Warfarin is useful to prevent blood clots from growing inside blood vessels. It is administered as a racemate, though the S- isomer is 6-fold more potent than the corresponding R-isomer. This is because dosage control of the pure S-isomer was difficult. Figure 1 shows the chiral carbon at C9 (marked *) in warfarin, which results in two enantiomers: (R)-warfarin and (S)-warfarin. Separation of warfarin enantiomers using different chiral stationary phases (CSPs) under supercritical fluid chromatography (SFC), normal phase chromatography (NPC), or reversed phase chromatography (RPC) modes was reported in many scientific literatures, oral and poster presentations, and application notes published by various CSP suppliers. Chromatographic conditions mentioned in these reports are mostly suitable for analytical chromatography purposes only.
We report the separation of racemate warfarin into its individual R- and S- enantiomers, under a specific chromatographic condition. We briefly discuss some of the factors that must be considered while developing a bench-scale chromatographic method suitable for preparative chromatography. In case there is an industrial need to purify the R- or S- enantiomer, this report is expected to be useful.
Experimental:
Figure 2 shows the separation of the R- and S- isomers of warfarin with high resolution from its racemate using a Daicel CHIRALPAK® IG (4.6 mm ID × 25 cm L, 20 µm particle size) column. CHIRALPAK® IG stationary phase was composed of an amylose polymer derivatized with phenyl moieties (chloro- and methyl-groups) in meta positions. Methanol was used as a single solvent. No chemical was used as an additive. The separation was carried out at 25°C with a flow rate of 1mL/min. A UV detector wavelength of 220 nm was used for detection.
The results in Figure 2 show that the two enantiomers were very well resolved from each other, with complete baseline separation. Pure enantiomer peaks were not identified as R- or S- enantiomers using corresponding reference standards or markers. Both the peaks were Gaussian shaped, and no significant fronting or tailing was observed. The individual enantiomer peaks (enantiomer 1 and enantiomer 2) were individually collected from a preliminary loading study of approximately 2 mg warfarin load (figure 3) and re-analyzed under the same chromatographic condition.
The results in Figure 3 show that the two individual peaks were still well resolved at ~2mg load in a preliminary loading study. This shows that the method can be used for the full-fledged process development (PD) study to estimate the productivity value. The productivity value will ultimately determine the cost-effectiveness of large-scale production.
The limit of detection (LOD) and limit of quantitation (LOQ) are important from an analytical perspective. LOD and LOQ values depend on the method developed. As per USP’s definition, the LOD should be the concentration at which the signal-to-noise ratio is 3:1. A signal-to-noise ratio of 10:1 was considered as the LOQ for the corresponding method.
As shown in Figure 4, the LOD and LOQ of warfarin were found to be 77 ng and 266 ng, respectively, under this chromatographic condition. The analysis was reproducible within a day and from day to day (data not shown here) and was stable at ambient temperature.
Discussion:
The biological significance of the enantiomeric purity of drugs is now scientifically well-established [1]. A report mentions the separation of warfarin on 11 different CSPs—including CHIRALPAK® IA, IB, IC, AD, AS, CHIRALCEL® OJ, Lux Cellulose 1,2,3, and 4, Lux Amylose-2 columns—using the 2.5 min method with methanol as the co-solvent [2]. In another report, the DAICEL CHIRALPAK® IG-U (3 mm ID x 100 mm L, 1.6 µm particle size) column was used with a mixture of water and acetonitrile in the ratio of 60:40 with formic acid as an additive to maintain a pH of 2.0 [3]. Similarly, a LiChroCART® 250-4 ChiraDex® (4 mm ID × 250 mm L, 5 µm particle size) column was used to separate the R- and S- enantiomers using a mobile phase consisting of acetonitrile, glacial acetic acid, and triethylamine in the volume ratio of 1,000:3:2.5 at a flow rate of 1 ml/min at 300 nm at UV detection wavelength and room temperature [4]. The separation of warfarin enantiomers was mostly carried out under supercritical fluid chromatography (SFC), normal-phase chromatography (NPC), or reversed-phase chromatography (RPC) modes. In these chromatographic methods developed in analytical (µg injection load) scale, either the temperature was not ambient, the pH was low, or the mobile phase composition contained acid or base as additives. Thus, most of these chromatographic conditions mentioned in these literature reports, application notes, etc., are suitable for analytical separation and not suitable for scale-up to preparative scale or large-scale purification for a variety of reasons. At large-scale purification, these factors not only add up to increase the production cost but also may be impractical in an industrial setup. Moreover, in this world of growing green consciousness, many solvents traditionally used by the pharmaceutical industry for large-scale purification, for example, dichloromethane (DCM), are either no longer allowed or strictly regulated (even with the potential to be completely banned soon). This obviously represents a challenge for separation scientists.
