Integration of supercritical fluid chromatography into drug discovery as a routine support tool. II. investigation and evaluation of supercritical fluid chromatography for achiral batch purification

Supercritical fluid chromatography (SFC) has recently been implemented within our analytical technologies department as a purity assessment and purification tool to complement HPLC for isomer and chiral separations. This report extends the previous work to achiral analysis and purification. This internal evaluation explores the potential impact SFC can have on high throughput, batch purification. Achiral methods have been optimised and batches of compounds purified using a retention time mapping strategy. Here the preparative retention time is predicted from a standard calibration curve and fraction windows set to ensure the peak of interest is collected in one of the four available fraction positions. In this contribution, a completely indirect scale up strategy is applied using totally independent analytical and preparative methods. This novel approach allows for fast analytical purity analysis without compromising the ability to scale up to the preparative system. The benefits and limitations of SFC for batch purification are described in comparison to HPLC across a set of standard compounds and a set of 90 research compounds.     

Screening approach for chiral separation of pharmaceuticals. Part III. Supercritical fluid chromatography for analysis and purification in drug discovery

High-throughput and performance analysis and purification of enantiomers are important parts of drug discovery and provide high-quality compounds for pharmacological testing. We have previously reported two parts describing chiral chromatographic screens using normal-phase (NPLC) and reversed-phase (RPLC) liquid chromatography, in order to cope with increasing numbers of new compounds generated by chemistry programs. We present in this part the development and implementation of a third faster screen using supercritical fluid chromatography (SFC) to maximize chance in achieving rapid enantiomer resolution of large numbers of compounds in a minimum of time. The SFC screen utilizes a narrow combination of only four columns (Chirlapak AD and AS, and Chiralcel OD and OJ) and two solvent modifiers (methanol and isopropanol). A modifier and column-switching setup was employed to allow the entire screening process to be serially run in the order AD> OD > OJ > AS and methanol > isopropanol, so that the screening for a given molecule can be stopped when separation is achieved. The switching system was fully automated for unattended operation of multiple compounds. An optimization procedure was also defined, which can be performed if needed for unsuccessful separations in the screening step. The chiral SFC strategy proved its performance and robustness in resolution of hundreds proprietary chiral molecules generated by drug discovery programs, with a success rate exceeding 95%. In addition, the generic capability of the strategy was evaluated by applying the screen and optimization methodology to a test set comprising 40 marketed drugs differing from proprietary compounds in terms of chemical diversity, revealing a similar high success rate of 98%. Chiral separations developed at the analytical scale work easily and equally well at the semi-preparative level, as illustrated with an example. The SFC screen allows resolution of compounds that were partially separated by NPLC or not separated at all by RPLC, demonstrating the utility of implementing complementary chromatographic techniques. The SFC screen is currently an integral part of our analytical support to discovery chemical programs and is considered the first try for chiral separations of new compounds, because it offers a higher success rate, performance and throughput.     

Aroma WaterLOGSY: a fast and sensitive screening tool for drug discovery

One-dimensional NMR spectroscopy has proven to be a powerful technique for screening compound libraries in drug discovery. We report a novel water ligand-observed gradient spectroscopy (WaterLOGSY) pulse sequence, named Aroma WaterLOGSY, that selectively detects aromatic WaterLOGSY signals from compounds or ligands. In the Aroma WaterLOGSY, water magnetization is untouched after water excitation and utilizes the whole period of the remaining pulse sequence to relax back to the +z direction. Due to the phase cycling design, the water magnetization is allowed to relax for the period of two full scans before it gets inverted again. Therefore, the recycle delay can be significantly shortened. Within similar experimental time, Aroma WaterLOGSY shows approximately two times higher sensitivity than the standard scheme. This method also allows the use of non-deuterated reagents, thereby accelerating experimental set-up time for ligand-binding studies.     

Development of a mass-directed preparative supercritical fluid chromatography purification system

In this paper, we report the development of a mass-directed supercritical fluid chromatography (SFC) purification system. We have addressed issues on software compatibility, the interface between the preparative SFC and the mass spectrometer, and fraction collection. Good peak shape and signal were achieved in the mass spectrometry (MS) trace, allowing accurate peak detection and reliable fraction collection. Simple modifications on a commercially available fraction collector enabled fractionation at atmospheric pressure with high recovery. The SFC/MS purification system has been used in support of high-throughput library purification and has been proven to be a valuable tool in complementing our reversed-phase high-performance liquid chromatograph (RP-HPLC/MS)-based technology platform.     

