in Acinetobacter baumannii has gradually become a global challenge. To identify the genes involved in carbapenem resistance in A. baumannii, the transcriptomic responses of the completely sequenced strain ATCC 17978 selected with 0.5 mg/L (IPM-2 m) and 2 mg/L (IPM-8 m) imipenem were investigated using RNA-sequencing to identify differences in the gene expression patterns. RESULTS: A total of 88 and 68 genes were differentially expressed in response to IPM-2 m and IPM-8 m selection, respectively. Among the expressed genes, 50 genes were highly expressed in IPM-2 m, 30 genes were highly expressed in IPM-8 m, and 38 genes were expressed common in both strains. Six groups of genes were simultaneously expressed in IPM-2 m and IPM-8 m mutants. The three gene groups involved in DNA recombination were up-regulated, including recombinase, transposase and DNA repair, and beta-lactamase OXA-95 and homologous recombination. The remaining gene groups involved in biofilm formation were down-regulated, including quorum sensing, secretion systems, and the csu operon. The antibiotic resistance determinants, including RND efflux transporters and multidrug resistance pumps, were over-expressed in response to IPM-2 m selection, followed by a decrease in response to IPM-8 m selection. Among the genes over-expressed in both strains, blaOXA-95, previously clustered with the blaOXA-51-like family, showed 14-fold (IPM-2 m) to 330-fold (IPM-8 m) over-expression. The expression of blaOXA-95 in IPM-2 m and IPM-8 m cells was positively correlated with the rate of imipenem hydrolysis, as demonstrated through Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, suggesting that blaOXA-95 plays a critical role in conferring carbapenem resistance. In addition, A. baumannii shows an inverse relationship between carbapenem resistance and biofilm production. CONCLUSION: Gene recombination and blaOXA-95 play critical roles in carbapenem resistance in A. baumannii. Taken together, the results of the present study provide a foundation for future studies of the network systems associated with carbapenem resistance.

HILIC LC-MS/MS method for the quantification of cefepime, imipenem and meropenem.

A high performance hydrophilic interaction chromatography method combined with tandem-mass spectrometry for the quantification of cefepime, meropenem and imipenem in plasma and cerebrospinal fluid is presented. A solution of 0.5 M 3-Morpholinopropanesulfonic acid and ethylene glycol (1:1) was added to the samples before analysis to ensure stability of analytes during work up and storage. Deuterated forms of cefepime and meropenem were used as internal standards. Protein precipitation prior to injection into the LC-MS/MS system provided a fast and easy sample preparation. For online extraction, a Turboflow Cyclone-MCX column was used and the chromatographic separation was carried out on a Hypersil GOLD HILIC column. Linear calibration curves were obtained in the concentration range of 0.4-40 mg/l, 0.6-60 mg/l and 1-100 mg/l for meropenem, imipenem and cefepime, respectively. The intra- and interday imprecision and inaccuracy values were below 10 % for plasma and 13 % for cerebrospinal fluid using a calibration in plasma. The method was employed for therapeutic drug measurements in a university hospital.

Photochemical degradation of the carbapenem antibiotics imipenem and meropenem in aqueous solutions under solar radiation.

This paper deals with the photochemical fate of two representative carbapenem antibiotics, namely imipenem and meropenem, in aqueous solutions under solar radiation. The analytical method employed for the determination of the target compounds in various aqueous matrices, such as ultrapure water, municipal wastewater treatment plant effluents, and river water, at environmentally relevant concentrations, was liquid chromatography coupled with hybrid triple quadrupole-linear ion trap-mass spectrometry. The absorption spectra of both compounds were measured in aqueous solutions at pH values from 6 to 8, and both compounds showed a rather strong absorption band centered at about 300 nm, while their molar absorption coefficient was in the order from 9 × 103-104 L mol-1 cm-1. The kinetics of the photochemical degradation of the target compounds was studied in aqueous solutions under natural solar radiation in a solar reactor with compound parabolic collectors. It was found that the photochemical degradation of both compounds at environmentally relevant concentrations follows first order kinetics and the quantum yield was in the order of 10-3 mol einsten-1. Several parameters were studied, such as solution pH, the presence of nitrate ions and humic acids, and the effect of water matrix. In all cases, it was found that the presence of various organic and inorganic constituents in the aqueous matrices do not contribute significantly, either positively or negatively, to the photochemical degradation of both compounds under natural solar radiation. In a final set of photolysis experiments, the effect of the level of irradiance was studied under simulated solar radiation and it was found that the quantum yield for the direct photodegradation of both compounds remained practically constant by changing the incident solar irradiance from 28 to 50 W m-2.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry rapid detection of carbapenamase activity in Acinetobacter baumannii isolates.

