Surviving Matrix Effects Experiments. Grace van der Gugten St. Paul s Hospital, Vancouver, BC, Canada

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1 Surviving Matrix Effects Experiments Grace van der Gugten St. Paul s Hospital, Vancouver, BC, Canada gvandergugten@providencehealth.bc.ca

2 Outline What are matrix effects Post Column Infusion Phospholipid transition monitoring Matrix Mixing Quantitative matrix effects experiment How does it work How to calculate the pre and post spikes Examples

3 Matrix effects Extraction of analyte from matrix: Liquid/Liquid Extraction Supported Liquid Extraction Protein precipitation Solid Phase Extraction Dilute and shoot extraction method optimized for the best recovery of analyte of interest but many other compounds extracted too!

Matrix effects biological samples What else are we extracting? Endogenous compounds: phospholipids, metabolites, hormone steroids Exogenous compounds: blood, serum, plasma, urine.. http://www.

4 Matrix effects biological samples What else are we extracting? Endogenous compounds: phospholipids, metabolites, hormone steroids Exogenous compounds: blood, serum, plasma, urine.. medications, materials absorbed into matrix from collection tube (e.g. gel tubes) or extraction containers Other matrix effects: Buildup on curtain plate Mobile phase additives

5 Matrix effects So, what are matrix effects? Anything that is present in the sample extract and your system that will either decrease or enhance signal of your analyte of interest at its elution time

6 Matrix effects More significant in ESI: Co-extracted molecule Another co-extracted molecule charge competition ion suppression

7 Matrix effects assessment 1) Post Column infusion Qualitative indicates areas of ion suppression Quantitative compare infusion with no matrix to infusion with matrix 2) Phospholipid monitoring Qualitative indicates where phospholipids elute in LC gradient 3) Matrix Mixing Quantitative how alternate matrix will affect analyte recovery 4) Quantitative ME Experiments Quantitative % matrix effects

8 Post Column Infusion HPLC Autosampler COLUMN MASS SPEC

9 Post Column Infusion HPLC HPLC MP s and gradient Pure Analyte or IS in solution Infusion Pump Autosampler COLUMN MASS SPEC Extracted Sample (no analyte or no IS) T-connector

10 Post Column Infusion Protein Precipitation anticoagulation drug panel Printing Time: 1:33:05 PM Printing Date: July I n t e n... XIC of +MRM (9 pairs): / Da ID: Apixaban 1 from Sample 4 (Sample 1) of PostColumnInfusion.wiff (Turbo Spray e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e Max. 2.8e6 cps XIC of +MRM (9 pairs): / Da ID: Rivaroxaban 1 from Sample 4 (Sample 1) of PostColumnInfusion.wiff (Turbo Spr... Max. 3.1e6 cps. I n t e n e6 2.0e6 1.0e XIC of +MRM (9 pairs): / Da ID: Dabigatran 1 from Sample 4 (Sample 1) of PostColumnInfusion.wiff (Turbo Spra... Max. 6.2e6 cps. I n t e n e6 4.0e6 2.0e Collected by: QTRAP5500\5500 Electronic Signature: no Operator: 5500

