Efficient Synthesis of Potential Impurities in Levonadifloxacin (WCK 771).

Levonadifloxacin (WCK 771) is a novel broad-spectrum anti-methicillin-resistant Staphylococcus aureus (MRSA) agent recently launched in India. Five process impurities and one degradation impurity were synthesized as reference standards for their quantification by high-performance liquid chromatography (HPLC) methodology in drug substance and drug product. These compounds are not easily commercially available. The synthesis and characterization of these impurities are discussed in detail.

Enantiomeric Separation and Thermodynamic Investigation of (R)-N-Tert-Butoxy Carbonyl-Piperidine-3-Carboxylic Acid Hydrazide, a Key Starting Material of Zidebactam.

A new selective, accurate and precise chiral high-performance liquid chromatography method for the separation of (R)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (RE) and its enantiomer was developed. RE is a key starting material of novel ß-lactam enhancer drug Zidebactam. Chiral resolution of more than 10 was achieved on Chiralpak IA column using mobile phase consisting of n-hexane, ethanol in the ratio of 70:30, v/v. The flow rate of the mobile phase was 1.0 mL min-1 and the column oven temperature was 30°C. Detection was carried out at 225 nm. The developed method was validated as per the International Conference on Harmonization guideline. Limit of detection and limit of quantification of the enantiomeric impurity (S)-N-tert-butoxy carbonyl-piperidine-3-carboxylic acid hydrazide (SE) was 2.5 and 7.5 µg mL-1, respectively. Mean recovery of the SE was 96.83 ± 1.4%. The effect of thermodynamic parameters on the chiral separation was evaluated.

Impurity Profiling of a Novel Anti-MRSA Antibacterial Drug: Alalevonadifloxacin.

Alalevonadifloxacin (ALA, formerly described as WCK 2349) is a novel antibacterial drug developed to treat infections caused by Gram-positive bacteria. The current study was aimed to identify and quantify impurities in ALA. Mass spectrometry (MS) compatible reverse phase liquid chromatographic method was developed to identify the impurities in ALA. Three impurities were identified based on the molecular ion peak and their product ions. These impurities were synthesized and characterized using MS and nuclear magnetic resonance spectrometry. However, this method was unable to resolve the diastereomeric impurity, which is likely to be formed during synthesis. Therefore, another method was developed using YMC Chiral NEA-[S] as a chiral stationary phase and validated for quantification of all impurities including diastereomeric impurity. The developed method was employed for quality control and stability studies of the ALA drug substance used in pre-clinical and clinical studies.

Liquid Chromatographic Separation and Thermodynamic Investigation of Ethyl Nipecotate Enantiomers on Immobilized Amylose-Based Chiral Stationary Phase.

Ethyl nipecotate enantiomers are widely used as chiral building blocks in the synthesis of drug substances. An efficient and economic chiral high-performance liquid chromatographic method for determination of enantiomeric purity of ethyl nipecotate is developed and validated. Chiral separation was achieved on immobilized amylose-based stationary phase using a mixture of n-hexane: ethanol: diethylamine (80:20:0.1, v/v/v) as mobile phase. Resolution between R-(-)-ethyl nipecotate (REN) and S-(+)-ethyl nipecotate (SEN) peaks was found to be 3.59. The detector response of SEN exhibited an excellent linearity over the concentration range of 4.5-120 µg mL-1. The limit of detection and limit of quantification for SEN were 0.016 and 0.045 µg and REN were 0.015 and 0.043 µg, respectively. The influence of column oven temperatures on chiral retention and separation was studied in the range of 25°C to 50°C; the thermodynamic parameters ΔH°, ΔS° and ΔG° were evaluated from van't Hoff plots and used to explain the strength of interaction between analyte and stationary phase.

Identification of metabolites of novel Anti-MRSA fluoroquinolone WCK 771 in mouse, rat, rabbit, dog, monkey and human urine using liquid chromatography tandem mass spectrometry.

WCK 771 is an l-arginine salt of levonadifloxacin (LND) being developed in intravenous dosage form and has recently completed a phase III trial in India. The pharmacokinetics of WCK 771, a novel anti-MRSA fluoroquinolone, were examined in mice, rats, rabbits, dogs, monkeys and humans after systemic administration during pre-clinical and clinical investigations. Urine and serum were evaluated for identification of metabolites. It was observed that LND mainly follows phase II biotransformation pathways. All of the species showed a different array of metabolites. In mice, rabbit and dog, the drug was mainly excreted in the form of O-glucuronide (M7) and acyl glucuronide (M8) conjugates, whereas in rat and human major metabolite was sulfate conjugate (M6). Monkeys exhibited equal distribution of sulfate (M6) and glucuronide conjugates (M7, M8). In addition to these three major phase II metabolites; five phase I oxidative metabolites (M1, M2, M3, M4 and M5) were identified using liquid chromatography tandem mass spectrometry. Out of these eight metabolites M2, M3, M5, M7 and M8 are reported for the first time.

Development and Validation of the Chiral Liquid Chromatography Method for Separation of Enantiomeric Impurity in Novel Oxazolidinone Antibacterial Agent WCK 4086.

A highly stereo-specific liquid chromatographic method was developed and validated for the quantification of enantiomeric impurity (R-enantiomer) in novel oxazolidinone antibacterial agent (WCK 4086), a drug substance. The separation was achieved on Chiralpak AD-H (amylose-based chiral stationary phase) using a mobile phase consisting of n-hexane:2-propanol:methanol:trifluoroacetic acid (80:10:10:0.4, v/v/v/v) at a flow rate of 1.0 mL min-1. Chromatographic resolution between two enantiomers was found to be more than 2.0. Method was extensively validated for the quantification of R-enantiomer in WCK 4086 and proved to be robust. Method was found to be highly specific as all other related impurities were separated from the enantiomers. The calibration curve for R-enantiomer showed an excellent linearity over the concentration range of 1-5 µg mL-1. Limit of quantitation (LOQ) and limit of detection (LOD) for R-enantiomer were 0.009 µg and 0.003 µg, respectively. Average recovery of the R-enantiomer was in the range of 94.55-109.67%. Analytical solutions were found to be stable up to 70 h at room temperature. Developed method was found to be specific, sensitive, precise and accurate for quantitative determination of R-enantiomer in WCK 4086 and useful for controlling the enantiomeric impurity in drug substance used for preclinical studies.

Liquid chromatographic separation and thermodynamic investigation of lorcaserin hydrochloride enantiomers on immobilized amylose-based chiral stationary phase.

A novel liquid chromatographic method was developed for enantiomeric separation of lorcaserin hydrochloride on Chiralpak IA column containing chiral stationary phase immobilized with amylose tris (3.5-dimethylphenylcarbamate) as chiral selector. Baseline separation with resolution greater than 4 was achieved using mobile phase containing mixture of n-hexane/ethanol/methanol/diethylamine (95:2.5:2.5:0.1, v/v/v/v) at a flow rate of 1.2 mL/min. The limit of detection and limit of quantification of the S-enantiomer were found to be 0.45 and 1.5 µg/mL, respectively; the developed method was validated as per ICH guideline. The influence of column oven temperatures studied in the range of 20°C to 50°C on separation was studied; from this, retention, separation, and resolution were investigated. The thermodynamic parameters ΔH°, ΔS°, and ΔG° were evaluated from van't Hoff plots,(Ink' versus 1/T) and used to explain the strength of interaction between enantiomers and immobilized amylose-based chiral stationary phase.