Current methodologies for DoA whole blood testing (section 3)

Abstract

Extraction of drugs of abuse panels from whole blood samples can be challenging, especially when analyzing a broad panel of analytes from different classes. This white paper studies the analysis of 56 common illicit drugs in whole blood, focusing on various aspects of sample preparation, including pre-treatment of whole blood samples, extraction techniques, and automation using Biotage® Extrahera™ sample preparation workstation.

Section 1 examines recovery and matrix effects for a panel of 56 drugs in whole blood when extracted by the following extraction techniques: protein precipitation (ISOLUTE® PPT+), protein and phospholipid depletion (ISOLUTE® PLD+), supported liquid extraction (ISOLUTE® SLE+), and the following solid phase extraction (SPE) sorbents: reverse phase (EVOLUTE® EXPRESS ABN), mixed-mode strong cation exchange (EVOLUTE® EXPRESS CX), and mixed-mode weak cation exchange (EVOLUTE® EXPRESS WCX). The retention behaviors of various drug classes with different sorbent chemistries are discussed and key aspects crucial to method development are highlighted.

Section 2 focuses on whole blood sample pre-treatment comparing different hemolysis approaches, including osmotic breakdown, inorganic ZnSO4 denaturing, and bead homogenization, to release analytes bound to erythrocytes. It also addresses how to select the appropriate hemolysis techniques considering the subsequent extraction and cleanup procedures outlined in section 1.

Section 3 details the automation of the sample preparation methods described in sections 1 and 2. It provides the settings and parameters required for optimal performance with each sorbent, highlighting best practices for transferring and mixing whole blood.

Section 3: Automation of whole blood sample preparation on the Biotage® Extrahera™ Classic

Part I. Biotage® Extrahera™ Classic sample preparation workstation

Laboratory automation streamlines analytical testing workflows, including sample preparation, instrumentation, data reporting, and interpretation. The goals are to achieve higher throughput, improve data quality and traceability, enhance efficiency in labor and material costs, and ensure safer laboratory operations. Popular automation systems integrate multiple procedures and functions within a single device. However, establishing such a platform requires significant investments in both hardware and software, and skilled personnel. While automation promises labor cost saving and improved efficiency, the return on investment is a critical factor in making purchasing decisions. The ideal automation should be user-friendly and cost-effective, flexible, and plug-and-play ready. This is especially important for mid-sized laboratories where flexibility and cost saving are essential for maintaining and growing the business. An alternative to integrated “all-in- one” systems is to automate specific workflow functions using individual workstations. These workstations often outperform “all-in-one” systems because each is optimized for a specific task. Additionally, their independence and flexibility reduce the complexity of integration and maintenance.

The Biotage® Extrahera™ Classic is a sample preparation workstation designed to automate the tedious and repetitive steps involved in sample extraction and cleanup before LC-MS analysis. It supports various sample preparation techniques and methodologies (e.g., PLD, PPT, SLE, SPE), and offers standardized, pre-programmed templates for each technique. Method development and editing are easily accomplished through touch-screen operation, eliminating the need for programming skills. As an independent workstation, Biotage® Extrahera™ Classic seamlessly integrates into a complete workflow alongside the Biotage Lysera bead mill homogenizer and the TurboVap® evaporator (Figure 16). This automated setup meets the performance and efficiency needs, while the independence of each workstation simplifies programming requirements and provides operational flexibility.

This section provides tips and tricks for transferring manual extraction protocols (described in Table 2 Section 1) to the Biotage® Extrahera™ Classic sample preparation workstation.Figure 16. Streamlined sample preparation workflow.

Part II. Important considerations for successful automation with Biotage® Extrahera™ Classic

Fine-tuned protocols are essential for successful automation. While the Biotage® Extrahera™ Classic workstation enhances efficiency and reproducibility, it cannot alter chemistry-related performance, such as analyte retention and partitioning behav- iors. Therefore, users must first develop a workable protocol, selecting the appropriate sorbent chemistry and developing loading and elution procedures before automating the process.

Key parameters controlling the system’s liquid transfer and delivery performance are crucial for ensuring accuracy, preci- sion, and reproducibility. The Biotage® Extrahera™ Classic is designed with sample preparation in mind and considers physiochemical properties of commonly used solvents, sample matrices, and sorbent chemistries. The system provides recom- mended parameters, including aspiration and dispensation flow rates, Z-axis position, air gaps, and processing pressure, pre-programmed to be used as a starting point.

