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

PRODUCTION

1. Sample turnaround time (TAT) 2. Prioritize time savings Increase throughput & decrease FTE time 3. Maintain data accuracy and quality

Clinical Laboratory Challenges

Different UDT Goals

CORPORATE

1. Reimbursements 2. Billable does not equal payable 3. Lower production costs But in the end....

TIME IS MONEY

Workflow Production Model

Comprised of 4 Stages

Data Analysis

4

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• Review
• Release

LC Method

3

• LC Injection
• Run Time

Sample Processing

2

• Hydrolysis
• Pre-treatment

Accessioning

1

• Automation Difficult
• FTE Intensive
• Sample Cups to tubes
• LIMS/barcoding
• Offline by FTE
• Chromatography
• Isobars
• Automation Difficult
• Input Manually with FTE

Stage Processes Obstacles

Sample Processing

• From Hands-On to Hands-Free

Sample Preparation

Hydrolysis

Accessioning

Sample Processing

LC-MS Method Data Analysis

LC-MS Injection WHY HYDRO - cleaving the glucuronides provide

+ Increased abundances (sensitivity) + Reducing complexity = fewer target analytes, faster LC methods faster data review + Lowering cost (Gluc analytes Gluc ISTDs) + Availability

Sample Processing

• From Hands-On to Hands-Free

Sample Preparation

Hydrolysis

Accessioning

Sample Processing

LC-MS Method Data Analysis

LC-MS Injection WHY Clean UP the Urine

+ Data Integrity + Chromatography Robustness + Data Review + Instrument maintenance + Column longevity

Sample Processing

• From Hands-On to Hands-Free

Sample Preparation Hydrolysis

Accessioning

Sample Processing

LC-MS Method Data Analysis

LC-MS Injection Automation

Take advantage of your liquid handler

Accessioning

Sample Processing

LC-MS Method Data Analysis

Load and Go Strategy

Liquid Handler B-One Tip on Tip

Nick! What is B-One?

❖ 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).

B-One is the Solution, the All-in-ONE Hydrolysis Solution

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

• Prep ---------------------------------------
• Time ---------------------------------------
• Offline (means and FTE)------------
• Offline Heat-----------------------------
• Offline Spin------------------------------
• Throughput------------------------------
• Mastermix Waste ---------------------

Solution

Comes in Reaction buffer In as little as 5 min Incubation Leave it on the Deck Incubation is Room temp Online DPX filtration 96 in <2 mins Room Temp Stability = never throw away enzyme or IS

Load and Go Strategy

Liquid Handler B-One Tip on Tip

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

Study Design

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

• ther 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

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.

Plate Prep

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

❖ 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

DPX Filtration procedure on the Liquid Handler takes less than 2 minutes to complete for up to 96 samples.

Results

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

Incubation Time 500 ng/mL 5000 ng/mL 25000 ng/mL % 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

Nicholas.chestara@dpxlabs.com

Jack Andrews j.andrews@kurabiotech.com