To successfully scale up the bench-level chromatography method, the mobile phases in the bench scale and industrial scale must be identical, and the column chemistry should be the same. Smaller particle sizes, such as sub-two microns, are good for analytical separation but may not be suitable for large-scale purification due to high back pressure issues. A particle size of 10–20 µm was preferred. When the method needs to be transferred from bench scale to large scale, the length of the column should remain the same as the internal diameter is increased to raise the loading capacity of the column. In the case of a simulated moving bed (SMB) chromatography technique, the back pressure issue becomes an even more important factor—in this method, 5–6 columns are used in series, and the back pressures from each individual column are additive in nature. Separation at ambient temperature is economical, compared to higher temperature, but higher temperature may be necessary due to viscosity and diffusivity issues of the mobile phase. Robustness of the method is critical during scale-up, as well as during production, so a single solvent mobile phase is preferred. For a mixture of solvents, azeotropes are preferred since they behave like a single solvent with a constant boiling point and composition throughout the distillation process. Ternary mixtures are not preferred since the composition is difficult to maintain within the window of robustness of the method during large-scale purification.
The method reported here has used 100% methanol as a single solvent. This is considered a ‘green solvent’, particularly if produced from renewable sources. Methanol is very commonly used as a mobile phase, with its low boiling point proving useful during isolation of the product. Some solvents such as tetrahydrofuran (THF) are not preferred as they may need a stabilizer, which might not be removed easily during isolation of the product. Both enantiomers are eluted within a short run time of 10 minutes. A shorter run time increases the number of cycles per hour and, thereby, the productivity. This method meets the preliminary requirements for the subsequent process development (PD) study to further optimize and establish a method for large-scale purification. The high resolution between the two enantiomers during analytical scale of separation or during the preliminary loading study of ~2 mg doesn’t necessarily mean the compound will be separable at large scale with high purity and yield. This will depend on whether the two enantiomer peaks will have L-behavior or anti-L behavior, where “L” stands for Langmuir separation (in short, how they behave with the increased load). There are many other aspects that are not discussed here, but the basic points covered are very important to remember during method development.
FDA regulations state that chiral compounds must be tested in their enantiomerically pure state as the individual enantiomers may have vastly different pharmacological and toxicity profiles. This report shows that the DAICEL CHIRALPAK® IG (4.6 mm ID × 25 cm L, 20µm particle size) column can separate warfarin R- and S- enantiomers with high resolution using 100% methanol as a single solvent at ambient temperature without the addition of any additive. The chromatographic condition developed and reported here is expected to be suitable for further process development (PD) studies for large-scale purification.
References:
- Aisha Qayyum et.al., Determination of S- and R-warfarin enantiomers by using modified HPLC method, Pak. J. Pharm. Sci., 2015 Vol.28, No.4, pp.1315-1321
- Waldeck B, Biological significance of the enantiomeric purity of drugs, Chirality 5 (1993) 350. https://doi.org/10.1002/chir.530050514
- Hamman C, et.al. A high throughput approach to purifying chiral molecules using 3μm analytical chiral stationary phases via supercritical fluid chromatography, Journal of Chromatography A 1218(22):3529-36; DOI: 10.1016/j.chroma.2011.03.066
- Application ID 2138, https://search.daicelchiral.com/cas/name/detail.html?application_id=2138
Meet the Experts
Atis Chakrabarti
Atis Chakrabarti, PhD, is currently working at Wilmington PharmaTech (WPT). As a Director of the division of “Advanced Purification and Chiral Separation,” he leads a team of scientists providing innovative and customized CDMO liquid chromatography solutions for both chiral and achiral molecules. He has more than 25 years of experience in the field of different modes of analytical chromatography and process chromatography and has supported separation scientists in the pharmaceutical and biopharmaceutical industries.
Kamran Falahatpisheh
Kamran Falahatpisheh is a Sr. Analytical Scientist at Wilmington PharmaTech (WPT). He has more than 25 years of experience in chromatographic QC, method development, and purification by liquid chromatography.
