WELCOME Load and Go Urine Drug Testing How Hands-Free Hydrolysis - PowerPoint PPT Presentation

WELCOME

Load and Go Urine Drug Testing How Hands-Free Hydrolysis and Sample Filtration can Transform your Workflow

Discussion Topics Key Takeaways Challenges in Urine Drug Testing (UDT) ❖ Workflow Model and Limitations ❖ Overcoming the Automation Conundrum with ❖ B-One™ and INTip™ Filtration Online Competitive Hydrolysis Study ❖ Achieving a Hands-Free Solution ❖ Q and A ❖

Clinical Laboratory Challenges Different UDT Goals CORPORATE PRODUCTION 1. Reimbursements 1. Sample turnaround time (TAT) 2. Billable does not equal payable 2. Prioritize time savings 3. Lower production costs Increase throughput & decrease FTE time 3. Maintain data accuracy and quality But in the end....

TIME IS MONEY

Workflow Production Model Comprised of 4 Stages Stage Processes Obstacles Sample Cups to tubes Automation Difficult 1 ● ● Accessioning LIMS/barcoding FTE Intensive ● ● Sample Hydrolysis 2 ● Offline by FTE ● Processing Pre-treatment ● LC Injection 3 ● Chromatography ● LC Method Run Time Isobars ● ● Donec risus dolor porta ● Review Automation Difficult 4 ● ● Data Analysis Release Input Manually with FTE ● ●

Sample Processing -From Hands-On to Hands-Free Accessioning Sample LC-MS Preparation Injection Sample Hydrolysis Processing WHY HYDRO - cleaving the glucuronides provide + Increased abundances (sensitivity) LC-MS Method + Reducing complexity = fewer target analytes, faster LC methods faster data review + Lowering cost (Gluc analytes Gluc ISTDs) Data Analysis + Availability

Sample Processing -From Hands-On to Hands-Free Accessioning Sample LC-MS Preparation Injection Sample Hydrolysis Processing WHY Clean UP the Urine + Data Integrity LC-MS Method + Chromatography Robustness + Data Review + Instrument maintenance Data Analysis + Column longevity

Sample Processing - From Hands-On to Hands-Free Automation Accessioning Accessioning Hydrolysis Sample LC-MS Sample Sample Preparation Injection Processing Processing Take advantage of your liquid handler LC-MS Method LC-MS Method Data Analysis Data Analysis

Load and Go Strategy Tip on Tip Liquid Handler B-One

Nick! What is B-One? B-One is the Solution, the All-in-ONE Hydrolysis Solution B-One is an enhanced/purified recombinant ❖ β-glucuronidase, developed for speed and thermal stability. B-One comes stabilized in its reaction buffer. In fact this ❖ product is actually a reagent. Leave it on the bench. B-One is stable at room temperature ❖ for 3 months or at 4°C for 12 months. (NO need to worry about storing it after using).

Tip-on-Tip Technology (ToT) High efficiency sub-micron matrix filtration ToT Filtration - Combines a conductive wide bore top tip (Hamilton) with a bottom Filtration tip (DPX) coupled to provide an automated alternative to traditional filtration/centrifugation. Advantages : Particulate removal down to less than 1 μm ● No positive pressure/vacuum manifold or centrifuge ● High throughput workflows that can process up to 96 samples in under 2 minutes ● This methodology is also applied for high-throughput protein precipitation and ● filtration. Filtration tip options: Low porosity, dual phase or single phase varieties. ● Low cost/ high efficiency / cleaner samples ● Matrices include urine, blood, serum and homogenized cannabis products. ●

B-One & Tip-on-Tip Technology Creates The “Load and Go” Hurdle Solution - Prep --------------------------------------- Comes in Reaction buffer - Time --------------------------------------- In as little as 5 min Incubation - Offline (means and FTE)------------ Leave it on the Deck - Offline Heat----------------------------- Incubation is Room temp - Offline Spin------------------------------ Online DPX filtration - Throughput------------------------------ 96 in <2 mins - Mastermix Waste --------------------- Room Temp Stability = never throw away enzyme or IS

Load and Go Strategy Tip on Tip Liquid Handler B-One

Study Design Codeine-6B-Glucuronide - the ultimate analyte in UDT Negative urine (Surine) 500, 5,000, 25,000 ng/mL ❖ 5, 15, 30, 45 min ❖ Room Temperature ❖ Competitive Conditions ❖ Tip on Tip filtration ( MeOH quenching ) ❖ LC MS/MS ❖

Competitive Hydrolysis Acetaminophen glucuronide was prepared @ 100,000 ng/mL in each of the Codeine-6β-D-glucuronide Samples for this study. This challenges the enzyme in a more “realistic” condition as in a production environment. Is the enzyme working efficiently to cleave our target analytes in the presence of other highly concentrated glucuronides in a given sample.

Key Components For Sample Preparation Sample preparation is carried out using ❖ robotics and DPX Technology. Quenching and filtering of each cohort ❖ time segment is done simultaneously Sample filtration Tip-on-Tip technology ❖ > 2 min 96 Online DPX filtration, Low Porosity ❖ Filtration Tip

Hydrolysis protocol Plate Prep Sample prep procedure was as follows: 5 Calibrators and 3 QCs 50uL of each Cal, QC and study sample ● 10uL of Codeine-D6 @ 1,000 ng/mL ● 100uL of B-One ● Samples were incubated for 5, 15, 30 and 45 min at room temperature on the deck of the Hamilton robot.

Clean up protocol Plate Prep Post-Incubation ● Each hydrolysis reaction was crashed and mixed with 275uL of ● methanol using a 300uL Hamilton wide-bore CO-RE tips. Crashed samples were aspirated and carried to the low pedestal ● to attach the DPX Low Porosity Filtration Tips (Tip on Tip Technology). The crashed sample was then dispensed through the filtration tip, ● into a collection plate. 100 uL of the filtrate was transferred to an injection plate ● containing 400 uL of water. Injected on an AB Sciex 6500 Q-Trap detector. ●

LC-MS/MS Validated Comprehensive/UDT Method DPX Filtration procedure on the Liquid Handler takes less than 2 minutes to complete for up to 96 samples. LC: 6 min run time (comprehensive) ❖ MP: FA in Water and FA in ACN ❖ C-18 2.1 x 50mm ❖ LOD 1.25 ng/mL ❖ LOQ 5 ng/mL ❖ ULOL 20,000 ng/mL ❖ 1200 Agilent front end with AB 6500 Q-Trap ❖ Regression, Linear 1/x ❖

Results

B-One's performance at different incubation times and analyte concentrations 500 ng/mL 5000 ng/mL 25000 ng/mL Incubation Time % Conversion %CV % Conversion %CV % Conversion %CV 5 min 94.1 2.4 60 9.7 68.8 4.7 15 min 99.6 1.8 95.7 3.8 78.1 2.5 30 min 102.0 3.3 96.6 5.1 79.0 3.2 45 min 101.3 3.5 97.8 4.1 80 2.4

B-One's performance at different incubation times & analyte concentrations

Conclusions

Conclusions Automating a production method with 15 min room temp ❖ hydrolysis is possible DPX Tip on Tip Filtration replaces offline centrifuging allowing ❖ for automated clean up Reaches acceptable recoveries for Codeine ❖ Load and Go workflow ❖

Questions?

Nicholas Chestera Jack Andrews Nicholas.chestara@dpxlabs.com j.andrews@kurabiotech.com