Identification of related impurities in an oral pharmaceutical formulation of tebipenem pivoxil using ultra-high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry.

RATIONALE: Tebipenem pivoxil (TBPM-PI) has been developed as the first oral carbapenem drug in the world to treat otolaryngological and respiratory infections in pediatric patients. Due to its structural properties and external factors, some related impurities, which may cause side effects in patients, might be formed during the synthesis and storage of TBPM-PI. It was vital to rapidly separate and identify the related impurities to guarantee the safe use of TBPM-PI. METHODS: A method using ultra-high-performance liquid chromatography (UHPLC) coupled with quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) was developed to separate and detect TBPM-PI and related impurities in an oral pharmaceutical formulation. LC/MS and MS/MS spectra of these compounds in the formulation were acquired to confirm their elemental compositions and propose their structures based on LC/MS data and fragmentation pathways of available reference substances. RESULTS: LC/MS parameters and MS/MS fragmentation pathways of reference substances of TBPM-PI and related impurities were summarized in detail. Based on this, a total of 23 related impurities were found and characterized in the oral pharmaceutical formulation. Eight of these were verified by comparison with reference substances and the structures of the other 15 were proposed for the first time. In addition, four of these compounds were produced by the reaction of excipients and pre-existing related impurities. CONCLUSIONS: A UHPLC/QTOF-MS method was established and used for the separation and identification of 23 related impurities in a TBPM-PI oral pharmaceutical formulation. Moreover, it was proved that new related impurities could be produced by the reaction of excipients in the pharmaceutical formulation and related impurities in the corresponding active pharmaceutical ingredient (API).

Determination of carbapenems in water samples by UHPLC-MS/MS.

A rapid and reliable method for the detection of five carbapenems (biapenem, imipenem, doripenem, meropenem, and faropenem) in water was developed and validated. After acidification of water samples with acetic acid, carbapenems were isolated using a Bond Elut PPL cartridge. The target compounds were separated using ultra high performance liquid chromatography with a chromatographic run time of 5 min and detected on a triple quadrupole mass spectrometer operated in positive electrospray ionization and multiple reaction monitoring mode. Mean recoveries were in the range of 76.6-106.5%, with satisfactory intraday and interday relative standard deviations lower than 10.0 and 10.8%, respectively. The limits of detection and quantification were in the ranges of 0.05-0.2 µg/L and 0.1-0.5 µg/L, respectively, depending on the analyte. The proposed method was applied to the analysis of river samples and wastewater samples from swine farms, and no carbapenems were detected in the collected samples.

Enhanced antimicrobial de-escalation for pneumonia in mechanically ventilated patients: a cross-over study.

BACKGROUND: Antibiotics are commonly administered to hospitalized patients with infiltrates for possible bacterial pneumonia, often leading to unnecessary treatment and increasing the risk for resistance emergence. Therefore, we performed a study to determine if an enhanced antibiotic de-escalation practice could improve antibiotic utilization in mechanically ventilated patients with suspected pneumonia cared for in an academic closed intensive care unit (ICU). METHODS: This was a prospective cross-over trial comparing routine antibiotic management (RAM) and enhanced antimicrobial de-escalation (EAD) performed within two medical ICUs (total 34 beds) at Barnes-Jewish Hospital, an academic referral center. Patients in the EAD group had their antibiotic orders and microbiology results reviewed daily by a dedicated team comprised of a second-year critical care fellow, an ICU attending physician and an ICU pharmacist. Antibiotic de-escalation recommendations were made when appropriate based on microbiologic test results and clinical response to therapy. RESULTS: There were 283 patients evaluable, with suspected pneumonia requiring mechanical ventilation: 139 (49.1%) patients in the RAM group and 144 (50.9%) in the EAD group. Early treatment failure based on clinical deterioration occurred in 33 (23.7%) and 40 (27.8%) patients, respectively (P = 0.438). In the remaining patients, antimicrobial de-escalation occurred in 70 (66.0%) and 70 (67.3%), respectively (P = 0.845). There was no difference between groups in total antibiotic days ((median (interquartile range)) 7.0 days (4.0, 9.0) versus 7.0 days (4.0, 8.8) (P = 0.616)); hospital mortality (25.2% versus 35.4% (P = 0.061)); or hospital duration (12.0 days (6.0, 20.0) versus 11.0 days (6.0, 22.0) (P = 0.918). CONCLUSIONS: The addition of an EAD program to a high-intensity daytime staffing model already practicing a high-level of antibiotic stewardship in an academic ICU was not associated with greater antibiotic de-escalation or a reduction in the overall duration of antibiotic therapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02685930 . Registered on 26 January 2016.