11 Post Column Infusion Supported Liquid Extraction Aldosterone/Cortisol Printing Time: 10:52:19 AM Printing Date: September Printing Time: 10:53:22 AM Printing Date: September Printing Time: 8:12:23 AM Printing Date: September XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 3 (NO IS for PostColumnInf... Max cps. XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 7 (NO IS for PostColumnInfu... Max. 1.6e5 cps. XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 6 (NO IS for PostColumnInf... Max cps. 9.5e4 1.6e e4 9.0e4 8.5e4 1.5e5 1.5e5 1.4e5 1.4e5 5.2e4 5.0e4 4.8e4 8.0e4 1.3e5 4.6e4 7.5e4 1.3e5 1.2e5 4.4e4 4.2e4 7.0e4 1.2e5 4.0e4 6.5e4 6.0e4 1.1e5 1.1e5 1.0e5 9.5e4 3.8e4 3.6e4 3.4e4 5.5e4 9.0e4 3.2e4 8.5e4 3.0e4 Intensity, cps 5.0e4 4.5e4 4.0e4 3.5e4 Intensity, cps 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 Intensity, cps 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 3.0e4 5.0e4 1.8e4 4.5e4 1.6e4 2.5e4 4.0e4 1.4e4 2.0e4 1.5e4 1.0e e4 3.0e4 2.5e4 2.0e4 1.5e e Printing Time: 10:53:05 AM Printing Time: 10:52:56 AM Printing AM 5.73 Time: :54: September Printing Date: September September Printing Date: Printing Date: Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 5 (NO IS for PostColumnInf... Max cps. Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 4 (NO IS for PostColumnInf... Max cps. Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 8 (NO IS for PostColumnInf... Max cps. Electronic Signature: no Electronic Signature: no Electronic Signature: no Operator: 5500 Operator: 5500 Operator: e5 1.15e5 1.05e5 1.4e e4 1.0e e5 1.00e5 1.4e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 9.50e4 9.00e4 8.50e4 8.00e4 1.3e5 1.3e5 1.2e5 1.2e5 1.1e5 1.1e5 8.00e4 7.50e4 1.0e5 7.50e4 7.00e4 7.00e4 6.50e4 9.5e4 9.0e4 8.5e4 6.50e4 6.00e4 8.0e4 Intensity, cps 6.00e4 5.50e4 Intensity, cps 5.50e4 5.00e4 Intensity, cps 7.5e4 7.0e4 6.5e4 5.00e4 4.50e4 6.0e4 4.50e4 4.00e4 5.5e4 4.00e4 3.50e4 3.00e4 2.50e4 3.50e4 3.00e4 2.50e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.00e4 2.00e4 2.5e4 1.50e4 1.50e4 2.0e4 1.00e e e4 1.0e Collected by: QTRAP5500\5500 Electronic Signature: no Operator: Collected by: QTRAP5500\5500 Electronic Signature: no Operator: Collected by: QTRAP5500\5500 Electronic Signature: no Operator: 5500

12 Post Column Infusion Supported Liquid Extraction Aldosterone/Cortisol Printing Time: 10:55:24 AM Printing Date: September Printing Time: 10:55:50 AM Printing Date: September Printing Time: 10:56:01 AM Printing Date: September XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 3 (NO IS for PostColumnInf... Max cps. XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 4 (NO IS for PostColumnInf... Max cps. XIC of -MRM (6 pairs): / Da ID: Aldo 1 from Sample 5 (NO IS for PostColumnInf... Max cps. 9.5e4 1.05e5 1.15e5 9.0e4 1.00e5 1.10e5 8.5e4 9.50e4 1.05e5 8.0e4 9.00e4 1.00e5 9.50e4 7.5e4 8.50e4 9.00e4 7.0e4 8.00e4 8.50e4 6.5e4 7.50e4 8.00e4 6.0e4 7.00e4 7.50e4 6.50e4 7.00e4 5.5e4 6.00e4 6.50e4 Intensity, cps 5.0e4 4.5e4 Intensity, cps 5.50e4 5.00e4 Intensity, cps 6.00e4 5.50e4 4.0e4 4.50e4 5.00e4 4.50e4 3.5e4 4.00e4 4.00e4 3.50e4 3.0e4 3.50e4 3.00e4 2.5e4 3.00e4 2.50e4 2.0e4 2.50e4 2.00e4 2.00e4 1.5e4 1.50e4 1.50e e4 1.00e4 1.00e Printing Time: 8:11:53 AM Printing Time: 10:56:23 AM 6.59 Printing Time: 10:56:42 AM 6.60 Printing Date: September Printing Date: September Printing Date: September Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 6 (NO IS for PostColumnInf... Max cps. Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 7 (NO IS for PostColumnInfu... Max. 1.6e5 cps. Collected XIC of by: -MRM QTRAP5500\5500 (6 pairs): / Da ID: Aldo 1 from Sample 8 (NO IS for PostColumnInf... Max cps. Electronic Signature: no Electronic Signature: no Electronic Signature: no Operator: 5500 Operator: 5500 Operator: e4 1.6e5 1.4e5 1.4e5 5.2e4 1.5e5 1.4e5 1.5e5 5.0e4 1.4e5 1.3e5 4.8e4 1.4e5 1.3e5 4.6e4 1.3e5 1.2e5 4.4e4 1.3e5 1.2e5 4.2e4 1.2e5 1.1e5 4.0e4 1.2e5 1.1e5 3.8e4 1.1e5 1.0e5 3.6e4 3.4e4 3.2e4 3.0e4 1.1e5 1.0e5 9.5e4 9.0e4 8.5e4 9.5e4 9.0e4 8.5e4 8.0e4 Intensity, cps 2.8e4 2.6e4 2.4e4 Intensity, cps 8.0e4 7.5e4 7.0e4 Intensity, cps 7.5e4 7.0e4 6.5e4 2.2e4 2.0e4 1.8e4 1.6e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 1.4e4 1.2e4 1.0e e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e e4 3.0e4 2.5e4 2.0e4 1.5e e4 1.0e Collected by: QTRAP5500\5500 Electronic Signature: no Operator: Collected by: QTRAP5500\5500 Electronic Signature: no Operator: Collected by: QTRAP5500\5500 Electronic Signature: no Operator: 5500

