Zhihong Ge

Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases

The enantioselectivity of a collection of neutral pharmaceutical compounds on six different types of polysaccharide chiral stationary phases (CSPs), Chiralpak AD (and AD-RH), Chiralcel OD (and OD-RH), Chiralpak OJ (and OJ-R), Chiralcel AS (and AS-RH), Sepapak-2 and Sepapak-4 are compared using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). Screening strategies for maximizing the probability of achieving an initial chiral separation hit for neutral compounds using both RPLC and NPLC are described. Further method optimizations are demonstrated by modifying parameters such as organic modifier composition, eluent pH or CSP particle size. Several practical examples of the application of chiral methods for the study of synthetic reaction mixtures are presented. The most critical validation aspects, including limit of detection, specificity, and ruggedness, are also briefly presented.

Use of a Quality-by-Design approach to justify removal of the HPLC weight % assay from routine API stability testing protocols

Due to the high method variability (typically > or = 0.5%, based on a literature survey and internal Merck experience) encountered in the HPLC weight percent (%) assays of various active pharmaceutical ingredients (APIs), it is proposed that the routine use of the test in stability studies should be discouraged on the basis that it is frequently not sufficiently precise to yield results that are stability-indicating. The high method variability of HPLC weight % methods is not consistent with the current ICH practice of reporting impurities/degradation products down to the 0.05% level, and it can lead to erroneous out-of-specification (OOS) results that are due to experimental error and are not attributable to API degradation. For the vast majority of cases, the HPLC impurity profile provides much better (earlier and more sensitive) detection of low-level degradation products. Based on these observations, a Quality-by-Design (QbD) approach is proposed to phase out the HPLC weight % assay from routine API stability testing protocols.

Examination of rofecoxib solution decomposition under alkaline and photolytic stress conditions

Rofecoxib is a highly active and selective cyclo-oxygenase II inhibitor. A stability-indicating method for the assay of rofecoxib has been developed using reverse-phase high-performance liquid chromatography (HPLC). Stress testing of rofecoxib was conducted during the method development and validation. HPLC analysis of rofecoxib solutions stressed under alkaline and photolytic conditions revealed the presence of several degradates. Two main degradates were determined to be the cyclization product formed by photo-cyclization and the dicarboxylate formed by ring opening in the presence of base and oxygen. The identities of these degradates were confirmed by comparison of UV spectra and HPLC retention time with the independently synthesized products. The mechanistic pathways for the formation of these degradates are discussed. Further improvement of the HPLC methods ruggedness has been made based on these studies.

Determination of tungsten in bulk drug substance and intermediates by ICP-AES and ICP-MS.

A quick and sensitive method has been developed and validated for the determination of tungsten in bulk drug substance and intermediates using either Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES) or Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Sample preparation is by direct dissolution with a 80:20 (v/v) concentrated nitric acid:deionized water mixture and avoids labor intensive and potentially hazardous digestion techniques. Excellent agreement was found between ICP-AES and ICP-MS results and between Merck results and Microwave Induced Plasma Mass Spectrometry (MIP-MS) results provided by an independent raw material vendor.

Development and Validation of an HPLC Method for the Impurity and Quantitative Analysis of Etoricoxib

Abstract Etoricoxib (5‐chloro‐6′‐methyl‐3[4‐(methanesulfonyl)phenyl]‐2,3′‐bipyridine) is a highly active and selective cyclo‐oxygenase II inhibitor. A single, stability‐indicating HPLC method has been developed and validated for both the impurity and quantitative analysis of etoricoxib. Method development incorporated the optimization of stationary phase, pH, temperature, and mobile phase composition for the resolution of thirteen process impurities and three major degradation products. Further optimization of pH and mobile phase composition was aided by the use of DryLab®, a computer‐based simulation program. The stability‐indicating capability of the method was proven through the identification of photolytic and oxidative decomposition products. Method validation produced excellent results for linearity, precision, limit of quantitation and limit of detection, specificity, accuracy, recovery, and robustness. The identities of etoricoxib decomposition products were confirmed by UV, LC/MS, and NMR spectra.