Pharmacokinetics and brain penetration of carbapenems in mice.

An adverse effect associated with the administration of carbapenems is central nervous system (CNS) toxicity, with higher brain concentrations of carbapenems being linked to an increased risk of seizures. However, the pharmacokinetics and brain penetration of carbapenems have not yet been examined. Thus, the aim of this in vivo investigation was to determine the pharmacokinetics and brain penetration of carbapenems in mice. Blood samples and brain tissue samples were obtained 10, 20, 30, 60, and 120 min after the subcutaneous administration of carbapenems (91 mg/kg). We obtained the following values for the pharmacokinetic parameters of carbapenems in mice: 1.20-1.71 L/h/kg for CLtotal/F, 1.41-2.03 h(-1) for Ke, 0.34-0.51 h for T1/2, 0.66-0.95 L/kg for Vss/F, 0.49-0.73 h for MRT, 83.46-110.58 µg/mL for Cmax, plasma, and 0.28-0.83 µg/g for Cmax, brain tissue. The AUC0-∞ of the carbapenems tested in plasma were in the following order: doripenem > meropenem > biapenem > imipenem, and in brain tissue were: imipenem > doripenem > meropenem > biapenem. The degrees of brain tissue penetration, defined as the AUC0-∞, brain tissue/fAUC0-∞, plasma ratio, were 0.016 for imipenem, 0.004 for meropenem, 0.002 for biapenem, and 0.008 for doripenem. The results of the present study demonstrated that, of the carbapenems examined, imipenem penetrated brain tissue to the greatest extent.

Quantitative determination of the ß-methyl carbapenem doripenem in powder for injection by a stability-indicating capillary zone electrophoresis method.

A capillary zone electrophoresis method for quantitative determination of doripenem in synthetic matrix was developed. The stability-indicating capability was performed applying stress testing protocols. The selected analytical conditions include 100 mM sodium borate buffer (pH 8.0) as run electrolyte, voltage of +15 kV, hydrodynamic injection of 5s (50 mBar), detection at 298 nm and temperature of analysis of 25 degrees C. The electrophoretic separation was carried out in a fused silica capillary (effective length 40 cm, 50 µm i.d.), using procainamide hydrochloride as internal standard. The proposed method showed quickness and reproducibility, with an analytical run in a total time of 5 min. The percentage of drug amount estimated was 101.33% (RSD = 0.80), with satisfactory intra-day and inter-day precision. In the recovery test, the method was found to be reliable and accurate in the drug quantitation (mean recovery = 101.86%). The robustness was performed applying the Plackett-Burman experimental design which confirmed the assay reliability. Based on results from forced degradation study, the stability-indicating capability was established, being observed a major degradation in alkaline, photolytic and thermal conditions. In comparison to HPLC method previously developed, the proposed capillary electrophoresis assay is statistically equivalent.

A fluorescent carbapenem for structure function studies of penicillin-binding proteins, ß-lactamases, and ß-lactam sensors.

