Donald S. Risley

Novel Method for the Determination of Piperazine in Pharmaceutical Drug Substances Using Hydrophilic Interaction Chromatography and Evaporative Light Scattering Detection

Abstract A novel method for the determination of piperazine in pharmaceutical drug substances was developed using high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). This method uses the hydrophilic interaction chromatography (HILIC) mode on a cyanopropyl (CN) bonded stationary phase. Optimization of organic modifier and acid composition in the mobile phase resulted in robust chromatography conditions with excellent resolution, peak shape, and retention time for the piperazine peak. The method was further evaluated with respect to linearity, precision, selectivity, limit of detection (LOD), and reproducibility. Based on the data provided, this HPLC–ELSD method demonstrated acceptable levels of linearity, precision, LOD, and selectivity for determination of piperazine.

Simultaneous Resolution and Detection of a Drug Substance, Impurities, and Counter Ion Using A Mixed-Mode HPLC Column with Evaporative Light Scattering Detection

Abstract An alternative approach to developing individual potency, impurity, and counter ion methods is the simultaneous resolution and detection of the drug substance, impurities, and the counter ion in a single chromatogram. LY326315 hydrochloride was used as a model compound to demonstrate this concept. The separation was achieved using a conventional HPLC system with an Alltech mixed-mode column, a reversed phase eluant, and evaporative light scattering detection (ELSD). The mixed-mode column, which has both reversed phase and ion chromatography functionalities (e.g. phenyl/cation, C8/anion), coupled with ELSD offers a novel approach to simultaneously resolving and detecting pharmaceutical compounds and counter ions in a single chromatogram.

Evaluation of a new macrocyclic antibiotic as a chiral selector for use in capillary electrophoresis

Abstract A new macrocyclic antibiotic, LY307599, has been evaluated as a chiral selector for the separation of the enantiomers of flurbiprofen using capillary electrophoresis (CE). The effect of varying separation buffer parameters such as buffer strength, pH, LY307599 concentration and methanol concentration were assessed. Using the optimized CE conditions, the separation of flurbiprofen enantiomers can be achieved using LY307599 as a chiral selector.

AMINO ACID ANALYSIS OF PEPTIDES USING HPLC WITH EVAPORATIVE LIGHT SCATTERING DETECTION

A new procedure for the amino acid analysis of peptides has been devised utilizing high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). This procedure eliminates the need for complex derivatization schemes inherent of previous amino acid analysis procedures since the ELSD detects the amino acids directly. This quantitative method detects and separates 18 of the common amino acids in a one hour run time using cation exchange chromatography coupled with ELSD. The procedure was tested by analyzing the hydrolysate of human parathyroid hormone 1–34 (PTH), a synthetic polypeptide. A standard digestion consisting of 24 hour hydrolysis at 110°C in 6 N hydrochloric acid with 3% phenol was used. Validation data reveal this is an accurate and precise procedure for the amino acid analysis of peptides. The sensitivity of this technique is low compared to other state-of-the-art analytical techniques. Detection limits varied depending on the amino acid being analyzed and were...

A simple and efficient approach to reversed-phase HPLC method screening

The development and utility of an efficient HPLC method screening strategy using only four columns for the separation of pharmaceutical compounds and related impurities is presented. The strategy established a two-column approach to enable rapid early method development, along with a four-column approach for commercial method development of the analytical methods utilized to verify the quality of drug substance or drug product. Mobile phases consisted of acetonitrile or methanol with aqueous trifluoroacetic acid for low pH screening, and ammonium hydroxide for high pH screening. Examples are provided to demonstrate the practicality and orthogonality of the method screening process. A unique system suitability check, using commercially available compounds, was incorporated as a tool for troubleshooting and for ensuring adequate system performance prior to screening. Initial testing of the strategy revealed that the columns chosen were successful in leading to assay and impurity methods for 40 pharmaceutical compounds.

