Evaluation of chiral normal-phase liquid chromatography as a secondary tier in pharmaceutical chiral screening strategy.

Determination of stereoisomers is an integral part of pharmaceutical analysis. Chiral liquid chromatography (LC) method development is typically initiated through screening of chiral stationary phases (CSPs) and mobile phases (MPs) since chiral separation is difficult to predict. We have previously reported a screening strategy using chiral reversed-phase (RP) LC as two primary tiers due to its versatility for enantio­recognition and compatibility with diverse sample matrices. Here we focus on developing a normal-phase (NP) LC screening strategy as a secondary tier for chiral method screening. A database was constructed from 60 NPLC screens performed on up to 18 CSPs and 3 MPs using gradient elution. This was used to investigate the effectiveness of NPLC compared to RPLC screening, as well as the impact of MP composition and the selectivity of different CSPs in NPLC screening. A success hit rate of 90% was observed in NPLC compared to 84% in RPLC screening for Bristol Myers Squibb compounds. Importantly, NPLC screening generated successful hit(s) in 81% of the cases that failed in RPLC, demonstrating the value of NPLC as a complementary screening tier. After optimizing the CSP/MP selection, we proposed a NPLC screening workflow with several user-options according to method requirements and instrument capacity. Among these, the most comprehensive NPLC screening consisted of ten CSPs (AD, AS, AY, AZ, OD, OJ, IC, IE, IG, O1) with three MPs. When combined with RPLC, an overall success rate of 97% was achieved for the diverse set of pharmaceutical compounds.

Evaluation of a polysaccharide-based chiral reversed-phase liquid chromatography screen strategy in pharmaceutical analysis.

Chirality control plays a critical role in developing stereoisomeric drugs. Due to the complexity and lack of predictability in chiral separations, column screening remains the gold standard to initiate chiral method development for active pharmaceutical ingredients (APIs) and synthetic intermediates. Chiral reversed-phase (RP) liquid chromatography (LC) has gained favor over other modes due to its versatility and compatibility in analyzing a wide range of chiral compounds in various matrices. Herein, we established a tier-based chiral RPLC screen strategy by constructing and analyzing a database of 101 chiral screens with a total of 3,401 entries (unique LC runs) for proprietary APIs or intermediates at Bristol Myers Squibb. Up to 17 polysaccharide-based chiral stationary phases (CSPs) and four mobile phases (MPs) have been screened with gradient elution. A selection of ten CSPs with two MPs was found sufficient to achieve successful separation for 82% of the total screens. Two RPLC screen tiers (Tier 1: AZ, OD, ID, and IG) and (Tier 2: AY, OJ, OZ, IA, IC, and IH) were proposed along with two MPs (acidic and neutral) to target ~70% hit rate for Tier 1, and ~80% for the combined set. We also implemented a user-friendly workflow to enable walk-up chiral RPLC screening with automated reports and system suitability tests.

Measuring atropisomers of BMS-986142 using 2DLC as an enabling technology.

BMS-986142 has been developed as an innovative Bruton's tyrosine kinase inhibitor for treatment of several autoimmune diseases. The drug substance of BMS-986142 may contain three potential atropisomeric impurities due to its unique structural characteristics. Developing a single liquid chromatography (LC) method to separate all four highly structurally related atropisomers and other process impurities from each other turned out to be a daunting task. Two-dimensional LC (2DLC) was found to be an extremely powerful enabling technology for extracting purity information out of the complex sample impurity profile and facilitated process development before a final single dimension method was discovered. The off-the-shelf 2DLC instrument could be configured to allow injection of the targeted first dimension peak through either no-loss multiple heart-cutting fractions or as a large, single volume fraction with on-line dilution. Excellent precision (relative standard deviation of 0.3 %) and recovery (101.2 ± 0.2 %) was achieved for an atropisomer impurity at a 10 % monitoring level in the first configuration with sensitivity down to 0.2 % w/w. With the second instrument configuration, which eliminated the need for fraction recombination, similar figures of merit were maintained for the second dimension at the cost of losing the ability to collect and park multiple fractions.

Advancing stereoisomeric separation of an atropisomeric Bruton's tyrosine kinase inhibitor by using sub-2 µm immobilized polysaccharide-based chiral columns in supercritical fluid chromatography.

BMS-986142 is a Bruton's tyrosine kinase inhibitor under development to treat several disease types. The compound contains three chiral elements: one chiral center and two chiral axes, resulting in three potential atropisomeric impurities in its drug substance and drug products. Separation of BMS-986142 atropisomers has been successfully achieved on an achiral polar-embedded C18 column in reversed-phase liquid chromatography (RPLC) and on polysaccharide-based chiral columns in RPLC and supercritical fluid chromatography (SFC). Compared to the RPLC chiral separation, the SFC atropisomeric separation on a sub-2 µm immobilized cellulose-based column is much more efficient and environmentally friendly. The analysis time in SFC was reduced by 8-fold compared to that in RPLC, and the method sensitivity in SFC on the sub-2 µm chiral column in 3.0 mm I.D. was 2 to 4-fold better than that on 3 µm chiral columns in 4.6 mm I.D.. Furthermore, our study suggests that the contribution to band broadening from the extra column volume (ECV) of modern commercial SFC instrument was not negligible for a 3.0 mm I.D. × 100 mm column packed with 1.6 µm particles. This result reaffirms that there is a great need for further improvement of SFC instrument design in order to realize the full theoretical efficiency of both sub-2 µm achiral and chiral columns.