INTRODUCTION: Carbapenamase-producing Acinetobacter baumannii are an increasing threat in hospitals and Intensive Care Units. Accurate and rapid detection of carbapenamase producers has a great impact on patient improvement and aids in implementation of infection control measures. AIM: In this study, we describe the use of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF MS) to identify carbapenamase-producing A. baumannii isolates in up to 3 h. Isolates and Methods: A total of 50 A. baumannii isolates (of which 39 were carabapenamase producers) were tested using MALDI TOF MS. Isolates were incubated for 3 h with 0.25 mg/ml up to 2 mg/ml of imipenem (IMP) at 37°C. Supernatants were analysed by MALDI TOF to analyse peaks corresponding to IMP (300 Da) and an IMP metabolite (254 Da) using UltrafleXtreme (Bruker Daltonics, Bremen, Germany). RESULTS: All carbapenamase-producing isolates were evidenced by the disappearance or reduction in intensity of the 300 Da peak of IPM and the appearance of a 254 Da peak of the IPM metabolite. In isolates that did not produce carbapenamase, the IPM 300 Da peak remained intact. CONCLUSION: MALDI TOF is a promising tool in the field of diagnostic microbiology that has the ability to transfer identification and antimicrobial susceptibility testing time from days to hours.

Stability and kinetics of degradation of imipenem in aqueous solution.

In weakly acidic solution, the broad-spectrum antibiotic imipenem undergoes complex oligomerization initiated by intermolecular carboxyl group attack on the beta-lactam group. In weakly alkaline solution, intermolecular reaction between the beta-lactam and formimidoyl groups occurs instead. Both beta-lactam and formimidoyl groups also hydrolyze at pH-dependent rates. Complex decomposition schemes were determined in kinetic studies at pH 4.0 and 9.0-9.5 using HPLC and mathematical models. The rates of the several initial reactions, calculated as functions of pH and imipenem concentration by fitting the models to kinetic data, fully account for imipenem decomposition rates throughout the neutral pH range.

Stability of imipenem and cilastatin sodium in total parenteral nutrient solution.

The chemical stability and compatibility of imipenem-cilastatin sodium (Primaxin) in two different total parenteral nutrient (TPN) solutions was determined. TPN solutions consisted of 4.25% and 5% amino acids with 25% and 35% dextrose, respectively. Imipenem-cilastatin sodium was constituted with 10 ml of sterile water and admixed with 90 ml of TPN solution for a final concentration of 5 mg/ml of each drug. The final solutions were assayed at times 0 (immediately after admixture), 15 min, 30 min, 1, 4, 8, and 24 hr by a stability-indicating high-performance liquid chromatographic assay. Concurrently, test TPN solutions were monitored for pH changes, color changes, and precipitate formation. The potential effect of imipenem-cilastatin sodium on the stability of amino acids and other TPN additives was not evaluated. Imipenem and cilastatin sodium was stable (greater than or equal to 90% recovered) in each TPN solution at 15 min. A significant (greater than or equal to 10%) and steady decrease of imipenem recovery occurred at subsequent sampling times. Cilastatin appeared more stable than imipenem in both TPN solutions. A physical color change from colorless to dark orange appeared in each TPN solution over the 24-hr study period. Imipenem-cilastatin sodium is stable for 15 min in the TPN solutions studied; however, until the stability of the amino acids can be determined, the antibiotic should be administered through a separate line or Y-site while the TPN infusion is interrupted.

First- and second-derivative spectrophotometric determination of imipenem and cilastatin in injections.

First- and second-derivative spectrophotometry has been used for the quantitation of mixtures of imipenem and cilastatin sodium, compounds that have closely overlapping spectral bands. Beer's law was obeyed at concentrations up to 100 micrograms ml-1 of imipenem in both the first- and second-derivative modes and up to 75 micrograms ml-1 of cilastatin in the first-derivative mode. Detection limits at the P = 0.05 level of significance were calculated to be 0.40 and 0.52 micrograms ml-1 of imipenem and cilastatin sodium, respectively, in the first-derivative mode, and in a range from 0.45 to 0.68 micrograms ml-1 for imipenem in the second-derivative mode. The method, which is rapid, simple and does not require a separation step, has been successfully applied to the assay of commercial injections.

Determination of imipenem in human plasma, urine and tissue by high-performance liquid chromatography.

A simple and reliable HPLC method is described for the new beta-lactam antibiotic imipenem; suitable extraction procedures for the drug in human plasma, urine and prostatic tissue are described. The figures of merit for the assays are reported and examples given of their application.