Validation of an HPLC Method for the Determination of Sodium in LY293111 Sodium, a Novel LTB4 Receptor Antagonist, Using Evaporative Light Scattering Detection

Abstract Analysis of inorganic ions such as sodium or chloride in pharmaceutical compounds has traditionally employed ion-chromatography (IC) with conductivity detection. A new quantitative method for the determination of sodium in LY293111 sodium, a novel LTB4 receptor antagonist, using high performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD) is discussed. The separation of sodium from other ions and interferences was achieved using a Zorbax 300 SCX cation-exchange column suitable for use with organic solvents. Acceptable levels of precision, linearity, recovery, selectivity and limit of detection were achieved during the validation of the method. The results of this method were within 99.8% agreement when compared to the theoretical amount of sodium in LY293111 sodium. HPLC coupled with evaporative light scattering detection offers a practical alternative to IC using conductivity detection in pharmaceutical compounds.

A High-Performance Liquid Chromatography Method for the Quantitation of Impurities in an NMDA Antagonist Using Evaporative Light Scattering Detection

Abstract A reversed-phase high-performance liquid chromatography (HPLC) method utilizing an evaporative light scattering detector (ELSD) was developed for a new NMDA (N-methyl-D-aspartate) antagonist. This method permits quantitation of both the bulk drug substance purity and the related materials possible within the bulk drug substance. The method is compatible with LC/MS and the mass spectral data were obtained for each component in the bulk drug substance.

Drug-Excipient Interactions of Seproxetine Maleate Hemi-Hydrate: Isothermal Stress Methods

Seproxetine maleate hemi-hydrate was originally formulated with pregelatinized starch, to provide 1 and 20 mg free base equivalent gelatin capsule dosage forms for storage at 25°C and 40°C. HPLC analysis after 3 months revealed the formation of a 1,4 Michael addition adduct in each case. No additional degradation products were detected. To pursue a less interactive formulation, 5 mg formulation equivalent mixtures of seproxetine maleate hemi-hydrate were prepared with pregelatinized starch, lactose, and talc; thus, three distinctly different excipient classifications. These were evaluated in additional isothermal stress experiments at 25°C, 40°C, and 50°C. The results indicated that each excipient interacted with the drug in a unique chemically and thermally dependent manner. Thus, the drug-pregelatinized starch data may be represented by an Arrhenius type relationship, with activation energy of 32 kcal/mol, and formation of the previously described adduct. However, the drug-lactose data suggest reaction ...

Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC

The macrocyclic antibiotic LY333328 has been evaluated as a chiral selector for the enantioseparation of nine dansylated amino acids. This macrocyclic glycopeptide was used as a chiral mobile phase additive (CMPA) in conjunction with narrow bore high-performance liquid chromatography (HPLC). The key mobile phase parameters of LY333328 concentration and buffer pH were varied, along with variations in stationary phases consisting of C8, phenyl, cyano, and silica. After observing and plotting changes in retention and resolution based on corresponding variation in these parameters, a better understanding of the behavior of this chiral selector was obtained. The pKa values of the dansyl amino acid analytes and LY333328 were measured and used to gain a better understanding of the microenvironment in which these enantioseparations occur. Optimized conditions resulted in the baseline separation of eight of nine dansyl amino acids.

Capillary Electrophoretic Enantiomeric Separations of Nonsteroidal Anti-inflammatory Compounds Using the Macrocyclic Antibiotic Actaplanin A and 2-Methoxyethanol

Abstract Actaplanin A, a macrocyclic antibiotic, was examined as a chiral selector in capillary electrophoresis (CE) for the enantioseparation of several racemic nonsteroidal antiinflammatory compounds. The chiral selectivity of this macrocyclic antibiotic was evaluated as a function of the run buffer pH, chiral selector concentration, and organic modifier composition. Optimized conditions using 15-30% of 2-methoxyethanol and 0.5 mM actaplanin A in 40 mM phosphate buffer (pH 6) were successful in separating all of the enantiomers from the racemic compounds tested in this study.