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.
For drugs or metabolites that interact with red blood cells (RBCs), lysing the RBCs is necessary to release these compounds before extraction and sample cleanup process. Common hemolysis techniques include osmotic breakdown, ZnSO4 inorganic denaturation, and bead homogenization.
Water osmotic breakdown is a common technique for whole blood hemolysis. When whole blood is mixed with water in a 1:1 (v/v) ratio or higher, the RBCs become hypotonic. Water then moves into the cells by osmosis, causing them to swell and break down. In addition to water, any aqueous solution or buffer that can permeate into RBCs can also be used for hemolysis. Techniques that facilitate osmotic breakdown include freeze- thaw cycles, centrifugation, vortex, and sonication. The osmotic breakdown method is advantageous in its simplicity and cost-effectiveness, as it requires no additional reagents or specialized equipment.
Zinc sulfate (ZnSO4) is a commonly used lysing agent to break open RBCs in whole blood samples and release target analytes. The Zn2+ ions bind to RBC membrane proteins, forming insol- uble metal complexes that precipitate the membrane proteins and cause cell lysis. Most published protocols used 0.1M (about 2 %) ZnSO4/0.1 M ammonium acetate buffer for cell lysis. While this method is widely used, there is limited understanding of how Zn2+ and SO42- interact with matrices, affect downstream extraction and cleanup processes, and influence analyte ioniza- tion in LC-MS. Besides ZnSO4, acetonitrile or methanol are also used as hemolytic agents in sample preparation.
Mechanical disruption using a bead homogenizer, such as Biotage® Lysera, is an effective alternative for lysing RBCs. In this technique, small beads are added to blood samples and vigorously agitated to mechanically shear and rupture RBC membranes. Bead mill homogenization is an efficient and thorough technique which can be scaled for high throughput. Since no additional chemical reagents are introduced into the sample, the homogenate supernatant can be directly loaded onto the cartridge or plate for cleanup.
In our study, we systematically compared water osmotic breakdown, ZnSO4 inorganic denaturation, and bead homog- enization in their effects on RBC hemolysis and analyte release, while ensuring compatibility with the extraction and cleanup procedures.
Optical microscopy
Fresh whole blood sample (K2EDTA) (ZenBio) was kept in the refrigerator (4 °C) without freezing. Whole blood sample aliquots were treated with a 1:1 ratio (v/v) of the following
solutions: saline (as control), water, 2 % ZnSO4, 6 % ZnSO4, 50 mM NH4OAc buffer (pH 6.0), and saline (for bead homogeniza- tion). All samples were mixed and left at room temperature for 5-30 min except for the bead homogenization samples which were processed using the Biotage® Lysera with a programmed method (2.4 m/s, 30s). One set of hemolyzed samples was examined by direct visualization, while the other set of samples was diluted 1250-fold with saline and examined under a microscope.
To simulate the interaction between drug analytes and RBCs, the analytes were dissolved in saline and spiked into fresh whole blood aliquots at a low concentration level (5-20 ng/mL). The spiked aliquots were then incubated at 37 °C for 16 hours. A spiked fresh whole blood sample without incubation (t=0h) was used as the baseline.
Sample preparation with ISOLUTE® PLD+: Aliquots (100 µL) of the incubated samples were divided into four groups and treated with a 1:1 (v/v) of the following: saline (no lysis, control), water, 6 % ZnSO4, and saline (for bead homogenization), respectively. Each group includes five replicates. Samples treated with water, 6 % ZnSO4, and the saline control were left at room temperature for 5 minutes, then transferred onto pre-dispensed acetonitrile (ACN) in the ISOLUTE® PLD+ plate and extracted following the rest of the PLD procedure (Table 2 Section 1). The bead homogenization samples were transferred to the homogenization tube with beads, mixed with 800 µL ACN, and processed using the Biotage® Lysera with a programmed method (2.4 m/s, 30s). A volume of 900 µL of the homogenate was then transferred onto the ISOLUTE® PLD+ plate and extracted following the remaining steps described in the PLD procedure (Table 2 Section 1).