13 Post Column Infusion Analyte infusion with Printing Time: 8:03:17 AM Printing Date: September XIC of +MRM (4 pairs): / Da ID: Testosterone 1 from Sample 8 (E ) o... sample containing analyte I.S. Printing Time: infusion 8:03:58 AM with sample containing I.S. Printing Date: September Max cps. XIC of +MRM (4 pairs): / Da ID: Testosterone-d3 from Sample 14 (E )... Max. 1.4e5 cps e5 1.4e5 1.3e5 1.3e5 1.2e5 1.2e5 1.1e e5 1.0e5 9.5e4 9.0e e e4 Intensity, cps Intensity, cps 7.5e4 7.0e4 6.5e4 6.0e e e4 4.5e e e e4 2.5e e e4 1.0e Collected by: API5000\Administrator Electronic Signature: no Operator: Collected by: API5000\Administrator Electronic Signature: no Operator: 5500

14 Post Column Infusion Printing Time: 3:36:44 PM Printing Date: September Liquid-Liquid Extraction Testosterone QUANTITATIVE Printing Time: 3:37:01 PM Printing Date: September Printing Time: 4:09:25 PM Printing Date: September XIC of +MRM (4 pairs): / Da ID: Testosterone 1 from Sample 2 (WASH) of Max. XIC 5.9e4 of +MRM cps. (4 pairs): / Da ID: Testosterone 1 from Sample 3 (Blk) of PC... Max. 5.8e4 XIC of cps. +MRM (4 pairs): / Da ID: Testosterone 1 from Sample 4 (E ) of... Max. 5.9e4 cps. Intensity, cps 7.4e4 7.2e4 7.0e4 6.8e4 6.6e4 6.4e4 6.2e4 6.0e4 5.8e4 5.6e4 5.4e4 5.2e4 5.0e4 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Intensity, cps 7.8e4 Wash Blank Patient Sample 7.5e4 7.0e4 6.5e e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Collected by: API5000\Administrator Collected by: API5000\Administrator Collected by: API5000\Administrator Electronic Signature: no Electronic Signature: no Electronic Signature: no Operator: 5500 Operator: 5500 Operator: 5500 Internal Standard Testosterone Quant MRM Testosterone Qual MRM Intensity, cps e4 7.0e4 6.8e4 6.6e4 6.4e4 6.2e4 6.0e4 5.8e4 5.6e4 5.4e4 5.2e4 5.0e4 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e

15 Post Column Infusion Printing Time: 3:36:44 PM Printing Date: September Liquid-Liquid Extraction Testosterone QUANTITATIVE Printing Time: 3:37:01 PM Printing Date: September Printing Time: 4:09:25 PM Printing Date: September XIC of +MRM (4 pairs): / Da ID: Testosterone 1 from Sample 2 (WASH) of Max. XIC 5.9e4 of +MRM cps. (4 pairs): / Da ID: Testosterone 1 from Sample 3 (Blk) of PC... Max. 5.8e4 XIC of cps. +MRM (4 pairs): / Da ID: Testosterone 1 from Sample 4 (E ) of... Max. 5.9e4 cps. Intensity, cps 7.4e4 7.2e4 7.0e4 6.8e4 6.6e4 6.4e4 6.2e4 6.0e4 5.8e4 5.6e4 5.4e4 5.2e4 5.0e4 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e e4 Wash Blank Patient 7.5e4 6.6e4 7.0e4 Sample 6.4e4 6.2e4 6.5e4 6.0e e4 What if there is significant e4 ion suppression 5.6e4 at the RT of e e e e our analyte/is? e e e e e4 4.2e4 - adjust gradient lengthen gradient ramp/column e4 washout 3.8e4 4.0e e4 3.4e4 3.5e4 - extraction method changes? 3.2e4 3.0e4 3.0e4 2.8e4 2.6e4 2.4e4 2.5e4 2.2e4 Intensity, cps 2.0e4 1.5e4 1.0e Collected by: API5000\Administrator Collected by: API5000\Administrator Collected by: API5000\Administrator Electronic Signature: no Electronic Signature: no Electronic Signature: no Operator: 5500 Operator: 5500 Operator: 5500 Internal Standard Testosterone Quant MRM Testosterone Qual MRM Intensity, cps 7.2e4 7.0e4 6.8e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e

