Achiral SFC: Development of an Orthogonal SFC Method for Mometasone Furoate Impurity Analysis

Transcription

1 Achiral SFC: Development of an Orthogonal SFC Method for Mometasone Furoate Impurity Analysis Zhenyu Wang Merck Research Laboratories NJCG 2013

2 Outline SFC chronicle The journey of a half century Re-birth of SFC Chiral SFC in pharmaceutical industry New opportunity of SFC era Achiral SFC for complex analysis Summary

3 The chronicle of SFC : Packed Column SFC 1962: Ernst Klesper High pressure GC

4 The chronicle of SFC : Capillary SFC 1981: M. Novotny, and M.L. Lee. et.al.

5 1980s: the enthusiasm SFC should exhibit the combined advantages of GC and HPLC. SFC allows the separation of materials which are thermally labile and of much higher molecular weight than is possible using GC. High diffusivities, low viscosities SF CO 2 provide higher flow rates, shorter analysis, and higher efficiency separations.

6 1990s: the frustrations The extent to which SFC will be useful for relatively polar materials is still an open question and a matter for much future research. A decade debate: Open tubular SFC vs. Packed column SFC The winner: Packed column SFC: user friendly, quantitative injection, and apply to higher polarity compounds

7 The chronicle of SFC Late 1990s-present: Re-birth of parked column SFC Application: Chiral separation and purification Polar compounds even peptides Hardware: Dedicated SFC stationary phases New generation SFC instrumentation Conception: Green technology Cost effective

8 Key driver of SFC s Renaissance Chiral separation and purification in Pharm Ref.: Jeff Elleraas, Pfizer

9 Chiral Prep SFC : Stacked injections 16.5 g in 7 hours 2,100 2,000 1,900 1,800 1,700 1,600 1,500 1,400 1,300 1,200 1,100 1,

10 Chiral SFC for non-chiral compound separation Nobiletin (NOB): anti-inflammatory agent NOB is metabolized by hepatic p450 enzymes yielding 3 -demethyl- NOB and 4 -demethyl-nob No separation of the two metabolites on RPLC OMe OMe MeO O OH MeO MeO OMe OMe O O OMe OMe P450 MeO MeO OMe O OMe O 3 -demethyl-nob OH OMe Nobiletin (NOB) MeO OMe O 4 -demethyl-nob

11 RPLC separation of 3 -DNOB and 4 -DNOB Z ELSD An1 7.70e5 No Separation on Reversed Phase LC % 0 Time Mass Spectrum: 3 -DNOB 4 -DNOB

12 3 -DNOB and 4 -DNOB: Chiral LC vs. Chiral SFC Column: Chiralpak AD HPLC: 40/60 hexane/ethanol, 1.0 ml/min LC Column: Chiralpak AD Rs: 1.7 SFC: 20% MeOH as modifier, 2.0 ml/min, CO bar, 30ºC AD DATA [1] mau SFC Rs: RT [min] 20

13 Chiral SFC for non-chiral compound separation ID and quantitation of NOB metabolites in mice urine AD DATA [1] mau Demethyl-NOB (major metabolite) 28.9 µg/ml 10 NOB (parent) RT [min] 3 -Demethyl-NOB (minor metabolite) SFC: AD column, 20% MeOH as modifier, 2.0 ml/min, CO bar, 30ºC Z. Wang, S. Li, Biomed. Chromatogr., 20 (2006)

14 The new opportunity of SFC era: Achiral separation of complex analytes SFC achiral method development General practice for method development Mometasone furoate case study SFC achiral method validation Improve method sensitivity SFC sample prep for complex matrix (e.g. bio-fluids, formulated drugs)

15 Mometasone furoate franchise A highly potent glucocorticoid anti-inflammatory agent The active ingredient of several drug products

16 Mometasone furoate and its major impurities Cl O HO O O O Cl O Mometasone furoate

17 Current RPLC method for MF impurity analysis What if I use SFC Sample: MF with spiked impurities Column: Ultrasphere ODS ( mm, 5 µm) Mobile phase A: Water/Mobile phase B: Acetonitrile Gradient: 42% B to 52% B in 60 min Flow rate: 1.5 ml/min AU min! Minutes *Method slightly modified from USP MF monograph

18 SFC & HPLC instrument and software All SFC experiments were performed on: TharSFC Method Station Analytical System Solvent selector Column selector Waters 2998 PDA detector Instrument control and data collection: Empower 2 All HPLC experiments were performed on: Alliance 2690 HPLC System equipped with 2996 PDA detector Instrument control and data collection: Empower 2

19 Achiral SFC method development Fine tuning 2 Screening: P, T, Additive Preliminary Screening: Stationary Phase & Modifier

20 SFC achiral column screening Most time-consuming step Retention mechanism is not fully understood Normal phase, Reversed phase, Mixed mode Simplify column screening

21 SFC column selection: QSRR: Quantitative Structure Retention Relationship West, C.; Lesellier, E., J. Chromatogr. A, 2008, 1203, 105.

