Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

Comparison of chiral separations of aminophosphonic acids and their aminocarboxylic acid analogs using a crown ether column.

Crown ethers are capable of complexing with primary amines and have been utilized in chromatography to separate amino acid racemates. This application has been extended to resolve (1-amino-1-phenylmethyl)phosphonic acid and (1-aminoethyl)phosphonic acid racemates, along with their aminocarboxylic acid analogs (2-phenylglycine and alanine, respectively), via a ChiroSil RCA crown ether based chiral stationary phase. Effects of the organic modifier, temperature, and acid type and concentration on retention and selectivity were also investigated. Trends in retention and selectivity varied between aminophosponic acids and their aminocarboxylic analogs. Computer modeling and (1)H NMR analyses were performed to potentially gain a better understanding of interactions of the aforementioned molecules with the ChiroSil RCA chiral stationary phase. Theoretical predictions of the most stable conformations for (R)- and (S)-enantiomers were compared to elution order; it was found that the elution order agreed with molecular modeling such that the longest retention correlated with the predicted most stable complex between the enantiomer and crown ether. (1)H NMR demonstrated interactions of aminophosphonic and aminocarboxylic racemates with (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid in solution and was utilized to determine enantiomeric excess of (1-amino-1-phenylmethyl)phosphonic acid after its enantioenrichment via crystallization through diastereomeric salt formation with the crown ether followed by filtration.

Development of supercritical fluid extraction and supercritical fluid chromatography purification methods using rapid solubility screening with multiple solubility chambers.

Rapid solubility screening in diverse supercritical fluids (SCFs) was carried out via multiple solubility chambers with a trapping device and online ultraviolet (UV) detection. With this device, it was possible to rapidly study the solubility variations of multiple components in a mixture. Results from solubility studies have been used to develop efficient supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) methods. After the investigation of solubilities of theophylline and caffeine in several neat organic solvents and SCFs, advantages of SFE over conventional organic solvent extraction were demonstrated with a model mixture of theophylline and caffeine. The highest solubility ratio of 1:40 (theophylline:caffeine) was observed in the SCF with 20% acetonitrile (MeCN), where a ratio of 1:11 was the highest in the neat organic solvents. A model mixture of theophylline:caffeine (85:15 w/w, caffeine as an impurity) was successfully purified by SFE by leveraging the highest solubility difference. The SCF with 20% MeCN selectively removed caffeine and left theophylline largely intact. Rapid SCF solubility screening was applied to development of SFE and SFC methods in a drug discovery environment. Two successful applications were demonstrated with proprietary Amgen compounds to either remove an achiral impurity before chiral purification or enhance chiral chromatographic throughput.

Supercritical fluid chromatography comparison of the poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column with several derivatized polysaccharide-based stationary phases.

The poly(trans-1,2-cyclohexanediyl-bis acrylamide) (P-CAP) column has so far been primarily used with normal phase and polar organic mobile phase chromatography. Its use in supercritical fluid chromatography (SFC) was investigated via the analysis of 40 commercial and 100 proprietary compounds using a 12-min gradient with methanol as a modifier. Results were then compared against those obtained from the popular derivatized polysaccharide-based chiral stationary phases (CSPs) such as Chiralpak AD-H and Chiralpak AS-H as well as Chiralcel OD-H and Chiralcel OJ-H columns. P-CAP demonstrated separation of 25% of the 140 total compounds, while each of the derivatized polysaccharide-based CSPs separated at least 46%. A study that compared the loading of 1,1'-bi-2-naphthol with P-CAP and Chiralpak AS columns indicated a similar trend in resolution vs. amount injected, though AS appeared capable of allowing a greater loading of material. The P-CAP column was found to be beneficial in the separation of a complex mixture of enantiomers and achiral impurities, where the derivatized polysaccharide-based columns did not show as desirable of a separation. A key advantage of this type of chiral stationary phase is the fact that it is available in both enantiomeric forms, allowing manipulation of elution order of enantiomers, which is especially helpful for preparative applications. P-CAP also demonstrated that it could resolve an achiral impurity from the desired compound in a different mixture, while the same impurity co-eluted on the Chiralpak AD-H column. Overall, the synthetic polymer-based P-CAP showed less chiral discrimination power compared to the derivatized polysaccharide-based CSPs under the conditions explored in this study.

Supercritical fluid chromatography tandem-column method development in pharmaceutical sciences for a mixture of four stereoisomers.

A tandem-column method using Chiralpak AD-H and Chiralcel OD-H columns was achieved for baseline separation of a mixture of chiral pharmaceutical compounds (i.e., four stereoisomers) via supercritical fluid chromatography (SFC) with a mobile phase consisting of 90% liquid carbon dioxide and 10% ethanol:isopropanol (50:50 v/v). On the contrary, this mixture (mixture A) could not be baseline separated by SFC conditions explored with individual Chiralpak AD-H and Chiralcel OD-H columns. The effects of various mobile phases on elution order, capacity factor, selectivity, and resolution were determined with mixture A on the individual aforementioned columns to develop the tandem-column method.