Dried Blood Spot Analysis From The Clinic To The Laboratory

Dried Blood Spot Analysis From The Clinic To The Laboratory Joanne Mather, Pharmaceutical Life Sciences Waters Corporation 211 Waters Corporation 1

From The Clinic To The Lab Blood Spot Sampling First Used In Neonatal Screening 211 Waters Corporation 2

Dried Blood Spots Timeline of Implementation >1 yrs ago DBS were exclusively used for neonatal, infectious disease screening, therapeutic drug monitoring Dried Blood Spots have been around for over 4 years 5 Years Ago 2 manuscripts DBS to discovery-stage PK and metabolite ID (Merck Frost) Present Day Potential application to routine drug development was realized within GlaxoSmithKline in 26 Quickly saw the broadly applicable advantages offered by DBS Other pharmaceutical companies (sanofi-aventis et. al.) becoming heavily involved with DBS (as well as CROs and vendors) 211 Waters Corporation 3

On Board DBS Uptake To date GSK has developed >15 validated DBS methods for nearly 75 compounds >2 studies supported using DBS >1 compounds have reached phase 1 pre-clin GLP studies 211 Waters Corporation 4

Issues and Advantages of Dried Blood Spot Analysis [Card based sample collection] Reduced animal usage/ cost/ 3 R s Lower shipping costs Reduced compound needed for tox studies Improved data- PK data from toxicology animals [Small sample volumes 1-2µL] Assay sensitivity [Background card interference] More complicated methods development [Resolution from metabolites and matrix needed] High LC separation efficiency 211 Waters Corporation 5

Analytical Issues Non liquid format Current work flow will not work with this format Current approach requires punching, either manual or automated Card background may cause issues Matrix effects from blood components Resolution from metabolites and matrix needed for quality 211 Waters Corporation 6

Analytical Issues Non liquid format Current work flow will not work with this format Current approach require punching, either manual or automated Card background may cause issues Matrix effects from blood components 211 Waters Corporation 7

Blood Spotting Aliquot 15μL blood per spot 3 spots per sample plus spare Using a pipette or capillary o Do NOT allow tip to touch card surface! Dry for 2 hours at room temperature Ship & store in sealable bags containing desiccant 211 Waters Corporation 8

Sample Prep & Analysis Analytical sample obtained by punching small circular disc (typically 3 mm) from centre of DBS Manually Automation BSD1 Extract disc in organic solvent (typically methanol) containing internal standard Quantification by validated LC- MS/MS assay 211 Waters Corporation 9

Chromatography Requirements Long separation Short Separations 211 Waters Corporation 1

% % % % Analysis with LC/MS/MS Quantification by validated LC-MS/MS assay 1721_JoM_DBS_138.15.61.81 1.12 1.3 1.67 1.79 1.91 2: MS2 ES+ TIC 1.5e1 2.13 2.26..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.74 325 > 297 (Alprazolam Metabolite) 2.55e7..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.8 314.2 > 286.1 (AlprazolamD5) 4.15e7..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.81 39.2 > 281 (Alprazolam) 5.2e7 Use of UPLC: Increased sensitivity Increased resolution Increased speed of analysis..5 1. 1.5 2. Time 211 Waters Corporation 11

% % % % % Rapid Mode Switching Precursor Ion Confirmation Scan 1721_JoM_DBS_138.15.61.81 1.12 1.3 1.67 1.79 1.91 2: MS2 ES+ TIC 1.5e1 2.13 2.26..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.74 325 > 297 (Alprazolam Metabolite) 2.55e7 PIC Scan Acquisition 1721_JoM_DBS_4 3 (.79) 1 296.81 324.86 3.79e7..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.8 314.2 > 286.1 (AlprazolamD5) 4.15e7..5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.81 39.2 > 281 (Alprazolam) 5.2e7..5 1. 1.5 2. Time 175.81 251.76 226.76 278.81 8 1 12 14 16 18 2 22 24 26 28 3 32 Confirm peak identity in the presence of a complex matrix in one analytical run. m/z 211 Waters Corporation 12