By reacting fluorescein isothiocyanate with meropenem, we have prepared a carbapenem-based fluorescent ß-lactam. Fluorescein-meropenem binds both penicillin-binding proteins and ß-lactam sensors and undergoes a typical acylation reaction in the active site of these proteins. The probe binds the class D carbapenemase OXA-24/40 with close to the same affinity as meropenem and undergoes a complete catalytic hydrolysis reaction. The visible light excitation and strong emission of fluorescein render this molecule a useful structure-function probe through its application in sodium dodecyl sulfate-polyacrylamide gel electrophoresis assays as well as solution-based kinetic anisotropy assays. Its classification as a carbapenem ß-lactam and the position of its fluorescent modification render it a useful complement to other fluorescent ß-lactams, most notably Bocillin FL. In this study, we show the utility of fluorescein-meropenem by using it to detect mutants of OXA-24/40 that arrest at the acyl-intermediate state with carbapenem substrates but maintain catalytic competency with penicillin substrates.

Simultaneous determination of five carbapenems, highly polar antibiotics, in milk by LC-MS/MS.

The simultaneous determination of five carbapenems (biapenem, doripenem, ertapenem, imipenem, and meropenem) in raw and pasteurised bovine milk samples using LC-MS/MS was achieved and validated. Chromatographic separation was conducted on an InertSustain® AQ-C18 column using 0.1% formic acid in water and acetonitrile as the mobile phase. Target compounds were extracted using acetonitrile/water (20:80, v/v). After the removal of lipids with acetonitrile-saturated hexane, the dissolved protein was denatured with acetic acid. A portion of the supernatant was passed through an Oasis® PRiME HLB cartridge to remove the matrix. This novel method was validated in accordance with the Japanese validation guidelines and exhibited good trueness, ranging from 86.3% to 96.2%, using matrix-matched calibration curves. The relative standard deviation of repeatability ranged from 1.0% to 6.3%, and that of within-laboratory reproducibility ranged from 1.6% to 7.1%. The limit of quantification was 1.0 µg kg-1 for all analytes. None of the 60 milk samples commercially available in Tokyo contained any analytes. This novel method exhibited high-quality performance and can easily be implemented for the routine monitoring of carbapenems, which are highly polar antibiotics in milk.

Simultaneous determination of eight carbapenems in milk by modified QuEChERS and ultra high performance liquid chromatography coupled with high-field quadrupole-orbitrap high-resolution mass spectrometry.

A simple and accurate method of ultra high performance liquid chromatography (UHPLC) coupled with quadrupole-high field Orbitrap high-resolution mass spectrometry (QE HF HRMS) has been developed for analyzing 8 trace-level (µg/L) carbapenems in milk. Mass spectrometry conditions, chromatographic conditions, extraction solvent and QuEChERS procedures were optimized for determination of 8 carbapenems in milk. Samples were extracted and purified by modified QuEChERS procedure. Good separation for 8 carbapenems s was achieved with a PFP column at 8 min. Method validation results showed the linear ranges are 1-100 µg/L to 10-1,000 µg/L, the correlation coefficients are more than 0.995, and the recoveries of spiked samples are 79.3%-104% with a relative standard deviation less than 15%. This method successfully applied to monitor residue of carbapenems in real milk, four kinds of carbapenems have been detected in 8 sample of 79 collected milks with concentration ranged between 15 µg/L∼3,325 µg/L.

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan.

BACKGROUND: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clinical healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan. METHODS: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospitals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identified that had not been previously reported in patients from Japan. RESULTS: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As the ESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2. CONCLUSIONS: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary.

Determination of doripenem and related substances in medicinal product using capillary electrophoresis.

Doripenem, the latest carbapenem antibiotic licensed in the United States (15 October 2007) and the European Union (25 July 2008), has been implemented into therapeutic use along with imipenem, meropenem and ertapenem. The described method of zone electrophoresis in a low pH buffer for the separation of doripenem from its impurities has been successfully performed using field-amplified sample stacking (FASS), followed by UV absorption detection at 214 nm. The best results were obtained with phosphate buffer (100 mM) pH 2.9 containing 10% (v/v) of methanol, as the background electrolyte. Uncoated fused-silica capillary (60/52 cm; 75 µm id) with normal polarity, and voltage values of 25 kV, was used throughout the investigation. The optimised method of doripenem determination was validated in terms of linearity, accuracy and precision, and provides a detection limit of 3.0 µg/mL of doripenem. The repeatability, expressed by relative standard deviation (RSD) of the migration time, for doripenem and its degradation products varied from 1.37 to 2.51%, whereas the corrected peak areas were about 0.91-9.87%. Satisfactory separation was achieved within 20 min of electrophoresis; moreover, all carbapenems (imipenem, meropenem, ertapenem and doripenem) were well separated from each other during this time. The evaluated CZE method was applied in the analysis of a medicinal product containing doripenem Doribax(®) powder for solution for infusion.

