Current methodologies for DoA whole blood testing (section 1)

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 1: Practical considerations for sample preparation prior to LC-MS/MS analysis of drugs of abuse in whole blood

Whole blood is an important biospecimen for forensic, clinical, and toxicology testing. Drug analytes can be detected in blood samples within minutes to hours after intake and before they are rapidly eliminated from the body. Blood test results are easier to interpret as they generally measure the concentration of a drug in its parent form. This provides a more direct and accurate indication of current or recent drug use. Compared
to serum and plasma, whole blood samples provide broader exposure profiles and more accurate quantitative results, especially for drugs strongly interacting with red blood cells (RBCs) during transportation and metabolism.

Whole blood is a complex biological fluid comprised mainly of plasma and blood cells which impart its viscous nature. The blood cell portion contains large numbers of RBCs, leukocytes, and platelets, which may interfere with analysis. The main challenge in whole blood sample preparation for many clinical and forensic labs is efficiently extracting a large, broad panel of analytes, ensuring the release of those bound to proteins and\ or RBC, while removing matrix interferences. This is often also coupled with the need for a streamlined, scalable workflow that is both efficient and cost-effective. Here, we present optimized sample preparation methods for the analysis of a panel of 56 drug analytes in whole blood utilizing 6 different sample preparation techniques: protein precipitation (PPT), phospho- lipid depletion (PLD), supported liquid extraction (SLE), and polymer-based solid phase extraction (SPE) with reverse phase (ABN), strong cation exchange (CX), and weak cation exchange (WCX) mechanisms.

Part I. Importance and role of LogP and PH

The selection of sample preparation products and protocols should be based on the analytes’ chemical properties. Sample preparation techniques can be divided into matrix scavenging and selective extraction techniques. Matrix scavenging techniques, like protein precipitation (PPT) and phospholipid depletion (PLD) remove matrix components like protein and phospholipids but do not target specific analytes. Scavenging techniques require minimal method development but may not achieve sufficiently high recovery and concentration to meet satisfactory detection limits. In contrast, selective techniques, such as supported liquid extraction (SLE) and solid phase extraction (SPE), require more method optimization to selectively capture target analytes based on their chemical proper- ties. These properties determine how analytes are retained and eluted using different targeted extraction techniques. Understanding the analyte(s)’ structure and chemical features, including molecular weight, functional groups, hydrophobicity, and ionization behaviors, is crucial for method development. The octanol-water partition coefficient (LogP) is a measure of an analyte’s hydrophobicity and is an indication of the analyte’s reverse phase retention behavior and its ability to partition into an organic solvent. An analyte’s ionization behavior can be indicated by the dissociation constant (pKa), the pH at which the compound is 50 % ionized and 50 % non-ionized. The ionization status directly affects the compound’s polarity, with the ionized form being more polar than its neutral form. Figure 1 demonstrates the degree of ionization of an acidic or a basic compound with a pKa of 8.0 under different pH conditions. Sample pretreatment should be adjusted to ±2-pH units from the pKa to ensure compounds are fully ionized or neutral, depending on the retention mechanism of the extraction method used.
Figure 1. Ionization at different pH conditions for an acidic and basic analyte with a pK_a of 8.0.

Part II. Whole blood hemolysis

Unlike serum and plasma, whole blood samples contain large amounts of RBCs and a small portion of leukocytes and plate- lets. Most drugs are metabolized in the liver and circulate in the bloodstream as parent compounds and metabolites. However, some drugs, such as immunosuppressants, benzodiazepines, and certain new psychoactive substances, can cross the RBC membrane and distribute within the cells. To obtain compre- hensive drug exposure profiles and accurate quantitative results, it may be necessary to hemolyze whole blood to release drug analytes and metabolites, preventing their loss due to co-precipitation with proteins. The need for hemolysis depends on the target drug analytes in the sample, their metabolism, and interaction with blood cells. Hemolysis protocols must be simple, effective, and compatible with downstream extraction procedures. Section 2 will compare various hemolysis methods, their effectiveness and compatibility with different extraction techniques.

