1. Large Volume Parental (LVP) Application Biopharmaceutical Application
Analysis of Semi-Volatile Leachables by Dispersive Liquid Liquid Micro Extraction (DLLME)
Mark Stanford (PPD)
Extractables & Leachables, Stevenage
Conference: RAPRA Europe
Date: 10th to 11th Dec 2013
Introduction
Dispersive Liquid-Liquid Micro Extraction (DLLME) is a versatile and robust sample enrichment technique that can be applied to a variety of liquid samples.
DLLME is desirable due to its:
Ability to significantly enrich trace analytes
Ability to convert analytes into suitable solvents for analysis
Ability to remove interferences.
Figure 1 shows the relative proportions of each solvent within the DLLME system and emphasises the degree of enrichment for analytes moving from the aqueous to the organic phase.
DLLME on Aqueous Samples
DLLME on IPA Samples
Discussion
Biopharmaceutical Application
Semi volatile leachable analysis of complex drug product matrices can be challenging due to interference from the large amount of high molecular weight material and excipients making up the drug product matrix. This can cause interference in the sample preparation and subsequent analysis where very low level detection of target analytes is required (ppb levels).
The example given in column 3 involves a lyophilised drug product containing an active ingredient which is present at 1.3mg/mL, this being 2000 times the concentration of the leachable detection threshold (0.75 µg/vial for a two dose per day product) in order to align with regulatory requirements.
In the above case, a method was developed involving reconstitution of the DP, crashing of the monoclonal antibody using IPA and removal of the precipitate via centrifugation. The resulting IPA supernatant was then subjected to DLLME via method 2, figure 3 and the chloroform extract analysed by GC-FID. Quantification involved the use of predefined relative response factors against an internal standard. The method was successfully validated as per the data in column 3.
The significance of this particular biopharmaceutical method lies not in the enrichment and LOQ levels achieved but in overcoming the challenge of the very complex sample type.
For aqueous sample types, limits of quantification can reach sub ppb levels as per data shown in column 2.
Figure 2
Figure 3
Add
extraction
solvent
water
Cloudy
emulsion
formed
Withdraw
IPA
sample
Vortex
Centrifuge
Figure 1
Analytes are extracted from the aqueous sample into the organic solvent using the disperser to facilitate the extraction.
Analytes shift from the IPA sample into the extraction solvent using the water to create the two phases
Large Volume Parental (LVP) Application
Biopharmaceutical Application
Using DLLME System 5 in Table 1
Add 10 mL aliquot of sample to centrifuge tube
Add 1 mL of pre mixed disperser and extraction solvent
(875µL IPA & 125µL of chloroform) to form a cloudy solution of extraction solvent dispersed throughout sample
Vortex for 5 seconds & centrifuge for 5 minutes at 2500 rpm
Remove extraction solvent using a syringe and analyse
Using DLLME System 2 in Table 1
Add 0.75mL aliquot of IPA sample to centrifuge tube
Add 100µL of extraction solvent (Chloroform)
Vortex for 5 seconds, to mix
Add 5mL of water &vortex for 5 seconds.
Centrifuge for 5 minutes at 2500 rpm
Remove extraction solvent using a syringe and analyse
Table 1 illustrates some example DLLME solvent systems used within GSK for leachable analysis
Table 1
System
Aqueous Phase
Disperser
Extraction Solvent
Solvent Ratios (A/D/E) 1
H2O
ACN
CH3Cl
5.0/1.25/0.1 2
IPA
5.0/0.75/0.1 3
EtOH
5.0/0.60/0.1 4
0.05% PS-80 5
Glycine pH 12
10/0.88/0.13
Validation Data
This method was validated as a limit test for target leachables in an aqueous based parenteral drug product.
Sensitivity: - Signal to noise ratio’s of >75 were seen for all analytes spiked into the drug product at 15ng/mL.
Theoretical LOQs were calculated using a mean signal to noise ratio from n=9 standard injections throughout the validation sequence. These were extrapolated to a signal to noise ratio of 10:1.
Compound LOQ’s %RSD Recovery
P-Xylene ~ 0.8 ng/mL n/a n/a
2-Octanone ~ 0.6 ng/mL %
Tridecane ~ 1.1 ng/mL %
BHT ~ 0.9 ng/mL %
1-bromotridecane ~ 2.0 ng/mL %
Enrichment factors ranged from 40 to 115 taking into account the % recoveries for each analyte.
Validation Data
This method was validated as a quantitative method for target leachables in a lyophilised biopharmaceutical drug product.
Sensitivity: - Signal to noise ratios of >75 were seen for all analytes spiked at 0.19µg/vial in the lowest linearity standard injections.
Theoretical LOQs were calculated at <0.03µg/vial by extrapolating to give a signal to noise ratio of 10:1.
Compound R Accuracy %RSD
γ-nonanoic lactone % 1
2,4 di tert butyl phenol % 1
BHT % 4
N-N-dimethyltetradecylamine % 3
N-N-dimethylhexadecylamine % 3
Conclusions
Although more straightforward for aqueous sample types, DLLME has successfully been applied to the analysis of biopharmaceutical and other complex sample systems which are becoming more popular within the pharma industry.
The advantages of DLLME are listed below:
Versatile
Robust
Sensitive / High enrichment Factors
Convert analytes into GC Friendly extraction solvent
Low Solvent Consumption
Inexpensive
No need for adsorbing and desorbing of analytes
Acknowledgements
Edel Worley, Nick Morley, Mike Hodgson, PPD & the GSK E&L Team at Stevenage
Selection of Extraction Solvent
Chloroform has proved the superior choice as the extraction solvent for the applications presented in this poster due to it’s efficient extraction capabilities and density.
Compared to more conventional liquid-liquid extraction, which require ml of solvent per sample, DLLME is considered a greener alternative as typically only 100µl is required per sample.
GSK is committed to the reduction and replacement of halogenated compounds, hence where there’s a current option to utilise non-halogenated extraction solvents, GSK strongly encourages this approach; for other applications alternatives will continue to be sought.