Transition metal-induced degradation of a pharmaceutical compound in reversed-phase liquid chromatographic analysis.

Drug degradation that occurs in HPLC analysis, during either sample preparation or chromatographic separation, can greatly impact method robustness and result accuracy. In this work, we report a case study of drug dimerization in HPLC analysis where proximate causes were attributed to either the LC columns or the HPLC instrument. Solution stress studies indicated that the same pseudo-dimeric degradants could also be formed rapidly when the compound was exposed to certain oxidative transition metal ions, such as Cu(II) and Fe(III). Two pseudo-dimeric degradants were isolated from transition metal stressed samples and their structures were elucidated. A degradation pathway was proposed, whereby the degradation was initiated through transition metal-induced single electron transfer oxidation. Further studies confirmed that the dimerization was induced by trace transition metals in the HPLC flow path, which could arise from either the stainless steel frits in the LC column or stainless steel tubing in the HPLC instrument. Various procedures to prevent transition metal-induced drug degradation were explored, and a general strategy to mitigate such risks is briefly discussed.

Direct and indirect separations of five isomers of Brivanib Alaninate using chiral high-performance liquid chromatography.

Brivanib Alaninate is a novel chiral prodrug possessing two stereogenic centers. Simultaneous HPLC separation of five isomers of Brivanib Alaninate was systematically investigated on a wide variety of polysaccharide-based chiral stationary phases (CSPs) using underivatization and pre-column derivatization methods. The influence of derivatizing groups and mobile phase composition on the enantioseparation and retention behavior of Brivanib Alaninate compounds was studied. To better understand the chiral recognition mechanism, the temperature effect was also evaluated. The results of these studies led to the first complete HPLC resolution of all five isomers of Brivanib Alaninate as carbobenzyloxy (CBZ) derivatives on a cellulose benzoate CSP (OJ-H).

Determination of the interconversion energy barrier of 2,3-pentadienedioic acid enantiomers by HPLC. 2. On-column interconversion.

In this paper, an HPLC method is used to determine the enantiomerization barrier of 2,3-pentadienedioic acid enantiomers. The racemate of 2,3-pentadienedioic acid was separated by HPLC on a chiral CHIROBIOTIC T column with a 90:10 (100:0.5:0.5 MeOH/HOAc/TEA)/H2O mobile phase. Peak areas of enantiomers prior to (A(+)0, A(-)0) and after the separation (A(+), A(-)), were used for calculation of the rate constants and the enantiomerization barrier, as determined by computer-assisted peak deconvolution of the peak clusters on the chromatograms. The kinetic equation for irreversible reactions was used to determine the apparent enantiomerization rate constants and the interconversion energy barrier. The dependence of the apparent enantiomerization barrier (deltaG1(app), deltaG-1(app)) on temperature was used to determine the apparent activation enthalpy (deltaH1(app), deltaH(-1)app) and entropy (deltaS1(app), deltaS-1(app)) for the interconversion of 2,3-pentadienedioic acid enantiomers, where the coefficients 1 and -1 designate the interconversions (+) --> (-) and (-) --> (+), respectively.

Temperature and enantioseparation by macrocyclic glycopeptide chiral stationary phases.

Seventy-one chiral compounds were separated on four macrocyclic glycopeptide chiral selectors: teicoplanin, its aglycone, ristocetin A and vancomycin, using three possible separation modes: reversed phase with methanol/buffer mobile phases, normal phase with hexane/ethanol mobile phases and polar ionic mode (PIM) with 100% methanol mobile phase with trace amounts of acid and/or base. These 148 separations were studied in a 5-45 degrees C temperature range. Peak efficiencies always increased with temperature, but in only 17% of the separations studied a small increase of the enantioresolution factor was observed. In the majority (83%) of the cases, the enantioresolution decreased or even vanished when temperature increased. All 148 Van't Hoff plots were linear showing that the selector did not change in the temperature range studied. The calculated enthalpy and entropy variations showed that the interaction of the solute with the stationary phase was always enthalpy driven with normal and reversed mobile phases. It could be enthalpy as well as entropy driven with PIM mobile phases strongly dependent on the solute. The plots of delta(deltaH) versus delta(deltaS) were linear in most cases (enthalpy entropy compensation). This observation cannot be used to give clear information on chiral recognition mechanisms, but it allowed identifying specific stationary phase-solute interactions because the points corresponding to the respective thermodynamic parameters were clearly delineated from the general compensation lines.