Analytical Issues Non liquid format Current work flow will not work with this format Current approach require punching, either manual or automated Card background may cause issues Matrix effects from blood components 211 Waters Corporation 13

Dried blood spot microvolume sampling for DMPK Three types are available (Whatman, GE), which have different chemistries. FTA DMPK-A (treated) FTA DMPK-B (treated) FTA DMPK-C (untreated) FTA DMPK A & B cards contain proprietary chemical mixtures that lyse cells, inactivate pathogens and denature degradative enzymes and other proteins. FTA DMPK-C cards contain only cellulose. http://www.whatman.com/dmpk.aspx 211 Waters Corporation 14

Rapid MS to MRM Switching 211 Waters Corporation 15

% % % Using RADAR to Evaluate Card Type To Be Used 121_JoM_DBS_cards_13 Full scan blank card -background signal 1 1.18 1.55 2: MS2 ES+ TIC 5.98e9.22 1.3 1.29 1.42.32 1.91 2.41 1.76 2.8 2.24 DMPK A.94.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 121_JoM_DBS_cards_17 2: MS2 ES+ 1.54 1 TIC 1.63 5.98e9.98 1.28 1.78 2.34 DMPK B 2.41.22 1.4.63 1.88 2.5 2.24.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 121_JoM_DBS_cards_25 2: MS2 ES+ 1 TIC 5.98e9 1.54 2.34.23 1.73 1.45 1.28 1.3 1.87.9.58 2.7 DMPK C.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 211 Waters Corporation 16 Time

% % % Using RADAR to Evaluate Card Type To Be Used Analyte Response 221_JoM_DBS_cards_1 1 1.1 1: MRM of 3 Channels ES+ 39.2 > 281 (Alprazolam) 9.61e5 DMPK A.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 221_JoM_DBS_cards_2 1: MRM of 3 Channels ES+ 1 39.2 > 281 (Alprazolam) 9.61e5 1.9 DMPK B.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 221_JoM_DBS_cards_33 1: MRM of 3 Channels ES+ 1.9 1 39.2 > 281 (Alprazolam) 9.61e5 DMPK C.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 211 Waters Corporation 17 Time

Analytical Issues Non liquid format Current work flow will not work with this format Current approach require punching, either manual or automated Card background may cause issues Matrix effects from blood components 211 Waters Corporation 18

Matrix The Challenge Matrix effects from blood components Gain adequate resolution from matrix interferences and other analytes/ metabolites Maximize sensitivity Maintain or improve productivity Removal of matrix components 211 Waters Corporation 19

% % % % RADAR - Monitor matrix complexity Gradient A Gradient B Full Scan 1721_JoM_DBS_135 1.42 1.56 1.28 2: MS2 ES+ 2.1 TIC 1.54e1 1.9 2.24 1721_JoM_DBS_138.61.81 1.3 1.61 1.67 1.79 1.91 2: MS2 ES+ TIC 1.5e1 2.13 2.26.5 1. 1.5 2. 1721_JoM_DBS_135 1: MRM of 3 Channels ES+ 1.42 39.2 > 281 (Alprazolam) 6.36e7.5 1. 1.5 2. 1721_JoM_DBS_138 1: MRM of 3 Channels ES+.81 39.2 > 281 (Alprazolam) 5.2e7 MRM Resolved more effectively from the endogenous blood and card matrix peaks.5 1. 1.5 2. Time.5 1. 1.5 2. Time 211 Waters Corporation 2

Alternate Approach for Calculating Matrix Factors LC Column MRM Signal 1) Determine Analyte Profile AN and IS injected through the column 2) Inject Solvent Blank 3) Inject Extracted Matrix Blank Solvent Blank injected with post column infusion of AN and IS Extracted Blank injected with post column infusion of AN and IS Analyte = AN Internal Standard = IS AN and IS infused post-column 211 Waters Corporation 21

Automating Matrix Factor Calculations: Integrated Intellistart Fluidics 211 Waters Corporation 22