Sample preparation with ISOLUTE® SLE+: Aliquots (100 µL) of the incubated samples were split into three groups and processed with different lysis/pretreatment protocols: 150 µL 50 mM NH4OAc (pH 8.5), 50 µL 6 % ZnSO4 (wait for 5 minutes) followed by mixing with 100 µL 50 mM NH4OAc (pH 8.5), and 150 µL 50 mM NH4OAc (pH 8.5) then homogenized using the Biotage® Lysera (2.4 m/s, 30s). Each group included five replicates. The pretreated samples were transferred onto the ISOLUTE® SLE+ plate and extracted following the remaining steps described in the SLE method (Table 2 Section 1).
Sample preparation with EVOLUTE® EXPRESS CX: Aliquots (100 µL) of the incubated samples (16h) were divided into four groups and treated as follows: 100 µL 50 mM NH4OAc (pH 6.0), 3 % ZnSO4, 6 % ZnSO4, and 12 % ZnSO4, respectively. The lysed samples were then mixed with 300 µL 50 mM ammonium acetate (pH 6.0) and processed using the CX procedures described in Table 2. The baseline sample (100 µL) was processed under the same conditions as the samples treated with 50 mM NH4OAc. Corresponding post-spiked samples for different lysis methods were prepared by incubating and processing blood, without spiking, under the same conditions, then analytes were spiked at the reconstitution step. All prepared samples were analyzed using the same LC-MS method, and the extraction recovery, matrix effect, and extraction reproducibility (RSD, %) were evaluated as described in Section 1 Part V Methodologies.
RBC hemolysis can be directly indicated by the brightness of the red color and the presence of RBCs adhering to the container walls. As shown in Figure 1, the water and NH4OAc buffer treated whole blood samples appear more transparent, exhibiting a much brighter red color and fewer RBCs attached to the vial walls compared to other samples. In contrast, the ZnSO4- treated samples appear significantly cloudier, with noticeable precipitates. Microscopic examination clearly demonstrates how different lysis methods affect RBC breakdown in whole blood. While water osmotic breakdown, NH4OAc treatment, and bead homogenization are all effective in RBC lysis, NH4OAc showed the best performance (Figure 2). We investigated two ZnSO4 concentrations (2 % and 6 %, g/mL), and only the higher concentration (6 % ZnSO4) was effective in achieving RBC lysis.
Figure 12. Optical microscopy of RBCs homolyzed by water osmotic breakdown, 2 % ZnSO4, 6 % ZnSO4, 50mM NH4OAc, and bead homogenization (Biotage® Lysera). Whole blood sample aliquots were treated with a 1:1 (v/v) ratio of a) saline (as control), b) water, c) 2 % ZnSO4, d) 6 % ZnSO4, e) 50 mM NH4OAc (pH 6.0), and f) saline (for bead homogenization). Sample f) was processed by Biotage® Lysera (2.4 m/s, 30s). All samples were diluted 1250- fold with saline and then examined under the microscope.
The optimal hemolysis method should effectively release analytes that are bound to RBCs while being compatible with subsequent sample preparation procedures for LC-MS analysis. The lysing technique used cannot alter the physicochemical properties of the biomatrix, which could hinder liquid transfer and sample loading, or cause breakthroughs. Additionally, any reagents introduced during hemolysis should not interfere with analyte extraction or cause ion suppression. In this study, we evaluated the impact of different hemolysis techniques on analyte extraction recovery across various sample extraction techniques.
We spiked analytes into fresh whole blood samples and incubated them at 37 °C for 16 hours to simulate the interaction of drug compounds with RBCs during metabolism. The incubated samples were pretreated with saline and directly extracted by ISOLUTE® PLD+. The results showed most analytes in the panel did not bind to RBCs, except for xylazine, phencyclidine (PCP), trazodone, fentanyl, chlordiazepoxide, lorazepam, temazepam, triazolam, amitriptyline, cyclobenzaprine, and nortriptyline. These analytes showed lower extraction recoveries after a 16-hour incubation compared to the baseline (Figure 13A).
