Stability Indicating Assays for Dose Formulation Stability Testing

Teresa FullerGSK

Stability Indicating Assays for Dose Formulation Stability Testing

– Stability Studies for Dose Formulations

– What the white paper says

– What is a stability indicating assay?

– So do we need a stability-indicating assay?

– Closing thoughts

Stability Studies for Formulations

– Performed for every formulation to be used on a GLP study

– Test the formulation for concentration on day 0

– Test the formulation for concentration on further timepoints to assess stability, eg. day 1, day 7 and day 14

– Based on concentration results over time, assess stability

What the white paper says

“The analytical method should be suitable for the intended purpose. It is conceivable to have a rapid analytical method for test article only, which would not be suitable as a stability-indicating assay where degradents are separated from the test article or a more elaborate method which may have the additional advantage of identifying degradents. Both types of assays are acceptable and would require the appropriate level of validation for their intended purposes.”

What is a Stability Indicating Assay?

– According to the US-FDA stability guideline of 1998

‘validated quantitative analytical methods that can detect the changes with time in the chemical, physical, or microbiological properties of the drug substance and drug product, and that are specific so that the contents of active ingredient, degradation products, and other components of interest can be accurately measured without interference.’

– But this applies to drug substance and drug product not dose formulations!

– For dose formulations we do not necessarily need to quantify the degradents

– So what should the formulation analytical method look like?

Isocratic UPLC/UV

– Chromatography column, %organic solvent and wavelength chosen for analyte of interest

– K’ of at least 3 [K’ = (Vt – V0)/V0]

– Eg.

HPLC System Waters Acquity UPLC with TUV Detector using High Sensitivity Flow cell

Typical Injection Volume 1 µL

Flow Rate 1 mL/min

Wavelength 245 nm

Analytical Column 50 x 2.1 mm i.d. Waters BEH C18 1.7 µm

Column Temperature 60 ºC

Run Time 1 min

Typical Retention Time 0.8 min

Mobile Phase A 0.05% Trifluoroacetic Acid

Mobile Phase B Methanol

Isocratic Composition A:B 69:31

Isocratic UPLC/UV

- 0

.4

9

- 0

.7

0

GS

K3

00

83

48

- 0

.7

64.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95

GS

K3008348

- 0.7

5

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95

Reference standard

Stability sample day 7, ambient PFL0.02 mg/mL in water

Generic UPLC-UV Gradient

HPLC System Waters Acquity UPLC

Typical Injection Volume 0.5 – 5 uL

Flow Rate 0.6 mL/min

Wavelength Analyte specific

Analytical Column 100 x 2.1 mm i.d. Waters BEH C18 1.7 µm

Column Temperature 50 ºC

Run Time 12 min

Mobile Phase A 0.1% Trifluoroacetic Acid in water

Mobile Phase B 0.1% Trifluoroacetic Acid in acetonitrile

Gradient profile Time %B

0 3

10 100

10.5 100

11 3

Generic UPLC-UV Gradient

Isocratic and Gradient Compared

Isocratic Gradient

Run Time 1-2 mins 12 mins

Method development 1 day to several weeks Minimal

Visible degradents Significantly different degradents probably not visible

Most degradents and impurities likely to be visible

Stability-indicating possibly possibly

How can we assess how stability indicating the assay is?

– Forced degradation

– Store sample at 30°C, 65% RH or 40°C, 75% RH or 50°C, ambient RH

– UV light exposure

– Oxidation, eg. By addition of hydrogen peroxide

– Acid/alkali hydrolysis

– Compare forced samples to a fresh sample

– If any additional peaks are seen in the forced samples but these are well resolved from the analyte then assume assay is adequate

How can we assess how stability indicating the assay is?

– Peak purity analysis

– Acquire UV spectrum at several points (at least 3) across the chromatographic peak

– Compare spectra to look for differences which may indicate underlying peaks co-eluting with the analyte

How can we assess how stability indicating the assay is?

– Mass spectral peak purity

– Acquire mass spectrum at several points across the peak, similarly to UV peak purity

The ideal stability indicating method?

– UPLC-MS/MS

– Use specific parent and daughter ions to monitor the analyte

Analyte Precursor ion(m/z)

Product Ion (m/z)

GSK123456 539 290

Product 290

XIC of +MRM (2 pairs): 391.0/154.2 amu from Sample 3 (STD5) of GSK2245035KMSLIVALB-... Max. 1.2e4 cps.

0.5 1.0 1.5Time, min

0.00

2000.00

4000.00

6000.00

8000.00

1.00e4

1.20e4

Inte

ns

ity, c

ps

1.14

Pros and Cons

Technique Pros Cons

Forced Degradation

•No additional equipment reqd

•Time•Degradation may not be the same in real samples

UV Peak purity •Quick •Degradation products may have similar spectrum•Needs PDA detector

Mass spec •Specific •Expensive equipment•Mass spectral comparison difficult

So do we need a stability-indicating assay?

– White paper says we don’t!

– Unstable formulation considered to be greater than 10% drop from t0

– Complex oral and iv formulations are likely to have had previous development performed by a Product Development group

– storage stability already tested to some degree

– We need some confidence in our assay

Closing Thoughts

– Several factors to consider

– Balancing act between having confidence in the method and minimising method development time and effort

– Is a gradient better than isocratic?

– Is MS detection better than UV as it becomes more affordable?

Acknowledgements

– Emma Simmonds, GSK PD

– Tammy Clegg, GSK DMPK

– Louise Kay, GSK DMPK

Useful Reference

– Review of Development of Validated Stability Indicating Assay Methods

– Journal of Pharmaceutical and Biomedical Analysis 28 (2002) 1011–1040