Rapid method development for chiral separation in drug discovery using sample pooling and supercritical fluid chromatography-mass spectrometry

A novel strategy for rapid chiral method development has been implemented using sample pooling and supercritical fluid chromatography-mass spectrometry (SFC-MS) on four chiral stationary phases, namely Chiralpak AD and AS, and Chiralcel OJ and OD, and eight different modifier concentrations (5 to 40% methanol-0.2% isopropylamine). The screening is performed under an outlet pressure of 110 bar at 35 degrees C, and at a flow-rate of 2.5 ml/min for the initial 20 min and then ramped up to 4 ml/min and held for 4.5 min to elute all solutes from the column. The entire process is fully automated from injection to data processing, and operates unattended for 15 h overnight to obtain optimal chiral separation for multiple compounds. A unique feature of using SFC-MS to monitor chiral synthesis is the negligible interferences from achiral impurities. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than UV and much shorter analysis times compared to normal-phase/reversed-phase liquid chromatography.     

FastTrack to supercritical fluid chromatographic purification: Implementation of a walk-up analytical supercritical fluid chromatography/mass spectrometry screening system in the medicinal chemistry laboratory

Medicinal chemists often depend on analytical instrumentation for reaction monitoring and product confirmation at all stages of pharmaceutical discovery and development. To obtain pure compounds for biological assays, the removal of side products and final compounds through purification is often necessary. Prior to purification, chemists often utilize open-access analytical LC/MS instruments because mass confirmation is fast and reliable, and the chromatographic separation of most sample constituents is sufficient. Supercritical fluid chromatography (SFC) is often used as an orthogonal technique to HPLC or when isolation of the free base of a compound is desired. In laboratories where SFC is the predominant technique for analysis and purification of compounds, a reasonable approach for quickly determining suitable purification conditions is to screen the sample against different columns. This can be a bottleneck to the purification process. To commission SFC for open-access use, a walk-up analytical SFC/MS screening system was implemented in the medicinal chemistry laboratory. Each sample is automatically screened through six column/method conditions, and on-demand data processing occurs for the chromatographers after each screening method is complete. This paper highlights the “FastTrack” approach to expediting samples through purification.     

超临界流体色谱与高效液相色谱分离手性化合物的比较

超临界流体色谱(SFC)分离具有速度快、分离效率高、溶剂消耗少等优点,近年来在手性化合物的分离分析中得到诸多应用。本文对比研究了涂覆型多糖手性色谱柱在SFC和高效液相色谱(HPLC)上拆分24种手性化合物的差异。通过比较这些化合物在色谱柱上的保留时间和选择因子等发现多数化合物在SFC上的分离效率要高于其在HPLC上的分离效率,但HPLC对轴手性化合物的分离效率要优于SFC。SFC和HPLC的分离表现出一定的互补性,随着苯环侧链烷基的碳数增加,化合物在SFC上的保留逐渐增强,而在HPLC的保留却逐渐减弱。叶菌唑在使用SFC和HPLC分析时出现了洗脱顺序反转的现象。这些结果为SFC手性拆分提供了参考。     

Synthesis of cationic beta-cyclodextrin derivatives and their applications as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography

Four cationic beta-cyclodextrin derivatives, namely mono-6-(3-methylimidazolium)-6-deoxy-perphenylcarbamoyl-beta-cyclodextrin chloride (MPCCD), mono-6-(3-methylimidazolium)-6-deoxyper(3,5-dimethylphenylcarbamoyl)-beta-cyclodextrin chloride (MDPCCD), mono-6-(3-octylimidazolium)-6-deoxyperphenylcarbamoyl-beta-cyclodextrin chloride (OPCCD) and mono-6-(3-octylimidazolium)-6-deoxyper(3,5-dimethylphenylcarbamoyl)-beta-cyclodextrin chloride (ODPCCD), have been synthesized and physically coated onto porous spherical silica gel to obtain novel chiral stationary phases (CSPs). The performances of these CSPs are studied on high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) using 18 racemic aryl alcohols as test analytes. Among these four CSPs, OPCCD shows the best separation results for all analytes on both HPLC and SFC analyses. Chromatographic studies reveal that the CSPs consisting of an n-octyl group on the imidazolium moiety and phenylcarbamoyl groups on the cyclodextrin ring provide enhancement of analyte-chiral substrate interactions over CSPs bearing the methyl group on the imidazolium moiety and 3,5-dimethylphenylcarbamoyl groups on the cyclodextrin ring.     

