David J. Burinsky

Hydrophilic interaction chromatography on amino-silica phases complements reversed-phase high-performance liquid chromatography and capillary electrophoresis for peptide analysis

Hydrophilic interaction chromatography (HILIC) on amine bonded-phase silica columns provides separations of peptides that are complementary to those obtained with reversed-phase HPLC and free solution capillary electrophoresis. This is illustrated with the peptide drug atosiban and nine diastereomers. Moreover, one of the HILIC methods was suitable for coupling with electrospray mass spectrometry.

Separation and identification of retinoic acid photo-isomers

Abstract Retinoic acid was isomerized in ethanol—water (90:10) with fluorescent light. Reversed-phase high-performance liquid chromatography (HPLC) on a 3-μm ODS-2 column with a highly specific mobile phase allowed simultaneous determination of ten retinoic acid isomers that were produced during the photoisomerization. Nine of the isomers were isolated by HPLC and characterized by spectroscopic methods (1H NMR, mass spectrometry and UV). The variation of product distribution with time was determined over the course of the reaction (21 h).

Detection of choline and acetylcholine in a pharmaceutical preparation using high-performance liquid chromatography/electrospray ionization mass spectrometry

A sensitive, rapid, and specific method for the detection of choline and acetylcholine in a pharmaceutical preparation is described. The method employs a perfluorinated carboxylic acid as ion-pairing reagent, post-column addition of a surface tension reducing agent and mass spectrometric detection using either selected ion monitoring (SIM) or selected reaction monitoring (SRM) modes. The resulting chromatographic performance is comparable or superior to methods reported previously in both quality of the separation and sensitivity when using mass spectral detection, with the added advantage of reduced cycle time. Acetylcholine is easily and rapidly separated from its major decomposition product choline. The method was able to detect acetylcholine and its primary degradation product choline at the 30 fmol level, with an analysis time of less than 6 min.

Characterizationf of autoxidation products of retinoic acid

Abstract Retinoic acid underwent autoxidation in 90% ethanol at 25–85.5 °C to give epoxides, dioxetanes, an endoperoxide, and double-bond cleavage products. The majority of these products appear to have resulted from the initial direct oxidation of the olefinic carbons rather than from the expected allylic α oxidation process.

Mass spectrometric analysis of complex mixtures then and now: the impact of linking liquid chromatography and mass spectrometry

Abstract On-line compound isolation (using column switching) for the analysis of complex mixtures encountered in the pharmaceutical development process has been investigated. The strategy was used for the analysis of low-level compounds that responded poorly (or not at all) under standard atmospheric pressure ionization LC/MS conditions. Analytes were prepared using small secondary columns after the analytical separation. Subsequently, the retained compounds were eluted and interrogated using experimental conditions designed to maximize mass spectrometric information content; these conditions included optimized solvent systems, optimized flow rates, chemical manipulation of the sample, extended acquisition time, and other appropriate mass spectral techniques. The challenges of obtaining comprehensive qualitative information about a mixture component under the restrictive conditions of validated regulatory HPLC methods are discussed in the context of the historical framework of direct mixture analysis using mass spectrometric approaches.

Identification of a pharmaceutical packaging off-odor using solid phase microextraction gas chromatography/mass spectrometry

The use of a solid phase microextraction (SPME) sampling technique, in conjunction with gas chromatography/mass spectrometry (GC/MS) analysis, to identify an off-odor in a heat-stressed pharmaceutical packaging material is described. The ability of the commercially available polydimethylsiloxane (PDMS) coated microfiber to concentrate a trace volatile compound of interest enabled identification of the odor compound of interest. Despite being present at levels that defied detection using conventional headspace sampling techniques, ethyl-2-mercaptoacetate was determined to be the compound responsible for the offending odor. Formation of the thioester resulted from an unanticipated reaction (either esterification or transesterification) between a common residual solvent (ethanol), present in a commonly used pharmaceutical tablet dispersant, and low-level amounts of reactants or synthetic intermediates of an FDA-approved polyvinyl chloride (PVC)-resin thermal stabilizing agent.

Mass spectral fragmentation reactions of a therapeutic 4-azasteroid and related compounds

Mass spectra were acquired for a therapeutic 4-azasteroid (dutasteride), and some related compounds, using various ionization conditions (EI, CI, APCI and ESI) in both positive and negative ion modes. The ionization and fragmentation behavior of the compound dutasteride, its precursors and several analogs is reported. Positive atmospheric pressure chemical ionization (APCI+) and positive electrospray ionization (ESI+) produced distinctive collision-induced dissociation (CID) spectra for the respective [MH]+ ions of dutasteride. The spectral differences are attributed to ion populations having either different structures or different internal energy distributions (as a consequence of the method of ionization). Irrespective of their origin, the protonated molecules undergo interesting fragmentation reactions when collisionally activated. The identity of the major fragmentation products was confirmed by accurate mass measurement. The negative APCI mass spectrum of dutasteride displays extensive dehydrohalogenation, apparently due to the thermal component of the APCI process. Some of the resulting radical anions display remarkable stability toward collisional decomposition. Details of the fragmentation behavior for the negative ion species and their relationship to the positive ion results are discussed.

Quantification of naturally occurring pyrrole acids in melanosomes.

Three naturally occurring pyrrole acids were found in Sepia, human black hair, and bovine choroid and iris melanosomes using high‐performance liquid chromatography and mass spectrometry—pyrrole‐2,3‐dicarboxylic acid (PDCA), pyrrole‐2,3,5‐tricarboxylic acid (PTCA) and pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA). PDCA and PTCA are common markers quantified from oxidative degradation of eumelanins. Using standards, the amounts of naturally occurring PDCA and PTCA were determined and compared to those obtained following peroxide oxidation of the same samples. Because the naturally occurring acids are water soluble, these results indicate that care must be exercised when comparing PDCA and PTCA yields from the degradation analyses of melanins isolated and prepared by different methods. This work also establishes that PTeCA is a naturally occurring pyrrole acid in melanosomes.

Isolation of the components of a complex mixture by means of column switching for their enhanced detection by mass spectrometry.

Mass spectral characterization of low-level impurities in drug substances and formulations may be challenging when using a validated HPLC method developed for optimal chromatographic performance. In many cases, either the mobile phase contains non-volatile additives that are deleterious to the operation of the mass spectrometer, or some of the related substances fail to ionize effectively under electrospray ionization or atmospheric pressure chemical ionization conditions. This paper describes a way to capture these low-level compounds from an analytical HPLC column using a small trapping column. Mixture components are retained on the trapping column by means of reducing the solvent strength of the eluent. Subsequent elution of trapped compounds using mobile phases more amenable to mass spectral analysis yields improved detection and characterization of low-level compounds of interest. Possible applications of peak trapping and elution include: (1) analysis of compounds separated using a mobile phase containing high concentrations of non-volatile additives, (2) analysis of organic acids separated using a low-pH mobile phase (containing trifluoroacetic acid), and (3) improving the detection limit of a low-level compound of interest through multiple collections. The peak trapping apparatus and optimization experiments are described.

Characterization of autoxidation products of the antifungal compounds econazole nitrate and miconazole nitrate

Abstract Econazole nitrate 1 and miconazole nitrate 2 underwent autoxidation in 90% ethanol at 77 °C in the presence of AIBN and oxygen. The benzylic methylene carbons of 1 and 2 , the benzylic methine carbon of 2 , and the imidazole ring of 2 were sites of oxidation.