16 Phospholipid monitoring

17 Phospholipid monitoring MRM (+ESI) Class of PPL LysoPC (C18:0) LysoPC (C16:0) PC(C16:0/C18:2) PC(C16:0/C18:1) Plasmalogen PC (C18 (plasm)/c18:1) SM (dc18:1/c18:0) LysoPE (C16:0) PE (C18:0/C20:4) LysoPS (C18:1) Phospholipids in liquid chromatography/mass spectrometry bioanalysis: comparison of three tandem mass spectrometric techniques for monitoring plasma phospholipids, the effect of mobile phase composition on phospholipids elution and the association of phospholipids with matrix effects Yuan-Qing Xia and Mohammed Jemal* Bioanalytical and Discovery Analytical Sciences, Research and Development, Bristol-Myers Squibb, Princeton, NJ 08543, USA Received 3 February 2009; Revised 24 April 2009; Accepted 7 May 2009

18 Phospholipid monitoring Printing Time: 11:28:34 AM Printing Date: August Protein Precipitation XIC of +MRM (12 pairs): / Da ID: PPL-2 from Sample 3 (PPT) of PPT pos.wiff (Turbo Spray) Max. 7.0e6 cps. 7.0e e e6 5.5e e6 I n t e n s i t y, c p s 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e e6 5.0e Collected by: QTRAP5500\5500

19 Phospholipid monitoring Printing Time: 12:09:14 PM Printing Date: August Solid Phase Extraction XIC of +MRM (12 pairs): / Da ID: PPL-2 from Sample 6 (SPE) of PPT pos.wiff (Turbo Spray) Max. 6.9e6 cps. 6.9e e e6 5.5e6 5.0e I n t e n s i t y, c p s 4.5e6 4.0e6 3.5e6 3.0e e e6 1.5e e6 5.0e Collected by: QTRAP5500\5500

20 Phospholipid monitoring Printing Time: 12:25:52 PM Printing Date: August Liquid Liquid Extraction (with Hexane:Ethyl Acetate) XIC of +MRM (12 pairs): / Da ID: PPL-7 from Sample 8 (LLE (testo)) of PPT pos.wiff (Turbo Spray) Max. 2.8e4 cps. 6.3e6 6.0e6 5.5e6 5.0e6 4.5e6 I n t e n s i t y, c p s 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e Collected by: QTRAP5500\5500

21 Phospholipid monitoring Printing Time: 12:26:25 PM Printing Date: August Supported Liquid Extraction (with MtBE) XIC of +MRM (12 pairs): / Da ID: PPL-7 from Sample 9 (SLE (aldo)) of PPT pos.wiff (Turbo Spray) Max cps. 6.4e6 6.0e6 5.5e6 5.0e6 4.5e6 I n t e n s i t y, c p s 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e Collected by: QTRAP5500\5500

22 Phospholipid monitoring Printing Time: 12:26:25 PM Printing Date: August Supported Liquid Extraction (with MtBE) XIC of +MRM (12 pairs): / Da ID: PPL-7 from Sample 9 (SLE (aldo)) of PPT pos.wiff (Turbo Spray) Max cps. 6.4e6 6.0e6 5.5e6 I n t e n s i t y, c p s 5.0e6 What if there is buildup of PPLs on the column? - adjust gradient lengthen gradient ramp/column washout - extraction method changes - employ phospholipid removal strategies 4.5e6 4.0e6 3.5e6 3.0e6 2.5e6 2.0e6 1.5e6 1.0e6 5.0e Collected by: QTRAP5500\5500