Evaluation of the rCIM for carbapenemase detection in Enterobacterales and Pseudomonas aeruginosa and description of the TSBrCIM, an optimized variant.

OBJECTIVES: The rapid Carbapenem Inactivation Method (rCIM) was evaluated with a strain collection of 164 and 69 carbapenem-resistant Enterobacterales and Pseudomonas aeruginosa, respectively, that produced various carbapenemases. For an improved carbapenemase detection in Enterobacterales, an optimized variant of the rCIM named TSBrCIM was developed. METHODS: Bacterial isolates were incubated with two meropenem disks in distilled water (rCIM) or tryptic soy broth (TSBrCIM). After centrifugation, the supernatant was incubated with a susceptible E. coli indicator strain in tryptic soy broth. Growth of the indicator strain implied carbapenemase activity in the test strain. RESULTS: The rCIM detected 100/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 88.5% and a specificity of 94.1%. For P. aeruginosa, sensitivity and specificity were 96.0% and 100%, respectively. The TSBrCIM was able to detect 105/113 carbapenemase-producing Enterobacterales, resulting in a sensitivity of 92.9% and a specificity of 96.1%. CONCLUSION: This study shows that the TSBrCIM can be valuable tool for detection of carbapenemases in Enterobacterales in the clinical laboratory, while the rCIM showed the best results for carbapenemase detection in P. aeruginosa.

Determination of carbapenem antibiotics using a purpose-made capillary electrophoresis instrument with contactless conductivity detection.

In this study, the employment of a purpose-made capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C4D) as a simple and cost-effective approach for simultaneous determination of different carbapenem antibiotics is reported. The developed CE-C4D approach was for the first time applied for quality control of various pharmaceutical formulations in Vietnam, as well as for therapeutic monitoring of these antibiotics in plasma samples from patients under intensive care. Four of the most popular carbapenems in Vietnam, doripenem, meropenem, imipenem and ertapenem, were determined using an electrolyte composed of 10 mM Tris adjusted to pH 8.0 with acetic acid. The best detection limits achieved using the developed CE-C4D method were 0.36 mg/L and 0.45 mg/L for pharmaceutical and plasma samples, respectively. Good agreement between results from CE-C4D and the confirmation method (HPLC-PDA) was achieved, with a coefficient of determination (r2) for the two pairs of data of 0.9967.

Stability of doripenem in vitro in representative infusion solutions and infusion bags.

