Isolation of Pharmaceutical Impurities and Degradants Using Supercritical Fluid Chromatography - Pharmaceutical Technology

Latest Issue

Latest Issue
PharmTech Europe

Isolation of Pharmaceutical Impurities and Degradants Using Supercritical Fluid Chromatography
The authors demonstrate that using supercritical fluid chromatography offers distinct advantages in speed and in clean isolation of the desired peaks.

Pharmaceutical Technology
Volume 37, Issue 3, pp. 60-67

Case study 1: enrichment

Figure 1: Reversed-phase high-performance liquid chromatography (UV 260 nm, Method A) of the API showing known and unknown impurities. The unknown impurity (0.5% by relative absorbance) is observed at 9.7 min. [QA: please define AU]
A client API contained two process impurities—one identified and one unknown. Both were visible by UV detection at 260 nm, a wavelength at which the API absorbs light less strongly. Thus, the relative absorbance chromatogram observed in the straightforward gradient RP-HPLC method (Method A, see Figure 1) was known to overrepresent the true abundance of both impurities. The unknown impurity had a relative absorbance of 0.5% and a true abundance estimated at less than 0.2%. Preparative isolation by scaling up Method A was rejected due to time, solvent consumption, and the excessive fraction volumes that would be accumulated in the isolation of this low-abundance peak. It was hoped that, by using SFC methods, the target impurity could be recovered more expediently in the milligram quantities desired for a complete or partial structure elucidation by 2D NMR and MSn methods.

Figure 2: Preparative supercritical fluid chromatography (UV 260, Method B) chromatogram, showing the fractions collected to enrich the target impurity. The target elutes in fraction F1.
Under preparative SFC conditions with detection at 260 nm, the impurity peak signal was difficult to identify unambiguously. A preparative SFC method (Method B) was developed quickly and used to process several grams of API, with fractions collected before, during, and after the main peak eluted. These fractions were concentrated by rotary evaporation and analyzed using the RP-HPLC method for the presence of the desired peak. The desired peak was captured and enriched in a fraction collected immediately before the main peak, indicating that the chosen SFC method adequately resolved the peak from its neighbor. To accumulate the target peak in quantity sufficient for structural work, 50 g of the API were injected during a period of 7 h of automated stacked injection chromatography (see Figure 2) to produce a highly enriched fraction with a total mass of 240 mg (see Figure 3).

Figure 3: a (left): Reversed-phase high-performance liquid chromatography (Method A) chromatogram of enriched fraction. b (right): Analytical supercritical fluid chromatography (Method C) chromatogram of the enriched target, showing remaining active and other impurities. The mass spectrum for target at m/z 385 is identical for the indicated peaks in each chromatogram.
Method C, which involves direct isolation of the target peak from the enriched fraction, was developed by screening multiple SFC conditions in an automated method-development process. Typically, several hundred solvent and stationary phase pairings can be screened in an overnight series of gradient runs, and a promising separation may be converted within a few minutes to a preparative-scale isocratic process. In this case, the target peak was identified based on its relative abundance in the HPLC chromatogram in Figure 3a, and a method resolving the target completely from its near neighbors was chosen (see Figure 3b). The 240-mg enriched isolate was dissolved in the liquid cosolvent and processed in less than 1 h to isolate the desired compound. Evaporation of approximately 50 mL of collected solvent yielded 14 mg of the pure target peak.

The development of a controlled and well-characterized production process for a new drug is a complex task. The client producing this API estimated that the identification of this trace impurity, efforts to synthesize it based on various hypotheses about its source, attempts to remove it by chemical methods or through alternate synthetic approaches, and attempts to recover it through chromatography would take 12–14 months. Isolation of the compound using SFC required less than a week, and its structure was determined by NMR and MS.


blog comments powered by Disqus
LCGC E-mail Newsletters

Subscribe: Click to learn more about the newsletter
| Weekly
| Monthly
| Weekly

FDASIA was signed into law two years ago. Where has the most progress been made in implementation?
Reducing drug shortages
Breakthrough designations
Protecting the supply chain
Expedited reviews of drug submissions
More stakeholder involvement
Reducing drug shortages
Breakthrough designations
Protecting the supply chain
Expedited reviews of drug submissions
More stakeholder involvement
View Results
Eric Langerr Outsourcing Outlook Eric LangerTargeting Different Off-Shore Destinations
Cynthia Challener, PhD Ingredients Insider Cynthia ChallenerAsymmetric Synthesis Continues to Advance
Jill Wechsler Regulatory Watch Jill Wechsler Data Integrity Key to GMP Compliance
Sean Milmo European Regulatory WatchSean MilmoExtending the Scope of Pharmacovigilance Comes at a Price
New FDA Team to Spur Modern Drug Manufacturing
From Generics to Supergenerics
CMOs and the Track-and-Trace Race: Are You Engaged Yet?
Ebola Outbreak Raises Ethical Issues
Better Comms Means a Fitter Future for Pharma, Part 2: Realizing the Benefits of Unified Communications
Source: Pharmaceutical Technology,
Click here