It is critical to understand how to adjust these parameters for fluids with extreme viscosity or density such as whole blood or dichloromethane. Additionally, potential issues during the robotic process, like dripping, leaking, clogging, or bubbling, must be addressed. For example, transferring dichloromethane accurately requires increasing the lower air gap to prevent dripping, while transferring or mixing whole blood necessitates a slower aspiration flow rate to avoid bubbling. Here we provide a summary that outlines the physical properties of commonly used solvents and biofluids, along with suggested parameters for optimal liquid handling performance in the Biotage® Extrahera™ Classic operation (Table 1). Additionally, Table 2 provides a practical guide for maintaining smooth operation and minimizing downtown.

Positive pressure processing significantly affects extraction performance and consistency in automation. The Biotage® Extrahera™ Classic system offers default pressure settings for different sorbents based on their properties and packing density. However, the actual processing pressure must be tuned according to the specific method needs. For instance, SLE requires much lower pressure compared to SPE methods. When processing whole blood samples, higher pressure is needed for sample loading and washing compared to urine samples.

The processing pressure used in the Biotage® PRESSURE+ 96 manual positive pressure manifold (PPM) can serve as a reference when transferring methods to the Biotage® Extrahera™ Classic sample preparation workstation. Table 3 provides a side-by-side comparison of the processing pressure used in the Biotage® PRESSURE+ 96 and Biotage® Extrahera™ Classic for this study. These parameters can serve as starting conditions for automation development, but it is important to conduct experiments to assess each parameter and adjust the values based on the results.

Table 8. Suggested fluid transferring and mixing parameters for whole samples on the Biotage® Extrahera™ Classic

Table 9. Troubleshooting guidelines for processing whole blood samples on the Biotage® Extrahera™ Classic

Table 10. Suggested processing pressure parameters for whole blood samples for 96-well plates of different sample preparation techniques in Biotage® PRESSURE+ 96 positive pressure manifold and Biotage® Extrahera™ Classic

References

1. Hadland, S. E.; Levy, S. Objective Testing: Urine and Other Drug Tests. Child and Adolescent Psychiatric Clinics of North America. W.B. Saunders July 1, 2016, pp 549–565. https:// doi.org/10.1016/j.chc.2016.02.005.
2. Biagiotti, S.; Pirla, E.; Magnani, M. Drug Transport by Red Blood Cells. Frontiers in Physiology. Frontiers Media SA 2023. https://doi.org/10.3389/fphys.2023.1308632.
3. Borden, S. A.; Palaty, J.; Termopoli, V.; Famiglini, G.; Cappiello, A.; Gill, C. G.; Palma, P. Mass Spectrometry Analysis of Drugs of Abuse: Challenges and Emerging Strategies. Mass Spectrom Rev 2020, 39 (5–6), 703–744. https://doi.org/10.1002/mas.21624.
4. Xu, R. N.; Polzin, J.; Kranz, M.; Vaca, P.; Metchkarova, M.; Rieser, M. J.; El-Shourbagy, T. A. Strategies for
   Developing Sensitive and Automated LC-MS/MS Assays of a Pharmaceutical Compound and Its Metabolite from Whole Blood Matrix. Pharmaceutics 2010, 2 (2), 159–170. https:// doi.org/10.3390/pharmaceutics2020159.
5. Marin, S. J.; Menasco, D.; Neifeld, J.; Gairloch, E. Current Methodologies for Drugs of Abuse Urine: A White Paper from Biotage; 2019. https://www.biotage.com/documents/ current-methodologies-for-drugs-of-abuse-urine-testing-a-   white-paper-from-biotage
6. Goodhead, L. K.; Macmillan, F. M. Sourcebook of Laboratory Activities in Physiology Measuring Osmosis and Hemolysis of Red Blood Cells. Adv Physiol Educ 2017, 41, 298–305. https://doi.org/10.1152/advan.00083.2016.-Since.
7. Rodrigo Valero, A. M.; Quintela Jorge, O.; Serrano, B. B.; Tejedor, S. A. Optimization of a Rapid Method for Screening Drugs in Blood by Liquid Chromatography Tandem Mass Spectrometry. Advances in Laboratory Medicine 2023, 4 (4), 365–371. https://doi.org/10.1515/almed-2023-0154.
   
   

Literature number: PPS766