BACKGROUND: Doripenem, a new parenteral carbapenem with broad-spectrum antibacterial activity, is indicated for the treatment of complicated intra-abdominal infections and complicated urinary tract infections, including pyelonephritis. According to the US prescribing information, the carbapenems imipenem and meropenem are stable in sodium chloride for 4 hours. OBJECTIVE: The aim of this study was to assess the stability of doripenem following constitution (50 mg/mL) and in injection solutions in vitro (0.9% sodium chloride and 5% dextrose) at a concentration of 5 mg/mL in room and refrigerated conditions. METHODS: The stability of doripenem was assessed (1) under room-temperature conditions (25 degrees C +/- 2 degrees C, 60% +/- 5% relative humidity, under fluorescent light) immediately after constitution in vials with sterile water; (2) under room conditions and refrigerated conditions (5 degrees C +/- 3 degrees C, 60% relative humidity, protected from light) immediately after constitution with sodium chloride or dextrose in 3 different infusion bags (polyvinyl chloride [PVC], PVC with vial adapter, and polyethylene); and (3) under room and refrigerated conditions using 500-mg doripenem vials that had been stored for 12 months in room temperature and protected from light before constitution with water and dilution in sodium chloride or dextrose injection. Doripenem 5 mg/mL and related substances (degradation products and impurities related to doripenem) were measured using a validated high-performance liquid chromatography method. Doripenem was considered stable if its concentration remained within 90% to 110% of the initial concentration and the total concentration of degradation products and impurities related to doripenem was < or = 5%. Appearance of the solutions was assessed using clarity and comparison to color standards; pH, using standard methodologies; and particulate matter, using light-obscuration and microscopy. RESULTS: Three lots of doripenem were assessed. Doripenem potency in constituted suspension (50 mg/mL) remained unchanged (means, 99.2% and 99.3%, respectively, of initial doripenem amount) for up to 60 minutes in room or refrigerated conditions prior to transfer to infusion bags. The doripenem 5-mg/mL infusion solution retained its potency for 12 and 72 hours under room and refrigerated conditions, respectively, in 0.9% sodium chloride injection, and for 4 and 48 hours under room and refrigerated conditions, respectively, in 5% dextrose injection (mean percentages of initial doripenem concentration, 95.5% and 96.6% under room and refrigerated conditions, respectively). Comparable results were obtained with vials of doripenem that had been stored for 12 months at room temperature and protected from light and then constituted (50 mg/mL). Doripenem 5 mg/mL in 0.9% sodium chloride injection or 5% dextrose injection retained its potency in room conditions at the end of a 4-hour drip period when used with conventional infusion sets (mean, 96.6%) or Di(2-ethylhexyl)phthalate-free infusion sets (mean, 99.3%). CONCLUSIONS: Doripenem 5 mg/mL was stable for up to 12 hours in vitro in 0.9% sodium chloride at room temperature. Therefore, doripenem can be constituted, mixed with infusion fluids in the pharmacy, stored, delivered, and infused into a patient within a time frame suitable for 4-hour extended infusions.

Quantification of doripenem in human plasma and peritoneal fluid by high-performance liquid chromatography with ultraviolet detection.

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and peritoneal fluid protein was developed to determine doripenem concentrations in human plasma and peritoneal fluid. Doripenem was stabilized by immediate mixing of the plasma or peritoneal fluid with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Doripenem and an internal standard were detected by measuring their ultraviolet absorbance at 300 nm. The calibration curves for doripenem in human plasma and peritoneal fluid were linear from 0.05 to 100 microg/mL. For plasma, both the intra- and the interday precision were less than 3.41% (CV), and the accuracy was between 97.4 and 101.7% above 0.05 microg/mL. For peritoneal fluid, the intra- and the interday precision were less than 2.98% (CV), and the accuracy was between 94.4 and 103.9% above 0.05 microg/mL. The limit of detection was 0.02 microg/mL in both plasma and peritoneal fluid. The assay has been applied to the therapeutic drug monitoring of doripenem in both plasma and peritoneal fluid.

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.

Comparison of the in-house made Carba-NP and Blue-Carba tests: Considerations for better detection of carbapenemase-producing Enterobacteriaceae.

The in-house Carba-NP and Blue-Carba tests were compared using 30 carbapenemase- and 33 non-producing Enterobacteriaceae. Tests were read by three operators. 100% sensitivity was reported for both tests, but Carba-NP was slightly more specific than Blue-Carba (98.9% vs. 91.7%). We describe potential sources of error during tests' preparation and reading.

[Analysis of carbapenems by hydrophilic interaction chromatography and its application].