22 Primary screening: Column and Modifier Interaction Plot for Selectivity Data Means 2-PrOH EtOH MeOH Column Column 2-EP Cyano Si Modifier Modifier 2-PrOH EtOH MeOH EP Cyano Si

23 Separation on Silica column with Methanol as modifier MF AU Minutes

24 Achiral SFC method development Step Two Fine tuning 2 Screening: P, T, Additive Preliminary Screening: Stationary Phase & Modifier

25 Impact of temperature and pressure ºC, 100 bar AU M in u te s ºC, 100 bar AU Minutes Silica: mm, 5 µm, Kromasil Mobile phase: 4 ml/min, CO 2 Gradient: 5% MeOH to 20% MeOH in 15 min

26 Impact of temperature and pressure (Cont d) ºC, 100 bar AU M in u te s Silica: mm, 5 µm, Kromasil Mobile phase: 4 ml/min, CO 2 Gradient: 5% MeOH to 20% MeOH in 15 min

27 Impact of temperature and pressure (Cont d) ºC, 100 bar AU M in u te s ºC, 150 bar AU Minutes Silica: mm, 5 µm, Kromasil Mobile phase: 4 ml/min, CO 2 Gradient: 5% MeOH to 20% MeOH in 15 min

28 MF SFC AU Minutes RPLC AU Minutes

29 Good news for pharmaceutical chromatographers One of the most challenges for us: To develop stability indicating method to monitor DS & DP Most of stability indicating methods are RPLC based How to confirm method s specificity (detection or separation) SFC can be the 2 nd method with true orthogonal selectivity I get new tool

30 Stability indicating method: RPLC or SFC? Which one? Why don t we use SFC for the primary stability indicating method? But Is SFC sensitive enough?

31 Sensitivity: key to drug impurity analysis Drug safety and quality control Reporting threshold: 0.05% impurity in Drug Substance ( 2 g/day), ICH Q3A Reporting threshold: 0.1% degradation product in Drug Product ( 1 g/day), ICH Q3B

32 Why SFC-UV is less sensitive (vs. HPLC-UV) Three main sources of noise: Electronic: noise from detector system Mechanical: BPR, pump Thermal: endothermic process during depressurization Anton, K.et.al. Analusis, 1999, 27, 691 Helmy, R. et.al. Chirality, 2007, 19, 787 Wang, Z, et.al, Am. Pharm. Rev, 2009, 5, 59

33 How to get better sensitivity on SFC-UV Software filtering: reduce non-wavelength dependent noise 1,2 Hardware modification: reduce mechanical and thermal noise 3 Next generation SFC 1 Chen, R., LC-GC Application Notebook, 2009, Sep 2. Wang, Z, et.al, J. Chromatogr. A, 2011, 1218, Helmy, R. et.al. Chirality, 2007, 19, 787

34 Reference Wavelength Compensation to increase S/N PDA detector settings Without wavelength compensation With wavelength compensation Improvement in sensitivity Sampling rate Bandwidth Filter constant Peak width S/N a Peak width S/N b (S/N b )/(S/N a ) i Slow ii* Normal iii* Slow iv Normal v Slow vi Normal vii* Slow viii Normal ix Slow x Normal xi Slow xii Normal Detection wavelength: 245 nm Compensation wavelength: nm

35 AU Ref. Wavelength Compensation to increase S/N (Cont d) ii Sampling rate: 5 Bandwidth: 2.4 Filter cons.: Normal Peak width: S/N: AU Minutes S/N x3 increase iii Sampling rate: 5 Bandwidth: 3.6 Filter cons.: Slow Peak width: S/N: AU Minutes vii Sampling rate: 2 Bandwidth: 2.4 Filter cons.: Slow Peak width: S/N: Minutes

36 Comparison of RPLC and SFC method validation results RP-HPLC method SFC method Sample concentration 0.2 mg/ml 2.0 mg/ml Linearity Accuracy Assay level a 99.1% % 99.8% % Impurity level b 96.6% % c 88.3% % c Precision Assay level a 0.4% 0.7% Impurity level b 1.9% - 5.0% 1.4% - 5.4% Limit of Quantitation 0.05% (or 0.1 µg/ml) 0.05% (or 1.0 µg/ml) a. Six preparations (n = 6) b. Six preparations of spiked individual impurities c. The average recovery of each impurity was reported. Z. Wang, J. Chromatogr. A, 2011, 1218,

37 Sensitivity is not a major challenge for SFC anymore Can new instrumentation broaden SFC to complex sample analysis? Such as formulated drugs.

38 Using SFC in complex formulation analysis The Big Fact: SFC is rarely used for drug product analysis Chromatograph used for drug product analysis: RPLC, nearly 100% The Challenge: Drug product sample prep: working for RPLC working for SFC SFC prefers neat organic as sample solvent Direct injection of aqueous containing sample on SFC may result in: - Freezing of aqueous solutions - Precipitation of samples - Deteriorated separations

39 SFC Sample Preparation Strategies for Drug Products For solid dosage forms: - Dissolve sample in organic solvent - Filter to remove un-dissolved residue - Assay by SFC Extraction robustness need to be assessed For solutions and suspensions: - Direct injection might work (not recommended as 1 st choice) - Use Solid Phase Extraction or Liquid-Liquid Extraction

40 SPE-SFC: for an aqueous formulation SPE Cartridge Screen Study 120 SFC Recovery (%) (%) Strata X C18 E C8 Phenyl Methanol DMSO Acetonitrile SDB-L Z. Wang, Am. Pharm. Rev, 2013, 16(3), 28-25

41 Summary SFC dominated chiral separation and chiral purification The advantages of achiral SFC have not been fully recognized: Orthogonal selectivity Economical Faster SFC friendly sample prep is important for complex analysis (e.g. for formulated drugs)