23 Matrix mixing -Different native matrix samples mixed in several proportions - if sample-specific ME present, observed results will not agree with expected results - min. 5 native matrix admixing experiments should be performed - plot expected vs. observed

24 Matrix mixing Sample Name Apixaban Avg Expected (ng/ml) (ng/ml) (ng/ml) Accuracy Citrate Citrate Citrate Citrate:Serum 3: Citrate:Serum 3: % Citrate:Serum 3: Citrate:Serum 1: Citrate:Serum 1: % Citrate:Serum 1: Citrate:Serum 1: Citrate:Serum 1: % Citrate:Serum 1: Serum Serum Serum Citrate:LiHep 3: Citrate:LiHep 3: % Citrate:LiHep 3: Citrate:LiHep 1: Citrate:LiHep 1: % Citrate:LiHep 1: Citrate:LiHep 1: Citrate:LiHep 1: % Citrate:LiHep 1: LiHep LiHep LiHep `

25 CLSI Guidelines C62-A best practice: - At least 5 native matrix samples - comparing signal of samples spiked postextraction with pure analyte in solution refer to C50 and Matuszewski et al. European Guidelines 21 July 2011 EMEA/CHMP/EWP/192217/2009 Rev. 1 Corr. 2** - 6 lots of matrix for each analyte and IS at low and high - Comparing peak areas of pure analyte in solution (no matrix) with analyte spiked into extracts from matrix extractions.

26 Described by Matuszewski et al in 2003 Pure Analyte in solution Pure Analyte spiked into extracted blank Pure analyte spiked into blank and then extracted - no matrix - no extraction - all matrix constituents - no extraction - all matrix constituents - undergoes extraction

27 Described by Matuszewski et al in 2003 ME: Matrix Effect RE: Recovery PE: Process Efficiency A pure analyte in solution (at equivalent concentration to analyte in extract after extraction) B analyte spiked into the extract at equivalent concentration to analyte in extract after extraction C analyte spiked into matrix before extraction at specified levels (e.g. low and high)

28 Calculating the Pre and Post Spikes - 5 individual matrix sources or lots - Generally acceptable to do 2 levels low & high / clinically relevant levels PRE extraction spike (C): straightforward spike into 5 or 6 lots of matrix POST extraction spike (B): calculate through all the sample preparation steps to get the final equivalent concentration in the extract Pure solution (A): equivalent concentration same as B, but into reconstitution solvent (or other solution)

29 Endogenous Compounds: Is there an analyte free, truly commutable blank matrix? - Steroid free serum/treated serum/artificial serum - buffer mixtures (e.g. BSA in PBS) Each patient has unique matrix constituents therefore you want to take into consideration potential matrix effects for all patients.

30 Example 1: Testosterone assay sample prep: - 100uL serum - 40uL internal standard - 750uL extraction solvent - 500uL transferred and evaporated to dryness - reconstitute 200uL Range is nmol/l Low level 0.5 nmol/l High level 30 nmol/l

31 Example 1: Testosterone - LOW 0.5 nmol/l 0.1 ml sample ml IS. 0.5mL 0.75 ml 0.2 ml 0.23 nmol/l SO: for solution A prepare 0.23 nmol/l in reconstitution solvent (will also be used for B ) For C spike 0.5 nmol/l into 6 lots of blank* matrix. For B add 200uL of solution A to evaporated extracts

32 Example 1: Testosterone - LOW SPIKING CALCULATIONS: Pure Testosterone standard solution: 50 nmol/l A SAMPLE spike into reconstitution solvent inject straight 50 nmol/l x 46 ul to 10 ml reconstitution solvent = 0.23 nmol/l B SAMPLES spiking into reconstitution solvent add 200uL to evaporated extracts 50 nmol/l x 46 ul to 10 ml reconstitution solvent = 0.23 nmol/l C SAMPLES spiking into matrix samples before extraction: 50 nmol/l x 10 ul to 1 ml serum= 0.5 nmol/l C - 100uL serum - 40uL internal standard - 750uL extraction solvent - 500uL transferred and evaporated to dryness - reconstitute 200uL B

