High sensitivity assays using online SPE-LC-MS/MS -How low can you go? Mohammed Abrar Unilabs York Bioanalytical solutions, York, UK
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1 High sensitivity assays using online SPE-LC-MS/MS -How low can you go? Mohammed Abrar Unilabs York Bioanalytical solutions, York, UK
2 Background Unilabs YBS are a bioanalytical CRO based in York (Uk) Copenhagen and Sandwich (UK) There is an increase in the number of biological molecules in the drug development process There is a growing need for the capacity to analyse these molecules and quantify them at very low levels for bioanalytical purposes There is a large interest in inhaled, dermal and micro dosing applications New sampling techniques DBS and capillary microsampling All of these applications require high sensitivity bioanalytical assays (low pg/ml in plasma or urine)
3 Outline Introduction Column switching concept Offline vs online Symbiosis system Large molecule application Small molecule application Summary Conclusions
4 What is a Method for Bioanalysis? Step 1 Crude removal of biological material. Step 2 Fine separation based on molecule s physico/chemical properties. Step 3 Specific detection based on molecule s mass and charge.
5 Introduction High potency drugs require low level detection Detector sensitivity is fixed on our quadrupole instruments We need to manipulate either the sample extraction or chromatography or both to achieve the desired sensitivity levels
6 How can we increase sensitivity? Increase sample volume This will give us increase in sensitivity but not linear Increase in ion suppression Problems with lc column loading Sample concentration Will increase sensitivity but limited by what is practical Use nanospray Will give increased detector sensitivity Limited by lc column loadability Robustness? Extra cost?
7 Column switching concept Column switching Consists of a pre-column or trapping column for sample loading and a analytical column used for separation Pre-column used for multiple injections Online SPE Consists of a SPE cartridge for sample loading and a analytical column for used for separation SPE cartridge only used once (analogous to offline SPE)
8 Why online? Online SPE No concentration of sample by the use of temperature or pressure (Peptides are generally very susceptible to adsorption problems) Sample pre-treatment protein precipitation or offline SPE Sample extract diluted with water and surfactant to adjust polarity On-line SPE using symbiosis (To aid sample concentration and further sample clean-up) Allows higher sample loading Analytical column with reverse phase gradient This allows linear increase in sensitivity when sample volume is increased S e n s i t i v i t y Sample volume
9 Analytical Column Off-Line vs On-Line SPE S S S S S N 2 S S S S S S ON-LINE OFF-LINE Analytical Column S S S S Switch valve
10 Symbiosis Pharma System LC or XLC Peak focusing XLC or MD LC or HPD elution
11 Zero Extraction time Sample X+2 Sample X+1 Sample X
12
13
14 Large molecule applications On-line SPE using Spark Holland Symbiosis system allows extensive sample clean-up. Use of ph during the wash stages ie 0.1%-TFA or ammonia depending on the nature of the peptide (Allowing 2D sample clean-up) Utilising the reverse phase retention mechanism of peptides. Peptides are adsorbed on top of the SPE cartridge during the load and wash stages and are quickly back-eluted using critical organic percentage. 14
15 Large molecule applications Increased sample loading In our approach we load 0.1mL of sample extract consisting of offline protein precipitation extract, water to adjust polarity and surfactant to keep the peptides in solution. Allows sample concentration Peptides are very susceptible to adsorption and instability if concentrated by the use of temperature or pressure. 15
16 Small molecule application Parent compound and two 2 metabolites 400 amu Very polar, main functional groups tertiary amine and sulphonamide LLOQ required, low as possible CMAX predicted to be low pg/ml range. So, how low can we go?
17 Analyte Separation:- Online SPE-Reverse Phase Chromatography Symbiosis cartridges C2 18mg 7µm Sample load:- 100 µl of sample Sample wash: µl of 90:10:1:0.2 (water:meoh:1 M ammonium acetate: ammonia) Sample backward wash:-500 µl of 90:10:1:0.2 (water:meoh:1 M ammonium acetate: ammonia) Sample elution:-200 µl of 70:30:0.5 (water:meoh:formic acid)
18 SPE-Reverse Phase Chromatography Analytical column Agilent Poroshell 120 EC, 2.7 µm, 50 x 3 mm Column temperature* 50 ºC Mobile phase A Water-MeOH-Ammonium formate (1M)-Formic acid 90:10:0.2:0.2 v/v/v/v Mobile phase B Water-MeOH-Ammonium formate (1M)-Formic acid 10:90:0.2:0.2 v/v/v/v Flow rate 0.6 ml/min Gradient Time (minute) % A % B Flow rate Initial
19 Basics of the Focus Mode Focusing HPD
20 Small molecule application Offline mixed mode cation exchange SPE for compound isolation Orthogonal online SPE-LC-MS/MS for separation and detection Parent LLOQ:- 0.1 pg/ml M1 LLOQ: pg/ml M2 LLOQ: pg/ml
21 Validation data for Parent Nominal concentration of Parent (pg/ml) Mean intra-run statistics Mean intra-run mean (pg/ml) Mean intra-run precision (%CV) Mean intra-run bias (%) Mean intra-run accuracy (%) Inter-run statistics Inter-run mean (pg/ml) Inter-run SD (n-1) Inter-run precision (%CV) Inter-run bias (%) Inter-run accuracy (%) n Diluted ten-fold with human plasma prior to analysis
22 Validation data M1 Nominal concentration of M1 (pg/ml) Mean intra-run statistics Mean intra-run mean (pg/ml) Mean intra-run precision (%CV) Mean intra-run bias (%) Mean intra-run accuracy (%) Inter-run statistics Inter-run mean (pg/ml) Inter-run SD (n-1) Inter-run precision (%CV) Inter-run bias (%) Inter-run accuracy (%) n Diluted ten-fold with human plasma prior to analysis
23 Validation data for M2 Nominal concentration of M2 (pg/ml) Mean intra-run statistics Mean intra-run mean (pg/ml) Mean intra-run precision (%CV) Mean intra-run bias (%) Mean intra-run accuracy (%) Inter-run statistics Inter-run mean (pg/ml) Inter-run SD (n-1) Inter-run precision (%CV) Inter-run bias (%) Inter-run accuracy (%) n Diluted ten-fold with human plasma prior to analysis
24 Double Blank
25 Blank + IS
26 LLOQ
27
28
29
30
31 Small molecule application Method validation went through with no repeats Good method reproducibility and robustness Problems Each time standard weighing made large levels of contamination observed One week wash out period Several precautions taken Analysts change lab coats after weighing Stock solutions only handled in externally vented fume hood Samples analysed in a different lab
32 Overall Summary Summary Symbiosis instruments interfaced with API 4000, 5000 or Waters Xevo. Lc gradient chromatography developed for peptide or small molecule of interest Sample loading and wash optimised Back elution with standard gradient or HPD focusing (elution volume and time on SPE cartridge optimised) Asses carry over Method development initially 2 days and then decision made if online SPE is appropriate
33 Conclusion The use of Online SPE (Symbiosis system) for high sensitivity assays Can give significant improvements in sensitivity Precise heart cutting on the small particle SPE allows higher sample loading In general sensitivity can be increased by at least factor of ten compared to UHPLC. Analytical cycle times 5 min Same robustness as normal HPLC
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