Trials and Tribulations of Developing a Method for Quantification of Aerosol Chamber Concentration

Richard Snell,

Dose Concentration Analysis

GSK

Overview

Inhalation chamber concentration analysis

Meth Dev and Validation of methods for aerosol chamber concentrations

The problem Child Method Development and Validation Study Samples The Investigation We Have a Winner

Chamber Concentration Analysis - 1

7 or 14 day candidate selection studies supported including 2-3 weeks prelim phase

We perform: Concentration checks using GF/C filters Particle size distribution analysis using washings of stainless steel

foils and back-up GF/A filters

Samples quantified against calibration line

Quality control samples run to check performance of assay – must be within 10% of nominal concentration

UPLC is the preferred system due to faster run times leading to shorter turnaround times.

Chamber Concentration Analysis - 2

Particle size distribution (PSD) solutions and GF/A Filters:

Cascade impactor used in place of animal tube: contains up to 8 stainless steel foils + back-up GF/A filter

Foils trap different size particles - particle size gets progressively smaller towards the rear of the impactor with GF/A filter trapping smallest particles.

Foils from stages 3-8 are collected and washed with 3 mL of diluent

An aliquot is analysed from each 3 mL washing solution of foils 3-8

GF/C Filters: Aerosol chamber concentration check filters used in place of animal tubeGives representation of the dose animals will be exposed to

Method Development and Validation - 1

Solubility check - 3 to 5 mg weighed, analytical diluent added in 100 µL steps (increased if no signs of going into solution) Compound dissolution assessed visually.

λmax Determined by running samples prepared in diluent on a UPLC system equipped with a photo diode array (PDA)

Chromatography developed

Linearity investigated

Interference from filters checked

Method Validation Standard line bracketing QCs QCs at 2 Levels – HLQ and LLQ One QC solution prepared at each level, 10 mL added to each

filter type in replicates of 6 6 replicates of blank filters 10% acceptance criteria on standards and QCs

QCs re-injected with freshly prepared calibration line after storage at room temperature to establish stability

Method Development and Validation - 3

The Problem Child – Method Development and Validation - 1

Solubility in initial diluent (50/50 MeCN/Water) was good

Linear range of 0.25 to 30µg/mL

QCs at low level failed to meet acceptance criteria.

Re-run, as usual reason for failure is spiking error, or difference when spiking vastly different volumes

This time marked difference between QCs containing filters and those without, ≈ 15-20%, indicating drug binding to the filters

High and low filters not exhibiting same trend, leading to a change in diluent. Consistent with binding.

40/40/20 (MeCN/MeOH/Water) was tried, resulting in the same approximate 20% difference between QCs with and without filters

LLQ Raised to 0.5ng/mL

Investigate different Solvents as diluent Addition of 0.1% Formic acid to diluents Addition of 0.05% Triflouroacetic Acid (TFA)to diluents Aqueous Tertrahydrofuran (THF) 20%, 30% and 50%

The Problem Child – Method Development and Validation - 2

The Problem Child – Method Development and Validation - 3

Addition of acid had no effect

50%, and 30% THF both had detrimental effect on chromatography

20% no effect on chromatography and appears to stop the compound binding to filters

Validation run and stability runs successful

METHOD VALIDATED................. Or so we thought...

First submission of chamber characterisation sample GF/C Filters only - run fails standard line fails and QCs outside acceptance criteria

Repeated with fresh line and QCs, same issues, on closer examination line exhibits an exponential rather than linear correlation

The Problem Child – Study Samples - 1

The Problem Child – Study Samples - 2

Consistent with non-specific binding

Since study required Low levels, range truncated 0.5 to 8µg/mL

FAILURE! No improvement on linearity. Samples sent for analysis by another group to eliminate our system

The Problem Child – Study Sample - 3

Analysis shows – Compound sticking to glass as well as filters Worse at lower concentrations Worsens over time

Sample Name Content% Nominal

Claim

Recovery 01 8.87 ug/ mL 98.1

Recovery 02 6.63 ug/ mL 97.9

Recovery 03 4.24 ug/ mL 93.7

Recovery 04 2.04 ug/ mL 90.5

Recovery 05 0.97 ug/ mL 86.0

STD 0.5 0.17 ug/ mL 34.0

STD 1 0.38 ug/ mL 38.0

STD 2 1.57 ug/ mL 78.5

STD 4 3.58 ug/ mL 89.5

STD 8 5.44 ug/ mL 90.7

STD 7 6.63 ug/ mL 94.7

STD 8 7.69 ug/ mL 96.1

QC 0.5 0.18 ug/ mL 36.3

GF/ A 0.5 0.27 ug/ mL 53.0

GF/ C 0.5 0.34 ug/ mL 68.3

QC 8 7.90 ug/ mL 98.8

GF/ A 8 8.00 ug/ mL 100.0

GF/ C 8 8.00 ug/ mL 100.0

All glassware eliminated from extraction procedure, and replaced with polypropylene

Raised LLQ to 1µg/mL

Highlights compound still binding to filters

Binding worse at higher levels indicating that in the absence of glass compound has an affinity for itself

Options, Different Filter types Changes to diluent addition of NH3 or TEA

Change to the way compound is trapped

The Problem Child – The investigation

The use of Quartz filters were tried but did not offer any advantage

The addition of 1% NH3 to 40/40/20 (MeCN/MeOH/Water)

The Problem Child – We have a Winner

QC 1 QC 1 GF/A

QC 1 GF/C

QC 30 QC 30 GF/A QC 30 GF/C

1 1.036 1.020 1.026 29.669 29.844 29.867

2 1.028 1.023 1.037 29.579 29.896 29.544

3 1.039 1.018 1.042 29.681 29.758 30.422

4 1.044 1.018 1.036 29.722 30.191 30.172

5 1.035 1.024 1.033 30.631 29.950 30.136

6 1.043 1.026 1.029 29.567 30.180 30.283

Mean 1.037 1.022 1.034 29.808 29.970 30.071

S.D. 0.006 0.003 0.006 0.408 0.178 0.317

Precision (%CV)

0.6 0.3 0.5 1.4 0.6 1.1

Bias % 3.7 2.2 3.4 -0.6 -0.1 0.2

n 6 6 6 6 6 6

Acknowledgements

Jessica Benton

Aida Merchan

Bob Gibbon

Alan Ferrie

Kay Rush