33 Example 1: Testosterone - LOW SPIKING CALCULATIONS: Pure Testosterone standard solution: 50 nmol/l A SAMPLE spike into reconstitution solvent inject straight 50 nmol/l x 46 ul A to & B 10 are ml the reconstitution same: can solvent = 0.23 nmol/l B SAMPLES spiking just into make reconstitution 1 solution and solvent add 200uL to evaporated extracts use for both 50 nmol/l x 46 ul to 10 ml reconstitution solvent = 0.23 nmol/l C SAMPLES spiking into matrix samples before extraction: 50 nmol/l x 10 ul to 1 ml serum= 0.5 nmol/l C - 100uL serum - 40uL internal standard - 750uL extraction solvent - 500uL transferred and evaporated to dryness - reconstitute 200uL B

34 Example 1: Testosterone - HIGH 30 nmol/l 0.1 ml sample ml IS. 0.5mL 0.75 ml 0.2 ml 14 nmol/l SO: for solution A prepare 14 nmol/l in reconstitution solvent (will also be used for B ) For C spike 30 nmol/l into 6 lots of blank* matrix. For B add 200uL of solution A to evaporated extracts

35 Example 1: Testosterone - HIGH SPIKING CALCULATIONS: Pure Testosterone standard solution: 1000 nmol/l A SAMPLE spike into reconstitution solvent inject straight 1000 nmol/l x 140 ul to 10 ml reconstitution solvent = 14 nmol/l B SAMPLES spiking into reconstitution solvent add 200uL to evaporated extracts 1000 nmol/l x 140 ul to 10 ml reconstitution solvent = 14 nmol/l C SAMPLES spiking into matrix samples before extraction: 1000 nmol/l x 30 ul to 1 ml serum= 30 nmol/l C - 100uL serum - 40uL internal standard - 750uL extraction solvent - 500uL transferred and evaporated to dryness - reconstitute 200uL B

36 Example 1: Testosterone - HIGH SPIKING CALCULATIONS: Pure Testosterone standard solution: 1000 nmol/l A SAMPLE spike into reconstitution solvent inject straight A & B are the same: can just make 1 solution and 1000 nmol/l x 140 ul to 10 ml reconstitution solvent = 14 nmol/l B SAMPLES spiking into reconstitution solvent add 200uL to evaporated extracts use for both 1000 nmol/l x 140 ul to 10 ml reconstitution solvent = 14 nmol/l C SAMPLES spiking into matrix samples before extraction: 1000 nmol/l x 30 ul to 1 ml serum= 30 nmol/l C - 100uL serum - 40uL internal standard - 750uL extraction solvent - 500uL transferred and evaporated to dryness - reconstitute 200uL B

37 Example 1: Testosterone * Blank matrix: - Steroid free serum which we use to prepare calibrators: Not really like serum collected in red top tubes so what to do? - alternative: spike into real patient samples in this case, you must do an extra set of non-spiked samples: 1) Extract 1 set normally 2) Extract 1 set pre-spiked with known concentration (Sample C ) 3) Extract 1 set and spike with known concentration at reconstitution step (Sample B )

38 Example 1: Testosterone RESULTS Sample Name Average Analyte Peak Area (counts) Averag e Spiked Sample s minus straight ME (%) RE (%) PE (%) Sample Name Averag Averag e e Analyte Spiked Peak Sample Area s minus (counts) straight ME (%) RE (%) PE (%) LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PostSpk 'B' HighSample_PostSpk 'B' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample_PreSpk 'C' HighSample_PreSpk 'C' LowSample HighSample LowSample HighSample LowSample HighSample LowSample HighSample LowSample HighSample LowSample HighSample LowSample-A-1 Pure Solution HighSample-A-1 Pure Solution

39 Example 2: VitaminD assay sample prep (PPT): - 100uL serum - 350uL of precipitating reagent containing internal standard (total volume after addition = 450uL) - Vortex Mix, Centrifuge, direct inject supernatant Range is nmol/l Low level 25 nmol/l (<25 nmol/l = severe VitD deficiency) High level 75 nmol/l (75+ nmol/l = normal VitD levels)

40 Example 2: VitaminD assay sample prep - LOW: 25 nmol/l 0.1 ml 0.45 ml 5.56 nmol/l SO: For A We need to account for aqueous in the final extract: - Make a solution that is 1:3.5 DI water:ppt reagent - spike in VitD at 5.56 nmol/l For C spike 25 nmol/l into 6 lots of blank* matrix & extract as normal For B add 350uL of solution with equivalent [VitD] to 6 lots of blank* matrix: nmol/l x 450/350 = 7.14 nmol/l