Determination of imipenem and cilastatin sodium in Primaxin by first order derivative ultraviolet spectrophotometry.

A UV-spectrophotometric assay to measure the concentrations of the active drug components (imipenem and cilastatin) or Primaxin for routine release testing is described. The assay is based on the use of first order derivative spectrophotometry. The trough amplitudes in the first derivative spectrophotometric spectra at 243 and 318 nm were selected to determine cilastatin and imipenem, respectively. A linear relationship (R > 0.99) between the trough amplitudes and concentrations was demonstrated over the range 14-42 micrograms ml-1 for both drug components. Commercial IV formulations and laboratory prepared mixtures containing both drugs in different proportions were assayed using the developed method with good recoveries (ave. 100.6%). The method is rapid, precise, accurate and was shown to be equivalent to the more time consuming LC method; which is currently used for routine release testing. The specificity and stability indicating properties of the method will also be addressed.

In situ degradation: a new concept for system suitability tests in monographs of the European Pharmacopoeia.

Monographs of the European Pharmacopoeia describe in the LC-test for related substances usually a system suitability test in order to ensure the adequate separation of impurities. Since the reference substances required are often not available a recent approach to avoid this problem is the generation of the required impurity by 'in situ degradation' of the active principle. This paper describes some typical applications of this technique as well as recent examples, such as the controlled degradation of cefalotin sodium, imipenem and spiramycin.

[Determination of moxalactam, aztreonam and imipenem by the perchloric hydroxylamine method]. / Oznaczanie moksalaktamu, aztreonamu i imipenemu metoda hydroksyloaminowo-nadchloranowa.

The reaction of perchloric hydroxylamine was adopted for determination of new beta-lactam antibiotics within monobactam, carbapenem and oxazepam groups. Complexes formed in colour reaction are stable over 2 hours in the case of aztreonam and imipenem, and over 1 hour for moxalactam, statistical analysis proved that both hydroxylamine-perchloric and spectrophotometric methods can be used alternatively.

Comparison of disk diffusion, Etest and VITEK2 for detection of carbapenemase-producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems.

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase-producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended-spectrum ß-lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut-off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL-positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.

Selective determination of ertapenem and imipenem in the presence of their degradants.

Stability-indicative determination of ertapenem (E(RTM)) and imipenem (I(MPM)) in the presence of their corresponding open-ring degradation products, the metabolites, is investigated. The degradation products have been isolated via acid-degradation, characterized, and confirmed. Selective quantification of E(RTM) or I(MPM) singly in bulk form, pharmaceutical formulations, and/or in the presence of their corresponding degradants is demonstrated. The indication of stability has been undertaken under conditions likely to be expected at normal storage conditions. Among the chromatographic techniques adopted for quantification are coupled thin layer chromatography-densitometry and high-performance liquid chromatography.

Pharmacokinetic-pharmacodynamic correlation of imipenem in pediatric burn patients using a bioanalytical liquid chromatographic method / Correlação farmacocinética-farmacodinâmica de imipenem em pacientes queimados pediátricos utilizando um método bioanalítico de cromatografia líquida

A bioanalytical method was developed and applied to quantify the free imipenem concentrations for pharmacokinetics and PK/PD correlation studies of the dose adjustments required to maintain antimicrobial effectiveness in pediatric burn patients. A reverse-phase Supelcosil LC18 column (250 x 4.6 mm 5 micra), binary mobile phase consisting of 0.01 M, pH 7.0 phosphate buffer and acetonitrile (99:1, v/v), flow rate of 0.8 mL/min, was applied. The method showed good absolute recovery (above 90%), good linearity (0.25-100.0 µg/mL, r²=0.999), good sensitivity (LLOQ: 0.25 µg/mL; LLOD: 0.12 µg/mL) and acceptable stability. Inter/intraday precision values were 7.3/5.9%, and mean accuracy was 92.9%. A bioanalytical method was applied to quantify free drug concentrations in children with burns. Six pediatric burn patients (median 7.0 years old, 27.5 kg), normal renal function, and 33% total burn surface area were prospectively investigated; inhalation injuries were present in 4/6 (67%) of the patients. Plasma monitoring and PK assessments were performed using a serial blood sample collection for each set, totaling 10 sets. The PK/PD target attained (40%T>MIC) for each minimum inhibitory concentration (MIC: 0.5, 1.0, 2.0, 4.0 mg/L) occurred at a percentage higher than 80% of the sets investigated and 100% after dose adjustment. In conclusion, the purification of plasma samples using an ultrafiltration technique followed by quantification of imipenem plasma measurements using the LC method is quite simple, useful, and requires small volumes for blood sampling. In addition, a small amount of plasma (0.25 mL) is needed to guarantee drug effectiveness in pediatric burn patients. There is also a low risk of neurotoxicity, which is important because pharmacokinetics are unpredictable in these critical patients with severe hospital infection. Finally, the PK/PD target was attained for imipenem in the control of sepsis in pediatric patients...