A hydrophilic interaction chromatographic (HILIC) method has been developed for the determination of the four carbapenems in human urine and tap water. The parameters including acetonitrile amount, buffer concentration and pH on the retention behavior of the four carbapenem antibiotics on an XAmide column were explored and the possible HILIC retention mechanism was proposed. Good linearities were obtained over the mass concentration ranges of 0.1-250 mg/L for biapenem, doripenem and ertapenem with correlation coefficients (R2) = 0.999 9 and while it was 0.5-250 mg/L with R2 = 0.999 8 for meropenem. The limits of quantification (LOQs) of all carbapenems were 0.1-0.5 mg/L. The spiked recoveries were within 100.4%-111.9% (RSD < 1%) for urine samples and 79.6%-107.4% (RSD < 5%) for tap water samples all at the spiked levels of 5 mg/L and 25 mg/L. The proposed method is accurate, sensitive, simple and suitable for the determination of the four carbapenems in human urine samples and tap water samples.

The chromatographic approach to kinetic studies of tebipenem pivoxil.

A validated high-performance liquid chromatography-diode array detector (HPLC-DAD) method for stability studies of tebipenem pivoxil was developed. The separation of tebipenem pivoxil in the presence of main degradation product­tebipenem­was achieved by using a LiChrospher C-18 column (5 µm, 250 × 4.6 mm) with the mobile phase containing a mixture of 50 mmol L(-1) ammonium acetate-acetonitrile-triethylamine (68 : 30 : 2, v/v/v) adjusted to pH 3.5 with concentrated phosphoric acid (V). The column effluent was monitored by a photodiode array detector at 330 nm. The flow rate was 0.8 mL min(-1). Tebipenem pivoxil was subjected to degradation in aqueous solutions (acid-base hydrolysis, oxidation) and in the solid state (photolysis, thermolysis at an increased relative humidity and in dry air). The validated HPLC method was successfully applied to investigate the kinetics of conversion of tebipenem pivoxil to tebipenem (main metabolite). The other degradation products of tebipenem pivoxil were also monitored.

High-performance liquid chromatographic separation of beta-methyl ADC-13 enolphosphate diphenyl ester and its alpha-methyl diastereomer.

The development of a high-performance liquid chromatographic (HPLC) separation of beta-methyl ADC-13 enolphosphate diphenyl ester, an intermediate compound in the synthesis of a carbapenem antibiotic drug candidate, and its alpha-methyl diastereomer is reported. The method development involved separation on different columns in both normal- and reversed-phase modes. The use of normal-phase mode resulted in the desired elution order of the two diastereomers. The influence of different polar modifiers and their concentrations on resolution, capacity factor and separation factor was investigated. Different stationary phases were compared for their efficiency and resolution. The optimized separation was applied to the determination of the minor diastereomer in the bulk intermediate, and 0.1% minor diastereomer was detectable.

Identification by LC/MS(n) of degradates of a novel carbapenem antibiotic in an aqueous matrix.

Increased drug resistance in Staphylcocci and Enterococci to currently available antibiotics has significantly limited therapeutic options. Recently, a novel carbapenem antibiotic (Compound A) with a releasable side chain adjacent to the carbapenem was investigated to combat methicillin- and vancomycin-resistant Staphylococci and vancomycin-resistant Enterococci. The major advantage of Compound A over existing antibiotics can be attributed to the fact that cleavage of the side chain upon beta-lactam ring opening retained anti-bacterial activity while expelling the immunodominant epitope of the presumed beta-lactam hapten. In this work, LC/MS methods were developed to identify degradates of Compound A in an aqueous matrix utilized in assessing product safety and supporting analytical method and formulation development. A total of eight significant degradates were observed in this Compound A sample by LC/MS(n) and other techniques. Detailed structural analysis of degradates based upon LC/MS(n) data and other supporting results will be described in this work. Proposed molecular structures were confirmed by synthesis and use of authentic standards for several degradates. Degradates 1 and 4 were identified as degradates formed through the reversal of Michael reaction from Degradate 3 that is apparently formed by hydrolysis. Degradates 2 and 8 were found to be Hofmann elimination degradates. Degradates 5 and 6 are believed to be formed through dimerization of two parent molecules followed by the reversal of Michael reaction. Finally, Degradate 7 is attributed to a displacement reaction. Potential degradation pathways based upon these preliminary studies will also be discussed.