41 Example 2: VitaminD assay sample prep - LOW: 25 nmol/l 0.1 ml 0.45 ml 5.56 nmol/l SO: SPIKING For A CALCULATIONS: We need to account for aqueous in the final extract: Pure - Make VitDa standard solution solution: that is 1: DI nmol/l water:ppt reagent - spike in VitD at 5.56 nmol/l A SAMPLE spike into 1:3.5 mix of water:ppt inject straight 2500 nmol/l x 22 ul to 10 ml reconstitution solvent = 5.5 nmol/l For C spike 25 nmol/l into 6 lots of blank* matrix & extract B SAMPLES spike into PPT reagent add 350uL to blank matrix as normal 2500 nmol/l x 28.5 ul to 10 ml PPT reagent = 7.13 nmol/l C SAMPLES spike into matrix samples before extraction: For B add 350uL of solution with equivalent [VitD] to 6 lots of blank* 2500 nmol/l matrix: x 10 ul to 1 ml serum= 25 nmol/l nmol/l x 450/350 = 7.14 nmol/l

42 Example 2: VitaminD assay sample prep - HIGH: 75 nmol/l 0.1 ml 0.45 ml nmol/l SO: For A We need to account for aqueous in the final extract: - Make a solution that is 1:3.5 DI water:ppt reagent - spike in VitD at nmol/l For C spike 75 nmol/l into 6 lots of blank* matrix & extract as normal For B add 350uL of solution with equivalent [VitD] to 6 lots of blank* matrix: nmol/l x 450/350 = 21.4 nmol/l

43 Example 2: VitaminD assay sample prep - HIGH: 75 nmol/l 0.1 ml 0.45 ml nmol/l SO: SPIKING For A CALCULATIONS: We need to account for aqueous in the final extract: Pure - Make VitDa standard solution solution: that is 1: DI nmol/l water:ppt reagent - spike in VitD at nmol/l A SAMPLE spike into 1:3.5 mix of water:ppt inject straight 2500 nmol/l x 67 ul to 10 ml reconstitution solvent = For C spike 75 nmol/l into 6 lots of blank* matrix & extract nmol/l as normal B SAMPLES spike into PPT reagent add 350uL to blank matrix 2500 nmol/l x 85.6 ul to 10 ml PPT reagent = 21.4 nmol/l C SAMPLES spike into matrix samples before extraction: For B add 350uL of solution with equivalent [VitD] to 6 lots of blank* matrix: nmol/l x 450/ ul to 1 = ml 21.4 serum= nmol/l 75 nmol/l

44 Example 2: VitaminD assay sample prep (PPT): Blank matrix: Steroid free serum same issue as with testosterone... SO: spike into real patient samples in this case, you must do an extra set of non-spiked samples: 1) Extract 1 set normally 2) Extract 1 set pre-spiked with known concentration (Sample C ) 3) Extract 1 set and spike with known concentration at reconstitution step (Sample B )

45 Example 2: VitD - RESULTS Sample # Avg Peak Area un-spiked Avg Peak Area PRE SPIKE ("C") minus un-spiked PA Low Level (25 nmol/l) VITD3 Avg Peak Area POST SPK ("B") minus un-spiked PA Avg Peak Area Sample "A" % ME %RE %PE Sample % % % Sample % % % Sample % % 94.33% Sample % 98.43% 90.84% Sample % % % Sample % 96.92% 91.99% Steroid Free Serum NA % % % High Level (75 nmol/l) VITD3 Sample # Avg Peak Area un-spiked Avg Peak Area PRE SPIKE ("C") minus un-spiked PA Avg Peak Area POST SPK ("B") minus un-spiked PA Avg Peak Area Sample "A" % ME %RE %PE Sample % 86.64% 89.40% Sample % 95.69% 97.78% Sample % 99.25% 94.65% Sample % 94.86% 87.42% Sample % % % Sample % % 86.13% Steroid Free Serum NA % 98.24% 95.86%

46 Example 3: Plasma Renin Activity (Solid Phase Extraction) - 250uL EDTA plasma - 50uL of generation buffer - Vortex Mix, Incubate at 37C for 3 hours to generate Ang1 - Add 300uL of 10ng/mL 15N-Ang1 (I.S.) in 10% formic acid, mix. - SPE procedure: - load all of sample (total 600uL) - Wash with 500uL of 5/20/75 FA/MeOH/water (v/v/v) - dry under vacuum 10 minutes - elute Ang1 with 200uL of 100% MeOH Range is ng/l/second Low level 1 ng/ml (equivalent to 0.09 ng/l/second) High level 100 ng/ml (equivalent to 9.26 ng/l/second)