MassLynx TargetLynx Simplifies Calculating The Matrix Effects TargetLynx automatically calculates matrix effects from 6 injections Results Presented in Final Report 211 Waters Corporation 23

Removal of Phospholipids 211 Waters Corporation 24

Removal of Phospholipids 211 Waters Corporation 25

Removal of Phospholipids 211 Waters Corporation 26

Response % % Sensitivity and Robustness Compound name: Alprazolam Correlation coefficient: r =.99968, r^2 =.99936 Calibration curve:.11984 * x +.65461 Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None.55.5 Alprazolam (benzodiazepine) 1pg/mL - 5ng/mL 421_JoM_DBS_QC_216 1 Alprazolam 1pg/mL 1.47 MRM of 3 Channels ES+ 39.2 > 281 (Alprazolam) 2.57e4 1.82 1.64 1.35 1.9 1.21 2.25.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3. 421_JoM_DBS_QC_31 MRM of 3 Channels ES+ 1 TIC 2.57e4.45.4 Blank.35.3.25.37 2.37 2.62 Time.25.5.75 1. 1.25 1.5 1.75 2. 2.25 2.5 2.75 3..2.15.1.5. ng/ml 5 1 15 2 25 3 35 4 45 5 Concentration (ng/ml) Average Std Dev QC 1.25.23 2.9 QC 2.4.38 4.84 QC 3 3 3.25 2.56 QC 4 3 31.9 4.97 QC 5 3 3.3 3.81 Published- Journal Bioanalysis Feb 211, Vol 3:4 p411-42 Rapid Analysis of DBS samples with sub 2µm LC/MS/MS Mather J et al. 211 Waters Corporation 27

% Response Compound name: Sitamaquine Correlation coefficient: r =.99991, r^2 =.99982 From Dried Blood Spot Calibration curve: 15.233 * x + 38.3894 Response type: External Std, Area Curve type: Linear, Origin: Exclude, Weighting: Null, Axis trans: None 6 4 2 LLOQ of 5pg/mL with a linear dynamic range of 4 orders of magnitude. Sitamaquine (WR-626) is an orally active 8- aminoquinoline analog for the potential treatment of visceral leishmaniasis (also known as kala-azar, black fever, and Dumdum fever). ng/ml 5 1 15 2 25 3 35 4 45 5 22FEB21_PR_118 1.41 MRM of 2 Channels ES+ 344.3 > 271.2 (Sitamaquine) 5.44e3 LLOQ of 5pg/mL 1.84 1.53 1..78 1.12 1.31 Time.2.4.6.8 1. 1.2 1.4 1.6 1.8 2. 2.2 2.4 2.6 2.8 211 Waters Corporation 28

Heart Cut Configuration 2D UPLC/MS/MS Trapping Decrease Matrix Effects and Increase Sensitivity At-Column Dilution Inject Large Volume Samples in Strong Solvent No evaporation & reconstitution 211 Waters Corporation 29

2 Dimensional Dried Blood Spot Analysis of Rosuvastatin Direct injection of methanol extract from DBS sample 8uL injection of methanol At column dilution No At Column Dilution With At Column Dilution 211 Waters Corporation 3

2 Dimensional UPLC/MS/MS Bioanalysis 1pg/mL Blank 211 Waters Corporation 31

Conclusions Dried Blood Spots offer a significant scientific and expense advantage. The lower sample volume derived from the dried blood spots and the background card matrix poses a significant analytical challenge. Information rich LC/MS/MS (RADAR) can simplify the process of DBS analysis/ method development. Analysis times as low as 3 minutes possible. 2D LC to remove Matrix components (trap) and allow injection of large volume samples in high organic solvent. 211 Waters Corporation 32

Acknowledgements Paul Rainville Jennifer Simeone Robert Plumb David Little Jessalynn Wheaton Erin Chambers Christopher Evans GSK Neil Spooner - GSK Ian Wilson - Astra Zeneca 211 Waters Corporation 33