Osmotic breakdown was effective in hemolyzing RBCs, but it was less efficient compared to bead homogenization. The bead homogenization protocol, which combines RBC lysis, analyte extraction, and protein precipitation in a single process, demonstrated the best performance in binding disruption and analyte release (Figure 13B). Although ZnSO4 has been widely used in clinical analysis, introducing 6 % ZnSO4 lysis into the matrices caused sample breakthrough (Figure 13C).
Extraction by SLE requires sample pretreatment with a basic buffer (50 mM NH4OAc, pH 8.5) to facilitate partitioning of drug analytes into the organic layer. Optical microscopy indicated that this buffer also lysed the RBCs (Figure 12). In this study, we compared the analyte release performance of using the buffer alone, a combination of ZnSO4 and the buffer, and bead homogenization with the buffer, followed by extraction using ISOLUTE® SLE+.
The results indicate that the buffer pretreatment alone is sufficient for releasing the analytes (Figure 4). The bead homogenization process did not further improve extraction recovery of drug analytes. Lysing with ZnSO4 inhibited the efficiency of liquid-liquid extraction, resulting in low recovery across the analyte panel (Figure 14). The ZnSO4 treatment increased the sample’s viscosity, making it difficult to transfer and load.
Whole blood samples were spiked with drug analytes at 5 ng/ ml. Spiked samples without incubation (0h) were treated with 50 mM NH4OAc, pH 8.5. The spiked samples were incubated for 16h to simulate the analyte-RBC binding. The incubated samples (16h) were pretreated with 50 mM NH4OAc, ZnSO4(6 %) and 50 mM NH4OAc, and 50 mM NH4OAc followed by bead homogenization, respectively. All samples were loaded onto ISOLUTE® SLE+ plates and eluted with DCM-IPA (95:5).
Whole blood hemolysis for SPE methods is similar to that for SLE, where samples are first pretreated with buffer. The pretreated samples are then loaded onto the SPE sorbent for extraction. In this study, we aimed to understand how varying concentrations of ZnSO4 affect the extraction recoveries of drug analytes when samples are extracted by CX SPE.
The results, consistent with those from the SLE method, indicate that buffer pretreatment alone, without ZnSO4, is sufficient to lyse and release analytes (Figure 15A). The addition of ZnSO4 resulted in lower recovery across the panel (Figure 15B). Weakly basic analyte, such as benzodiazepines, particularly those with low pKa values, were more affected by ZnSO4 compared to those with higher pKa values (Figure 15B). This effect may be due to competition between Zn2+ ions and analytes for binding sites on the EVOLUTE® EXPRESS CX sorbent. In addition, ZnSO4 treatment caused protein precipitation, necessitating high-speed centrifugation to spin down protein pellets before sample loading.
Whole blood samples were spiked with drug analytes at 5 ng/ ml. A control set of spiked samples without incubation (0h) were treated with 50 mM NH4OAc (pH 6.0) without ZnSO4. Remaining spiked samples were incubated for 16h to simulate the analyte- RBC (or protein) binding. Incubated samples (16h) were lysed with the NH4OAc (without ZnSO4), ZnSO4(3 %), ZnSO4 (6 %), ZnSO4(12 %), respectively. All samples were then extracted on EVOLUTE® EXPRESS CX following the outlined procedure in Table 2.
Based on these results, ZnSO4 treatments should be avoided when extracting whole blood samples by SPE-CX. Instead, lysing and pretreatment for extraction by SPE-CX can be achieved by the addition of 50 mM ammonium acetate buffer (pH 6.0) in one step.
Hemolysis in whole blood sample preparation is necessary to break open RBCs and release bound analytes for extraction. The hemolysis protocol should be simple, effective, and compatible with the downstream extraction procedures. The selection
of hemolysis protocols depends on the binding behaviors of analytes, as well as the extraction and cleanup techniques that follow. For most drugs of abuse analytes, pretreating the samples with either water or ammonium acetate buffer is typically sufficient to lyse the cells and release the bound analytes. Bead homogenization is recommended for the PLD protocol. The addition of Zinc sulfate can negatively affect the performance of the downstream sample extraction process. In cases where strong bindings cannot be disrupted by the methods mentioned above, isopropanol/methanol (50:50, v/v), 2 % disodium EDTA, and acetonitrile (protein precipitation) can be considered.
Literature number: PPS766