47 Example 3: Plasma Renin Activity - LOW 1 ng ml 0.25 ml 0.2 ml 1.25 ng/ml SO: For A spike 1.25 ng/ml into starting conditions mobile phase composition (20:80 MeOH:Water with 0.2% formic acid) For B spike 1.25 ng/ml into MeOH extract after extraction For C spike 1 ng/ml into plasma samples after incubation, before SPE procedure

48 Example 3: Plasma Renin Activity - LOW 1 ng ml 0.25 ml 0.2 ml 1.25 ng/ml SO: SPIKING For A CALCULATIONS: spike 1.25 ng/ml into starting conditions mobile phase composition (20:80 MeOH:Water with 0.2% formic acid) Pure Angiotensin I standard solution: 5000 ng/ml A SAMPLE Dilute stock standard solution to 25 ng/ml 25 ng/ml x 50 ul to 1 ml 20:80 MeOH:water with 0.2% FA = For B spike 1.25 ng/ml into MeOH extract after extraction For 1.25 C ng/ml spike 1 ng/ml into plasma samples after incubation, before SPE procedure B SAMPLES use diluted standard solution 25 ng/ml x 10 ul to 0.2 ml MeOH extracts = 1.25 ng/ml C SAMPLES use diluted standard solution (25ng/mL) spike into matrix samples before SPE step: 25 ng/ml x 10 ul to 0.25 ml serum= 1 ng/ml

49 Example 3: Plasma Renin Activity - HIGH 100 ng ml 0.25 ml 0.2 ml 125 ng/ml SO: For A spike 125 ng/ml into starting conditions mobile phase composition (20:80 MeOH:Water with 0.2% formic acid) For B spike 125 ng/ml into MeOH extract after extraction For C spike 100 ng/ml into plasma samples after incubation, before SPE procedure

50 Example 3: Plasma Renin Activity - HIGH 100 ng ml ml ml ng/ml SO: SPIKING For A CALCULATIONS: spike 125 ng/ml into starting conditions mobile phase composition (20:80 MeOH:Water with 0.2% formic acid) Pure Angiotensin I standard solution: 5000 ng/ml A SAMPLE Spike into 20:80 MeOH:water with 0.2% FA For B spike 125 ng/ml into MeOH extract after extraction 5000 ng/ml x 25 ul to 1 ml 20:80 MeOH:water with 0.2% FA = For 125 C ng/ml spike 100 ng/ml into plasma samples after incubation, before SPE procedure B SAMPLES spike into SPE elution 5000 ng/ml x 5 ul to 0.2 ml MeOH extracts = 125 ng/ml C SAMPLES spike into matrix samples before SPE step: 5000 ng/ml x 5 ul to 0.25 ml serum= 100 ng/ml

51 Example 3: Plasma Renin Activity (SPE) - RESULTS Sample Name Angiotensin I peak area Minus nonspiked sample % ME % RE % PE Sample Sample Sample Sample Sample Sample1 POST L "B" % Sample2 POST L % Sample3 POST L % Sample4 POST L % Sample5 POST L % Sample1 PRE L "C" % 42.81% Sample2 PRE L % 30.21% Sample3 PRE L % 37.61% Sample4 PRE L % 40.38% Sample5 PRE L % 39.12% Sample1 POST H "B" % Sample2 POST H % Sample3 POST H % Sample4 POST H % Sample5 POST H % SampHe1 PRE H "C" % 40.17% SampHe2 PRE H % 42.20% SampHe3 PRE H % 42.66% SampHe4 PRE H % 42.56% SampHe5 PRE H % 43.35% SAMPLE A Low SAMPLE A High

52 uestions? thank you

Fast quantitative Forensic Analysis of THC and its Metabolites in Biological Samples using Captiva EMR- Lipid and LC/MSMS

Fast quantitative Forensic Analysis of THC and its Metabolites in Biological Samples using Captiva EMR- Lipid and LC/MSMS Christophe Deckers, M.Sc. Sample prep Application Scientist Types of ``Interferences``