“High sensitivity assays using online SPE-LC-MS/MS”-How low can you

go?

Mohammed Abrar

Unilabs York Bioanalytical solutions, York, UK

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)

Outline

• Introduction

• Column switching concept

• Offline vs online

• Symbiosis system

• Large molecule application

• Small molecule application

• Summary

• Conclusions

What is a Method for Bioanalysis?

Step 1

Crude removal of biological material.

Step 2Fine separation based on molecule’sphysico/chemical properties.

Step 3Specific detection based on molecule’smass and charge.

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

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?

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)

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

Sensitivity

Sample volume

Off-Line vs On-Line SPE

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Switch valve

Peak focusingXLC or

MDLC or HPD

elution

LC or XLC

Symbiosis Pharma System

‘Zero’ Extraction time

Sample X+2 Sample X+1 Sample X

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

Large molecule applications

• Increased sample loading

– In our approach we load 0.1mL of

sample extract consisting of off-

line 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

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?

Analyte Separation:- Online SPE-Reverse Phase Chromatography

• Symbiosis cartridges C2 18mg 7µm

• Sample load:- 100 µL of sample

• Sample wash:- 1000 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)

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 100 0 0.45

• 1.0 100 0 0.45

• 1.10 100 0 0.60

• 4.00 55 45 0.60

• 4.10 0 100 0.60

• 5.10 0 100 0.60

• 5.20 100 0 0.60

• 7.00 100 0 0.60

Basics of the Focus Mode

Focusing

HPD

Small molecule application

• Offline mixed mode cation exchange SPE for compound isolation

• Orthogonal online SPE-LC-MS/MS for separation and detection

• Parent LLOQ:-

• M1 LLOQ:-

• M2 LLOQ:-

0.1 pg/mL

0.25 pg/mL

0.25 pg/mL

Validation data for Parent

Nominal concentration of Parent (pg/mL)

0.100 0.300 4.00 16.0 1001

Mean intra-run statistics

Mean intra-run mean (pg/mL)

0.0896 0.289 3.87 15.6 100

Mean intra-run precision (%CV)

9.9 3.7 2.1 1.5 3.5

Mean intra-run bias (%) -10.4 -3.7 -3.3 -2.7 0.0

Mean intra-run accuracy (%) 89.6 96.2 96.8 97.3 100

Inter-run statistics

Inter-run mean (pg/mL) 0.0902 0.289 3.87 15.6 99.9

Inter-run SD (n-1) 0.00888 0.0157 0.0971 0.238 3.88

Inter-run precision (%CV) 9.8 5.4 2.5 1.5 3.9

Inter-run bias (%) -9.8 -3.7 -3.3 -2.5 -0.1

Inter-run accuracy (%) 90.2 96.3 96.8 97.5 99.9

n 22 18 18 17 17

1 Diluted ten-fold with human plasma prior to analysis

Validation data M1

Nominal concentration of M1 (pg/mL)0.250 0.750 4.00 16.0 1001

Mean intra-run statisticsMean intra-run mean (pg/mL)

0.253 0.745 3.89 15.8 99.3

Mean intra-run precision (%CV)

4.2 6.4 4.5 3.3 4.9

Mean intra-run bias (%) 1.3 -0.7 -2.7 -1.2 -0.7

Mean intra-run accuracy (%) 101 99.3 97.3 98.8 99.3

Inter-run statisticsInter-run mean (pg/mL) 0.253 0.745 3.89 15.8 99.5Inter-run SD (n-1) 0.0108 0.0551 0.183 0.639 5.09Inter-run precision (%CV) 4.3 7.4 4.7 4.0 5.1Inter-run bias (%) 1.2 -0.7 -2.8 -1.3 -0.5Inter-run accuracy (%) 101 99.3 97.3 98.8 99.5n 18 18 18 17 171 Diluted ten-fold with human plasma prior to analysis

Validation data for M2

Nominal concentration of M2 (pg/mL)

0.250 0.750 4.00 16.0 1001

Mean intra-run statistics

Mean intra-run mean (pg/mL)

0.226 0.740 3.83 15.3 97.9

Mean intra-run precision (%CV)

9.0 6.6 5.0 3.4 5.7

Mean intra-run bias (%) -9.7 -1.4 -4.2 -4.6 -2.1

Mean intra-run accuracy (%) 90.3 98.6 95.8 95.4 97.9

Inter-run statistics

Inter-run mean (pg/mL) 0.227 0.740 3.83 15.3 98.0

Inter-run SD (n-1) 0.0256 0.0490 0.216 0.645 5.78

Inter-run precision (%CV) 11.3 6.6 5.6 4.2 5.9

Inter-run bias (%) -9.2 -1.3 -4.3 -4.4 -2.0

Inter-run accuracy (%) 90.8 98.7 95.8 95.6 98.0

n 22 18 18 17 171 Diluted ten-fold with human plasma prior to analysis

Double Blank

Blank + IS

LLOQ

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

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

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