Jeffrey R. Mazzeo

Novel chiral surfactant for the separation of enantiomers by micellar electrokinetic capillary chromatography.

A novel chiral surfactant was prepared as both enantiomeric forms, (R)- and (S)-N-dodecoxycarbonylvaline, and employed for the separation of enantiomeric mixtures by micellar electrokinetic capillary chromatography (MECC). The enantioselectivities (alpha) obtained for twelve typical pharmaceutical amines using the (S)-surfactant were compared to those obtained with (S)-N-dodecanoylvaline, a chiral surfactant described in the literature. Higher enantioselectivities were seen for ten of the twelve compounds using (S)-N-dodecoxycarbonylvaline. Furthermore, (S)-N-dodecoxycarbonylvaline had significantly less background absorbance in the low UV. It is shown that exact enantiomer migration order reversal can be obtained by individually employing both enantiomeric forms of the surfactant. For ionizable compounds like the amines examined here, enantioselectivity can be optimized by changing the pH of the MECC buffer. Partitioning is optimized through surfactant concentration, organic additives and pH. The ability to achieve fast chiral separations is shown. A separation of ephedrine enantiomers in urine is shown, with the only sample preparation being filtration.

Electrospray ionization quadrupole ion-mobility time-of-flight mass spectrometry as a tool to distinguish the lot-to-lot heterogeneity in N-glycosylation profile of the therapeutic monoclonal antibody trastuzumab.

Monoclonal antibodies are typically glycosylated at asparagine residues in the Fc domain, and glycosylation heterogeneity at the Fc sites is well known. This paper presents a method for rapid analysis of glycosylation profile of the therapeutic monoclonal antibody trastuzumab from different production batches using electrospray quadrupole ion-mobility time-of-flight mass spectrometry (ESI-Q-IM-TOF). The global glycosylation profile for each production batch was obtained by a fast LC-MS analysis, and comparisons of the glycoprofiles of trastuzumab from different lots were made based on the deconvoluted intact mass spectra. Furthermore, the heterogeneity at each glycosylation site was characterized at the reduced antibody level and at the isolated glycopeptide level. The glycosylation site and glycan structures were confirmed by performing a time-aligned-parallel fragmentation approach using the unique dual-collision cell design of the instrument and the incorporated ion-mobility separation function. Four different production batches of trastuzumab were analyzed and compared in terms of global glycosylation profiles as well as the heterogeneity at each glycosylation site. The results show that each batch of trastuzumab shares the same types of glycoforms but relative abundance of each glycoforms is varied.

Validation of enantiomeric separations by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The first report on the feasibility of method validation for the separation of enantiomeric mixtures by chiral micelle electrokinetic capillary chromatography is described. Method validation data elements were investigated according to U.S. Pharmacopeia protocol and are reported for the separation of ephedrine enantiomers using the synthetic chiral surfactants ( S )- and ( R )-N-dodecoxycarbonylvaline as buffer additives. Excellent linearity from 1–150% of the target concentration was obtained. A 1.0% limit of quantitation for an enantomeric impurity with acceptable precision is shown. Method robustness and ruggedness is also presented. Migration order reversal was used for the determination of a trace enantiomeric impurity, and to identify the enaniomeric compound in a multiple active ingredient formulation. The capability to reverse migration order is shown to be a valuable tool to satisfy the U.S. Pharmacopeia specificity requirement as well as improving detection and quantitation limits.

Separation of piperidine-2,6-dione drug enantiomers by micellar electrokinetic capillary chromatography using synthetic chiral surfactants

Abstract The enantioselective separation of five racemic piperidine-2,6-dione compounds was accomplished using chiral micelle electrokinetic capillary chromatography. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The resolution of all five compounds with simultaneous enantioselective separation of four of the five was obtained in a single run, using the synthetic chiral surfactant (S)-N-dodecoxycarbonylvaline. An enantioselective separation of the fifth compound was obtained by a second synthetic chiral surfactant, (R)-dodecoxycarbonylproline. It was observed that surfactant type and concentration, pH, and sample matrix all effect enantiomeric resolution. The migration order of cyclohexylaminoglutethimide enantiomers was confirmed by injecting a sample comprised mostly of the (−)-enantiomer. In addition, the separation of a sixth related sample (a thalidomide mixture) is shown to be achiral and not enantiomeric, as verified by the lack of enantiomer migration order reversal.

Chapter 3 - Techniques for sample preparation using solid-phase extraction

This chapter describes techniques for sample preparation using solid-phase extraction (SPE). The 1-D reversed-phase sample clean-up method is a simple technique designed to remove major interferences such as plasma proteins or polar compounds. The 2-D reversed-phase solid-phase extraction method provides much cleaner backgrounds, but the development of the method is more complex. A range of useful methods for the sample preparation of samples of biologic origin (plasma and urine) prior to high-performance liquid chromatography (HPLC) or HPLC and mass spectrometry (MS) or MS analysis are discussed in the chapter, with an emphasis on the general principles of each method. Each method can carried out with multiple analytes and can be adapted to related sample preparation problems without difficulties. However, occasionally, the particular properties of a sample matrix, an analytical technique, or even simply the analytes require departures from the details of the approaches described in the chapter. Both offline and online SPE techniques have advantages depending upon the particular application, and additional progress in the future is anticipated with respect to the speed and efficiency of both approaches.