Desenvolveu-se e aplicou-se método bioanalítico para quantificar concentrações de imipenem livre para estudos de farmacocinética (PK) e de correlação PK/PD dos ajustes de dose requeridos para manter a efetividade antimicrobiana em pacientes pediátricos queimados. Utilizou-se coluna Supelcosil LC18 (250 x 4,6 mm 5 micra), fase móvel binária, consistindo de tampão fosfato 0,01M pH 7,0 e acetonitrila (99:1, v/v) e fluxo de 0,8 mL/min. O método mostrou boa recuperação absoluta (acima de 90%), boa linearidade (0,25-100,0 µg/mL, r²=0.999), boa sensibilidade (LLOQ: 0,25 µg/mL; LLOD: 0,12 µg/mL) e estabilidade aceitável. Os valores de precisão inter/intradia foram 7,3/5,9% e a exatidão média foi de 92,9%. O método bioanalítico foi aplicado para quantificar concentrações de fármaco livre em crianças com queimaduras, Seis pacientes pediátricos queimados (idade média de 7,0 anos, 27,5 kg), com função renal normal e 33% da superfície total queimada foram investigados prospectivamente. Lesões por inalação estavam presentes em 4/6 (67%) dos pacientes. O monitoramento plasmático e a as avaliações de PK foram efetuadas utilizando coleção de amostras seriais de sangue para cada série, totalizando 10 conjuntos. O alvo PK/PD alcançado (40%T>MIC) para cada concentração inibitória mínima (MIC: 0,5, 1,0, 2,0, 4,0 mg/L) ocorreu em porcentagem maior do que 80% dos conjuntos investigados e 100% após o ajuste de dose. Em conclusão, a purificação das amostras do plasma usando técnica de ultrafiltração seguida de quantificação das medidas do imipenem no plasma usando método de cromatografia líquida é bastante simples, útil e necessita de pequenos volumes para as amostras de sangue. Além disso, pequena quantidade de plasma (0,25 mL) é necessário para garantir a efetividade do fármaco nos pacientes pediátricos queimados. Há, ainda, baixo risco de neurotoxicidade, o que é importante, visto que as farmacocinéticas são imprevisíveis nesses pacientes...

[Presence of metallo beta-lactamases in imipenem-resistant Pseudomonas aeruginosa]. / Presencia de metalo-beta-lactamasas en Pseudomonas aeruginosa resistente a imipenem.

BACKGROUND: Metallo-beta-lactamases (MBL) confer high resistance to carbapenems in Pseudomonas aeruginosa (Psae). They are encoded in mobile elements of different genes (VIM, IMP, SMP, GIM), along with other resistance genes. AIM: To detect the presence of MBL in imipenem resistant Psae strains. MATERIAL AND METHODS: Fifty-nine imipenem resistant Psae strains isolated from January 2004 to August 2005 in a University Clinical Hospital, were included. The presence of MBL was studied by Etest (phenotypic) and genotypic polymerase chain reaction (PCR) methods. To rule out a nosocomial outbreak, MBL positive strains were studied by pulse field gel electrophoresis. RESULTS: The presence of MBL was detected in eleven strains. AH were type VIM and were not clonally related. There was no concordance between phenotypic and genotypic MBL detecting methods. All the strains were also multiresistant. CONCLUSIONS: The presence of MBL was detected in 19% of imipenem resistant Psae strains.

Imipenem stability in a predried susceptibility panel.

We performed a 15-month study using 11 clinical strains and 1 control strain (ATCC 27853) of Pseudomonas aeruginosa to determine whether changes in the manufacturing process of Sensititre predried panels result in a reliable test of susceptibility to imipenem. MIC and breakpoint susceptibility results remained stable during the manufacturer's recommended shelf life of 18 months and compared well with standard agar disk diffusion and broth macrodilution results. Imipenem concentrations measured by high-pressure liquid chromatography were acceptable through 15 months but declined in the breakpoint panels by approximately 50% at 18 months. Between 9 months and panel expiration, 13 of 141 (9%) of the MIC panel packages had moisture entry, as indicated by pink desiccants, with a resultant loss of imipenem activity of 32 to 100%. It appears that the new manufacturing process produces MIC panels that are reliable for imipenem susceptibility testing until the labeled expiration date, provided that packages containing pink desiccants are not used.