Eric Loeser

Water-immiscible solvents as diluents in reversed-phase liquid chromatography

It has recently been shown that the use of strong organic solvents as diluent is possible in RPLC, provided that the solvent used as diluent is retained more strongly by the column than the analytes in the sample. In this study, the phenomenon was further studied experimentally using several water-immiscible solvents (ethyl acetate, isopropyl acetate, and methyl isobutyl ketone) and several model analyte compounds. In all cases, analyte peak distortion was minimal provided the analyte eluted earlier than the diluting solvent, in contrast to substantial broadening and distortion when the analyte eluted after the diluting solvent. The potential analytical utility of this approach is discussed, and an example of a practical application is also presented.

SELECTIVE N-ALKYLATION OF PRIMARY AMINES WITH CHLOROACETAMIDES UNDER pH-CONTROLLED AQUEOUS CONDITIONS

ABSTRACT A practical method for selective alkylation of primary amines with chloroacetamides under modified Finkelstein conditions using stoichiometric amounts of sodium iodide in acetonitrile/water is described. An interesting finding in this study was that α-alkylaminoacetamides are less basic than the corresponding primary alkylamines by about 2 pKa units; this necessitated a pH of 12 during alkylation to optimize the monoalkylation selectivity. Depending on the amount of primary amine used, mono : dialkylation selectivities ranged from 2.7 to 10.

Mechanism of the pinacol–pinacolone rearrangement of 2,3-di-(3-pyridyl)-2,3-butanediol in sulfuric acid

Abstract The reaction of 2,3-di-(3-pyridyl)-2,3-butanediol ( 1 ) in H 2 SO 4 was studied. It was found that the meso and racemic forms give mono- and bis-SO 3 addition products, which rearrange to a ketone (Metopirone®) and two other major by-products. The formation of SO 3 addition products and a marked increase in reaction rates with greater amount of SO 3 suggest an alternate mechanism involving sulfonyloxy leaving groups.

Chemical interactions between an active pharmaceutical ingredient and its counterion in a tromethamine salt under forced degradation conditions

In this study, the tromethamine salt of an active pharmaceutical ingredient containing both a carboxylic acid and ethyl ester functionality was subjected to forced degradation conditions. Based on HPLC-MS analysis, it was found that tromethamine formed both amide and ester type condensation products with the API, with amide formation predominating over ester formation. Addition of tromethamine at the carboxylic acid group of the API was favored over addition at the ethyl ester group. Tromethamine condensation products were observed only under the harshest stress conditions (80 degrees and 75% relative humidity), in which the salt physically changed from a crystalline form to a deliquesced state. Under stress conditions in which the crystalline structure of the salt remained intact, good stability was observed. Thus, the interaction between tromethamine and API occurred only in cases where the crystallinity of the salt was compromised.

Correlations between conformational isomerism and chromatographic diastereoselectivity of bis amino amide s-triazine derivatives.

NMR spectroscopy was used to probe the conformational behavior of diastereomeric s-triazine derivatives containing two chiral amino amide substituents, in order to shed light onto the mechanism of chromatographic diastereoselectivity. Utilizing the amino hydrogen signals in the proton NMR spectrum, the population of the conformations caused by rotation about the bond between the amino nitrogen and aromatic carbon atoms could be observed. The population distribution between the three possible conformations was similar but not identical between the two diastereomers, with similar trends being observed for both bis alanine amide and bis valine amide derivatives. Based on a simple model in which it is assumed that adsorption to the hydrophobic RP-LC stationary phase occurs only for the conformations having both amino amide R-groups on the same side of the triazine ring plane, the different conformation populations between the two diastereomers obtained by NMR was consistent with the observed RP-LC elution order (L-L diastereomer followed by L-D). The predicted diastereoselectivity values from NMR data were compared to RP-LC diastereoselectivity values obtained using both C18 and polymeric columns, with both acetonitrile/water and DMSO/water mobile phases. Values obtained with the polymeric column were in better agreement with calculated values than those obtained with the C18 column, suggesting that the simple adsorption model used to calculate the diastereoselectivity is more relevant towards a simple hydrophobic polymeric surface rather than a more complex C18 stationary phase. This study indicates that proton NMR is a useful tool for studying the diastereoselective mechanism of these derivatives, due to the relatively slow C-N bond rotation caused by the significant sp(2) character of the amino nitrogen atoms.