Employment of on-line FT-IR spectroscopy to monitor the deprotection of a 9-fluorenylmethyl protected carboxylic acid peptide conjugate of doxorubicin

A method for accurately determining the end-point, >98% conversion, of the deprotection reaction of a highly toxic 9-fluorenylmethyl (Fm) ester 1b to its corresponding carboxylate 1d in real time by FT-IR spectroscopy is reported. Advantages of this method over analysis by conventional chromatographic means include real time determination of the end-point of a reaction that is time sensitive to by-product formation, and elimination of sampling a highly toxic reaction mixture. The FT-IR method is based on monitoring, in real time, the disappearance of the Fm ester carbonyl band for 1b at 1737 cm(-1), during deprotection by piperidine, and calibration models were established by Partial Least Squares (PLS) regression analysis with high performance liquid chromatography (HPLC) as reference. The best calibration model was built with 5 PLS factors in the spectral range of 1780-1730 and 1551-1441 cm(-1) and resulted in a standard error of cross validation (SECV) of 0.63 mM 1b and a standard error of prediction (SEP) of 0.51 mM 1b in the range of 0-25 mM. This error of prediction is approximately 0.8% of the initial concentration of 1b and is well within our specifications of <2% initial concentration.

On-line monitoring of the distillates of a solvent switch process by near-infrared spectroscopy

An on-line fiber-optic near-infrared spectroscopic method has been developed for real-time monitoring of distillate composition as a reliable way of assessing the degree of a solvent switch process from methanol and n-propanol to isopropyl acetate. Multivariate statistical methods of Multiple Linear Regression and Partial Least Squares were evaluated for quantitative determination of the total alcohol concentrations in the distillate samples. Robust models were obtained by incorporating temperature variance into the calibration set. Reliable prediction of total alcohol concentration was successfully achieved by near-infrared spectroscopy as judged by a comparison with gas chromatography data.

APPLICATION OF PACKED COLUMN SUPERCRITICAL FLUID CHROMATOGRAPHY FOR SEPARATION OF BROMOSULFONE FROM PROCESS RELATED IMPURITIES

Sub- or supercritical fluid chromatography (SFC) is a viable alternative method for separations that are not optimal under reversed phase conditions. 2-Bromo-4′-(methylsulfonyl) acetophenone (bromosulfone) was found to undergo on-column degradation under reversed phase conditions. An SFC separation was developed wherein bromosulfone could be resolved from seven process-related impurities within a run time of five minutes. The effect of column type, modifier type, temperature, and pressure were investigated during the method development.

DEVELOPMENT OF A DERIVATIZATION METHOD, COUPLED WITH REVERSE PHASE HPLC, FOR MONITORING THE FORMATION OF AN ENOLATE INTERMEDIATE

A sensitive liquid chromatographic method has been developed to monitor the formation of an enolate intermediate in a synthetic route to Etoricoxib, a drug candidate for the treatment of arthritis. The method requires the derivatization of the enolate with methyl iodide to form a stable methylketosulfone derivative followed by reverse phase HPLC analysis. Parameters affecting the derivatization, including the nature of derivatizing agent, reaction solvent, amount of derivatizing agent, reaction time, reaction temperature, and amount of excess base in the reaction were investigated. The derivatization reaction was shown to give selective C-alkylation. The linear range of the chromatographic method for the determination of the starting material, ketosulfone, and the derivative, methylketosulfone, was determined. Finally, the accuracy of the method was established based on recovery experiments.

Near infrared spectroscopy for the accurate measurement of crystallisation seeding point

T his article is condensed from the research paper by Zhou et al. which describes the use of near infrared (NIR) spectroscopy as an in-line method for measuring the concentration of Etoricoxib, a polymorphic drug compound, as well as detecting the presence of solids produced by premature crystallisation prior to seeding in the crystallisation process. In this NIR spectroscopic method, a spectrum was fi rst qualifi ed as that of clear solution and then the concentration of Etoricoxib was predicted by a NIR spectroscopy calibration model built with partial least squares (PLS) regression using off-line high-performance liquid chromatography (HPLC) analysis as the reference method.