This study demonstrates the use of hydrophilic interaction liquid chromatography (HILIC) for the separation of both active and inactive ingredients in pharmaceuticals from a single injection. Excipients commonly used in parenteral formulations were separated using a gradient method employing increasing aqueous composition. An evaporative light-scattering detector (ELSD) provided direct detection of inactive excipients and inorganic salts lacking UV chromophores. Analyses of Gemzar parenteral formulations using optimized isocratic HILIC-ELSD method conditions were performed based on retention time screening from the gradient assay. All of the components were efficiently separated using a TSK-Gel Amide 80 column including gemcitabine, mannitol, and sodium cation demonstrating the qualitative capability of the technique. The method was thoroughly validated for mannitol content to access the quantitative potential of the technique. Validation parameters included linearity, accuracy, specificity, solution stability, repeatability, and intermediate precision. Overall, the method described in this report proved to be very robust and represents a novel technique to conveniently separate and detect the active and inactive components in pharmaceuticals both quickly and accurately.

Seven macrocyclic antibiotics were evaluated as chiral selectors for the enantiomeric separation of 11 dansyl amino acids using narrow-bore high-performance liquid chromatography (HPLC). The macrocyclic antibiotics were incorporated as mobile phase additives to determine the enantioselective effects on the chiral analytes. The resolution and capacity factor (k') of each analyte were assessed while varying the structure of macrocyclic antibiotic and the mobile phase buffer pH. The selectivity of the chiral selectors was measured as a function of changes in these parameters. All 11 dansyl amino acids were separated by at least one of the chiral selectors. Three-dimensional computer modeling of the more effective chiral selectors illustrated the importance of macrocyclic antibiotic structure concerning stereospecific analyte interaction.

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.

The authors evaluated an evaporative light-scattering detector and a chemiluminescent nitrogen detector for high performance liquid chromatography analyses of compounds that lack a sufficient ultraviolet (UV) chromophore. They compared the detectors in three critical areas of performance: precision, linearity, and limit of detection. The four compounds used as test analytes included caffeine and three pharmaceutical compounds with weak UV chromophores. Based on these experiments, the authors discuss the advantages and disadvantages of these detectors.

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 degrees 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 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.

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.

The authors have developed new analytical methodology using reversed-phase high performance liquid chromatography with evaporative light-scattering detection for the determination of LY354740, an mGluR2 agonist, from cleaning validation swab samples. Analysts can use this method for low-level quantitation of pharmaceutical drug substances that lack a sufficient UV chromophore. The authors achieved acceptable levels of precision, linearity, recovery, selectivity, and limit of detection during method validation.

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.

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.

Abstract A thorough analysis of a new commercially available pepsin chiral stationary phase (CSP) has been completed using seproxetine (S-norfluoxetine) hydrochloride bulk drug substance and R-norfluoxetine hydrochloride as the test analytes. Chromatographic properties of this new Ultron ES-Pepsin column were investigated by varying key mobile phase parameters (pH, flow rate, buffer strength and organic concentration), column temperature and sample loading. After observing and plotting changes in retention, resolution and theoretical plates based on corresponding variation in these parameters, it is possible to choose conditions for the separation that are optimum and robust. The subsequent method validation demonstrated acceptable precision, linearity, recovery, selectivity, limit of detection and ruggedness for the determination of R-norfluoxetine in seproxetine hydrochloride bulk drug substance.

Researchers traditionally have analyzed inorganic ions such as chloride in pharmaceutical drug substances by ion chromatography (IC) with conductivity detection or titration methods. The authors have developed a new quantitative method for the determination of chloride in pharmaceutical drug substances using high performance liquid chromatography (HPLC) with evaporative light-scattering detection, They compare the analyses of chloride in 17 pharmaceutical drug substances (hydrochloride salts) using HPLC analysis with evaporative light-scattering detection (ELSD) against the theoretical chloride content based on empirical formulas. In addition, they statistically compare chloride results obtained by IC, capillary electrophoresis, and titration methods with results obtained by HPLC-ELSD.

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.

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 LTB(4) 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.

The stability of fluoxetine hydrochloride in fluoxetine solution diluted with five common pharmaceutical diluents was studied. Fluoxetine syrup, containing fluoxetine 4 mg/mL (as the hydrochloride salt), was diluted to 1 and 2 mg/mL in each of the following: deionized water; Simple Syrup, British Pharmacopeia; Simple Syrup, USP; Aromatic Elixir, USP; and grape-cranberry drink. Each solution was divided into eight 120-mL amber glass bottles: four stored at 5 degrees C and four stored at 30 degrees C. Samples were removed from each bottle at time zero and two, four, and eight weeks and assayed in triplicate with high-performance liquid chromatographic methods for determining fluoxetine concentration and concentration of its primary degradation product, alpha-[2-(methylamino)ethyl]benzene methanol. Stability was established if the fluoxetine concentration changed by < 10% and if the concentration of the degradation product was < 1% of the initial fluoxetine concentration. No test mixture dropped below 95% of the initial fluoxetine concentration or exceeded 0.5% degradation product during the study period. Fluoxetine hydrochloride was stable for eight weeks in fluoxetine solution diluted to 1 or 2 mg/mL with common pharmaceutical diluents and stored at 5 or 30 degrees C.