Analysis of sorbitan ester surfactants. Part II: Capillary supercritical fluid chromatography

A rapid, simple and quantitative approach to the separation and identification of sorbitan ester surfactants has been developed using capillary supercritical fluid chromatography (SFC). The sorbitan ester surfactants were well separated into five groups: starting materials and mono-, di-, tri-, and tetraesters, with each group consisting of a number of peaks representing different isomers. High purity glycerides of fatty acids were employed to estimate the relative response factors of sorbitan esters, and reliable group-wise integration served for quantitation of the distribution of sorbitan fatty acid esters. A very important parameter, hydrophilic–lipophilic balance (HLB), which describes the hydrophilic and hydrophobic characteristics of surfactants, could be correlated with the distribution of the sorbitan esters. A combination of solid-phase extraction (SPE) and SFC was used to separate, concentrate, and analyze Span-20 from salt-water samples. In comparison with the HPLC method, capillary SFC broadens the scope of the technique to encompass high molecular weight sorbitan polyesters while maintaining high separation efficiency.     

Development of an automated dual‐mode supercritical fluid chromatography and reversed‐phase liquid chromatography mass‐directed purification system for small‐molecule drug discovery

We report the development of a dual‐mode mass‐directed supercritical fluid chromatography and reversed‐phase liquid chromatography purification system. The addition of a third pump allows for flexible mobile phase control between the two techniques, and enables operation of either chromatography mode within minutes by activation of a set of switching valves on a single system. Software control, fluidic pathways, interface to the mass spectrometer, and fraction collection have been modified for compatibility between both separation methods. The conditioning solvent and tuning parameters for the mass spectrometer were adjusted to achieve an ideal signal trace in either mode with good linearity (r² > 0.970) over a range of concentrations and minimal noise for accurate peak detection and isolation. The registration success rate is 90% and overall sample recovery for either technique is 80?90%. Combining two orthogonal separation and purification modes in one single system has improved the purification throughput of complex mixtures and has been a valuable, cost‐saving tool in our laboratory.     

Packed column supercritical fluid chromatography of a peroxysome proliferator-activating receptor agonist drug. Achiral and chiral purity of substance, formulation assay and its enantiomeric purity

This paper describes packed column supercritical fluid chromatography (SFC) for the analysis of a peroxysome proliferator-activating gamma-receptor agonist that is a carboxylic acid. Evaluation of conditions for the separation of this candidate drug and related compounds in bulk substance is described. A Chiralcel OD column was used for this purpose due to its high selective retention of related substances and relative inertness, though the enantioselectivity was negligible, with methanol as polar modifier. A high enantioselectivity was obtained on Chiralpak AD and it was possible to determine the enantiomeric purity within 10 min on a 5 cm short column. Both the achiral and the chiral systems were run without acid additive in the mobile phase and the level of detection of impurities by area was about 0.1%. For the analysis of samples dissolved in water, without any isolation step, 2-propanol was used as modifier. Due to the column surface activity, evidently generated by injected water, citric acid 1 mM was included as additive in the 2-propanol in order to maintain symmetric and undistorted peak shape. The detection limit for the assay was 21 microg mL(-1) (50 nmol mL(-1)) for 5 microL injected (R.S.D. 6.4%, n = 8). A 5 cm short Chiralcel OD column was used. Determination of enantiomeric purity of the drug in aqueous samples required increased sensitivity. The sample was acidified and extracted into a small volume of 1-pentanol, out of which 25 microl was analyzed by SFC. The minor enantiomer at the 3% (w/w) level added could be confirmed. Its ratio remained constant during the procedure as measured relative to a reference solution in organic media.     