Possible factors influencing the separation mechanism of diastereomeric amino acid derivatives obtained from s-triazine type reagents.

Diastereomeric derivatives prepared from an amino acid and an amino amide using trichloro s-triazine as a coupling platform are known to produce good chromatographic diastereoselectivity for many amino acid analytes. The chromatographic diastereoselectivity of these derivatives is difficult to rationalize based on the possibility of four possible conformational isomers, which can interconvert by rotation about the C-N bonds between the amino substituents and the triazine ring. The observed diastereoselectivity implicates an unobvious but significant driving force which causes one of several conformations to be favored over the others. Several possibilities are discussed. Intramolecular hydrogen bonding between acid and amide substituents was explored using computer aided molecular modeling. While such hydrogen bonding may be geometrically possible between the amino acid and the amide substituents, it does not explain why derivatives produced from other chiral compounds which are not capable of the same hydrogen bonding interaction nevertheless exhibit substantial diastereoselectivity. Two other more general effects, steric hindrance to solvation and ion pairing, are therefore suggested as possible contributing factors to the chromatographic diastereoselectivity. Based on the conformational equilibrium behavior of related triazine compounds as reported in the literature, either one of these effects could influence the conformation of the diastereomeric derivatives even in the absence of intramolecular hydrogen bonding interactions between the two chiral substituents, and these effects may therefore be a contributing factor for the observed elution order of the diastereomers.

Effect of Tertiary Alcohol Additives on Enantioselectivity of the Chiral‐AGP Column

Abstract The chiral‐AGP column is a protein based HPLC column widely used for analysis of chiral compounds in pharmaceutical and pharmacological applications. Organic solvents are frequently used as mobile phase additives to control analyte retention. In many cases, switching from one solvent additive to another can influence the enantioselectivity as well as retention. The group of solvents typically used as mobile phase additives includes methanol, ethanol, 1‐propanol, 2‐propanol, and acetonitrile. In this study, the column was used to resolve four different N‐substituted amino acid derivatives. The mobile phase consisted of a pH 7 phosphate buffer with the addition of organic solvent to control retention. During method optimization, nine different organic solvent additives were compared, including two tertiary alcohols. For three of the four analytes, the tertiary alcohol additives provided significantly higher enantioselectivity than any of the commonly recommended solvent additives, affording enantioselectivities in the range of 1.4 to 3.8.

Feasibility of using gravimetry to measure excess adsorption effects of a binary solvent system in a reversed-phase high-performance liquid chromatography column

A modified method for weighing HPLC columns filled with solvent is described. The method prevents the loss of traces of solvent from within the threads of the column. The method was tested by obtaining the weights of a C18 column filled with 10 different organic solvents, showing a standard deviation on the order of 0.1%. A plot of gross column weight versus solvent density showed excellent linearity. The method was then used to weigh a column filled with several acetonitrile-water mixtures. The gross column weights were lower than would have been predicted from the density of the acetonitrile-water mixtures. A likely explanation is the existence of an adsorbed acetonitrile-rich liquid on the surface of the C18 adsorbent, which caused the lower than expected weights due to the lower density of pure acetonitrile relative to the bulk mixtures. The volume of pure acetonitrile required for the observed weight discrepancy was calculated. Based on the surface area of the column adsorbent, values of micromoles acetonitrile per square meter of surface area were determined. The values showed reasonable agreement with values obtained from published adsorption isotherm studies. This suggests that pycnometry may be a useful technique for adsorption studies. The limitations of the technique are discussed.

ANALYTICAL METHOD FOR 1-METHYL-4-AMINO-PIPERAZINE IN AN ACTIVE PHARMACEUTICAL INGREDIENT USING CHEMICAL DERIVATIZATION AND HPLC-UV

A method to form a UV-active derivative of 1-methyl-4-amino-piperazine (AMP) was developed. The method was based on the reaction of AMP with benzaldehyde, forming a stable derivative, which was UV-active. The method was used to analyze samples of an active pharmaceutical ingredient (API) for trace amounts of AMP. The derivatization approach allowed detection of AMP at low levels, using readily available HPLC-UV instrumentation. The method was validated in the range of about 30 to 190 ppm (on a mass to mass basis relative to API). It was shown through spiked recovery tests that the derivatization reaction occurred smoothly without interference from the API. The results suggest that other organohydrazine compounds may be amenable to the same derivatization technique.