A Novel Approach to Low Volume Sample Preparation

Transcription

1 A Novel Approach to Low Volume Sample Preparation Tony Edge, Jon Bardsley, Ken Meadows, Luisa Pereira International Symposium on Chromatography Salzburg, Austria September The world leader in serving science

2 Introduction Identifying the challenges associated with bioanalysis Matrix issues Regulatory Sample volumes Improving recovery using Improved design of SPE material Overcoming matrix and sample volume issues Troubleshooting a mixed mode SPE method Issues with recovery Effect of buffer 2

3 Chromatography of a Typical Matrix % Tim LC-MS of Mouse Urine, full scan (with kind permission of Ian Wilson) 3

4 Challenges within Bioanalysis Matrix Effects Polar head fragment=184.3m/z 4

5 Challenges within Bioanalysis Matrix Effects 1.20E+06 Detector Response 1.00E E E E E E PPT SOLA SOLA CX 496 5

6 Challenges within Bioanalysis Matrix Effects 100% 90% 80% 70% % 50% 40% 30% % % 0%

7 Challenges within Bioanalysis - Regulatory..Changes in the response function relationship between pre-study validation and routine run validation indicate potential problems. Internal standard response should be monitored for drift. An SOP should be developed a priori to address issues related to variability of the IS response. 7

8 Challenges within Bioanalysis Reproducibility of IS IS Response Sample Number SPE extraction Protein precipitation SPE gives better reproducibility than protein precipitation 8

9 Meeting the Challenges with Bioanalysis Thermo Scientific SOLAµ SPE plates No frits - no movement Fixed media position Improved flow characteristics Improved reproducibility Fewer failures Improved throughput and efficiency 9

10 SOLAµ Plates Improved Flow Through Characteristics How does SOLAµ plates provide this? Consistent manufacture ensures the same amount of material is packed every time. Frits act as a barrier to flow, removal aids flow. Space between media is not controlled by packing density (diagram). SOLA unit structure has increased porosity due manufacture control. Conventional loose packed SPE 10

11 Challenges within Bioanalysis Reproducibility of IS 1.4 Loosed packed SPE (i) 10mg Neostigmine Reproducibility Data Endrophonium Neostigmine Pyridostigmine %RSD (n=20) without IS %RSD (n=20) with IS No IS Correction IS Corrected

12 Challenges within Bioanalysis Reducing Sample Size Move away from composite animal studies Scientifically a little dubious Uses less animals / samples 1.5 mls of blood in a mouse 5-10 time points Allows lower quantification limits Neo-natal patients Current legislation means that new born children have to be tested with a drug that is tested on them Minimal sample volume available 12

13 Challenges within Bioanalysis - Blowing down a Sample Non specific binding Workflow efficiency Volatility 13

14 SOLAµ Plates - Sample Limited Extraction (25µL) Extraction of niflumic acid from human plasma 25µL of sample Equivalent results on 2mg SPE to higher sample volume extraction using 10mg SPE High reproducibility 14

15 SOLAµ Plates - Sample Limited Extraction (25µL) Sample pre-treatment 25 (250) μl of human plasma diluted with 1:1 with 4% phosphoric acid Sample preparation: Condition 200 μl methanol Equilibrate 200 μl water Load Apply sample at 0.5 ml/min Elute 2 x 12.5 (125) μl methanol with 2% ammonia Direct injection of eluent SOLAμ WAX plate used Text in red is original 10mg method volumes 15

16 SOLAµ Plates - Sample Limited Extraction (25µL) 500, μl sample size 500, μl sample size 16

17 SOLAµ Plates - Sample Enrichment (20x Pre-Concentration) Sample pre-treatment: 500 (500) μl of human plasma diluted with 1:1 with 4% phosphoric acid Sample preparation: Condition 200 μl methanol Equilibrate 200 μl water Load Apply sample at 0.5 ml/min Elute 2 x 12.5 (250) μl 50/50 methanol/acetonitrile with 2% ammonia Direct injection of eluent SOLAμ WAX plate used 17

18 SOLAµ Plates - Sample Enrichment (20x Pre-Concentration) Increase in signal response x 20 using smaller elution volume 18

19 What Can Go Wrong with a Mixed Mode SPE? WCX has a pk a ~ 4.5 Below ph 4.5 sorbent neutral Above sorbent negatively charged Do not assume that deionised water has a ph of 7 This will have a dramatic effect on the stability of a WCX method / or where the nature of the analyte is assumed based on the ph 19

20 Start Method Sample 250 μl plasma μl H 2 O Condition Equilibrate Load Sample Wash 1 Wash 2 Elute 250 μl MeOH 250 μl H 2 O 250 μl H 2 O 250 μl MeOH 2% formic acid in MeOH (v/v) 20

21 Affect of Loading Speed with Compound in Matrix Recovery Fast Load Slow Load 20 0 Loading Speed 21

22 Effect of Dilution Solvent on Recovery 110% 100% 1:40 Aqueous solution % Recovery 90% 80% 70% 1:1 1:2 1:3 1:10 1:20 60% 50% 1:1 1:2 1:3 1:10 1:20 1:40 P/S Plasma : dilution solvent ratio Dilution of sample can have an effect on recovery 22

23 Buffered Method Controlling the Sorbent Charge Sample 200 μl plasma μl ph7 buffer Condition Equilibrate Load Sample Wash 1 Wash 2 Elute 200 μl 80/20 MeCN/MeOH 2% formic acid 200 μl ph7 buffer 200 μl ph 7 buffer 200 μl 80/20 acetonitrile / methanol (v/v) 2x 50 μl 2% formic acid in 80/20 acetonitrile / methanol (v/v) 23

24 Use of Appropriate Buffering Conditions 100% 90% 80% 70% 60% 50% 40% Plasma Ex diln ph7 buffer Water Ex diln ph7 buffer 30% 20% 10% 0% %Rec Recovery increased from plasma samples following addition of buffer Aqueous solution maintained high recovery 24

25 Conclusion Identifying the challenges associated with bioanalysis Matrix issues Regulatory Sample volumes Improving recovery using Improved design of SPE material Overcoming matrix and sample volume issues Troubleshooting a mixed mode SPE method Issues with recovery Effect of buffer 25