Application of supercritical fluid extraction and supercritical fluid chromatography to the production of taxanes as anti-cancer drugs

The supercritical fluid extraction (SFE) of taxicin from the dried needles of the English yew tree, Taxus baccata is described. Analysis by supercritical fluid chromatography (SFC) and proton NMR spectroscopy was used to confirm identity and purity. The extraction efficiency of SFE is comparable with that obtained using liquid solvents. Such taxanes may be used semi-synthetically to prepare potential anti-cancer drugs.     

Separation of enantiomers of ibuprofen on chiral stationary phases by packed column supercritical fluid chromatography.

A packed column supercritical fluid chromatography (SFC) method for the separation of ibuprofen enantiomers on a chiral stationary phase and CO2 with modifier as mobile phase has been developed at an analytical scale. Among 11 different stationary phases the Kromasil CHI-TBB phase showed by far the best separation properties. The influence of different modifiers, injection solvents, temperature, and pressure, and density of the fluid, respectively, on the separation behavior has been studied. It was found that the separation behavior strongly depends on the type of modifier and the modifier content. Temperature and pressure are of less influence.     

Systematic evaluation of new chiral stationary phases for supercritical fluid chromatography using a standard racemate library

A systematic approach to the evaluation of new chiral stationary phases (CSPs) for supercritical fluid chromatography (SFC) using a standard library of racemic analytes is described. A standard library of racemic analytes representing a variety of functional group classes was assembled from a mixture of proprietary and commercial compounds. The library is dispensed and stored in a convenient 96-well microplate format to facilitate ease of use, and to minimize the amount of analyte required for analysis. Automated SFC screening was performed on both established CSPs in common use, as well as a group of six recently commercialized CSPs. Screening results were archived in a structure-searchable database that allows convenient comparison of performance data to determine which CSPs shows the best performance.     

Screening approach for chiral separation of pharmaceuticals. Part I. Normal-phase liquid chromatography

A strategy for rapid screening for the separation of chiral molecules of pharmaceutical interest by normal-phase liquid chromatography using three cellulose/amylose stationary phases is proposed. In a first step, the most important parameters for the separations were determined and studied for their effects by means of experimental designs. Results showed that the cellulose tris-(3,5-dimethylphenylcarbamate), the amylose tris-(3,5-dimethylphenylcarbamate) and the cellulose tris-(4-methylbenzoate) stationary phases have very broad and complementary enantiorecognition properties. The type of organic modifier used in the mobile phase appeared to have a dramatic influence on the quality of the separation. Based on the results of the preliminary study, a screening strategy was developed and successfully applied to a set of 36 diverse drugs. Enantiomeric separation was observed in 89% of cases and the analysis times were usually shorter than 20 min.     

A comparative study of chiral separation of proton pump inhibitors by supercritical fluid chromatography and high-performance liquid chromatography

A comparative study of chiral separation of pantoprazole and rabeprazole is carried out using supercritical fluid chromatography and high-performance liquid chromatography. The columns used were Chiralpak IA and Chiralpak IE. The best mobile phase in supercritical fluid chromatography was carbon dioxide-0.2% triethylamine in methanol (60:40) and 0.1% triethylamine in n-hexane-ethanol (50:50) in high-performance liquid chromatography. For supercritical fluid chromatography, values of the retention factor of pantoprazole enantiomers were 3.97 and 4.88. These values for rabeprazole enantiomers were 6.10 and 7.52. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.23 and 1.23 and 2.20 and 3.36, respectively. Similarly, for high-performance liquid chromatography, the values of retention factor for enantiomers of pantoprazole were 4.02 and 7.32. These values for rabeprazole enantiomers were 5.32 and 7.88, respectively. The values of separation and resolution factor for pantoprazole and rabeprazole were 1.82 and 1.48 and 9.22 and 6.58, respectively. A comparison was carried out, which confirmed supercritical fluid chromatography as the best method due to its fastness, eco-friendly, and inexpensiveness. The reported methods are effective, efficient, and reproducible and may be used to separate and identify pantoprazole and rabeprazole in any unknown samples.