Part III. Extraction techniques

ISOLUTE® PPT+ protein precipitation

Protein precipitation is a widely used matrix scavenging technique for removing proteins from biofluid samples before LC-MS/MS analysis. Biotage ISOLUTE® PPT+ protein precipitation plate offers a streamlined, high-throughput, easy-to-automate, and centrifugation-free approach for protein precipitation. In this workflow, proteins are precipitated by the "crash agent" and remain above the functionalized bottom frit. The super- natant flows through the depth filter by application of positive pressure or vacuum and is collected. This ‘solvent first’ method- ology is optimal for both high efficiency protein precipitation and automation, since the solvent first approach negates the need for vortex mixing (Figure 2). PPT+ is available in a 96-well plate format making it suitable for automated high-throughput assays. While it demands little method development, it may not provide sufficient matrix clean up as it only removes protein, and not phospholipids.
Figure 2. ‘Solvent first’ procedure using ISOLUTE® PPT+ protein precipitation plate.

ISOLUTE® PLD+ protein and phospholipid depletion

Phospholipids are a family of matrix components that can elute at various points throughout the chromatogram, interfering with the detection of target analytes and causing ion suppres- sion. ISOLUTE® PLD+ simultaneously removes proteins and phospholipids utilizing the same in-well (or in-column) protein crash followed by the passing of the supernatant through a multifunctional sorbent that retains phospholipids. ISOLUTE® PLD+ is simple, quick, cost-effective, automatable, and is scalable. It can be used to extract a wide range of analytes from complex matrices such as whole blood and provides a much cleaner sample extract than protein precipitation.

ISOLUTE® SLE+ supported liquid extraction

Supported Liquid Extraction (SLE) is a selective sample extrac- tion technique with a similar mechanism as liquid-liquid extrac- tion (LLE). However, instead of partitioning analytes between aqueous and organic phases in a tube or funnel, the partition occurs on the surface of an inert modified diatomaceous earth (Figure 3). First, the biofluid sample is pretreated with an aqueous solution (or buffer) and loaded onto the ISOLUTE®
SLE+ cartridge/plate. The aqueous sample solution then quickly spreads over the sorbent surface forming small droplets. When a water-immiscible organic solvent passes through the bed, the analytes of interest partition into the organic solvent and are eluted, while the aqueous matrix components remain on the surface of the sorbent. Common elution solvents in SLE include water-immiscible, such as ethyl acetate (EtOAc), dichloro- methane (DCM), or methyl tert-butyl ether (MTBE). SLE works well for acidic, basic and neutral analytes. To promote parti- tioning of analytes into the organic phase, the aqueous sample pH must be modified to ensure analytes are in their non-ionized form. Adding a polar modifier such as 2-propanol (IPA) can help elute more hydrophilic analytes.

Figure 3. ISOLUTE® SLE+ 3-step procedure.

EVOLUTE® EXPRESS Solid Phase Extraction

Solid-phase extraction (SPE) is a selective sample extraction technique that works by retaining analytes of interest, removing matrix interferences with aqueous and/or organic washes, and then releasing the target analytes with an appropriate elution solvent. Retention and elution mechanisms of SPE are based on normal phase, reverse phase, and ion exchange. EVOLUTE® EXPRESS ABN is a modified PS-DVB polymeric reverse-phase sorbent for extraction of Acidic, Basic, and Neutral analytes. EVOLUTE® EXPRESS CX is a mixed-mode SPE sorbent that utilizes reverse-phase and strong cation exchange mechanisms. EVOLUTE® EXPRESS ABN and CX are polymeric sorbents with hydrophilic, i.e. water-wettable, characteristics. This eliminates the need for the conditioning and equilibrating steps of tradi- tional silica-based SPE. The pore size of the solid phase sorbent also impacts the extract cleanliness, particularly the removal of proteins. EVOLUTE® EXPRESS polymeric sorbent material has a 40 Å pore size, which can effectively exclude large molecules, such as protein, from being retained on the sorbent.

Method development for SPE sample preparation needs to rely on a deep understanding of the analytes' chemical features, including Log P, pKa, charge, etc. In addition to the analytes’ characteristics, SPE sample preparation procedures also need to be optimized to different sample matrices. For example, strong acid pretreatment is recommended for drug urine analysis, while it needs to be avoided in whole blood samples to prevent the reduction of iron in the blood. Often, a significant amount of lab work is required to optimize the wash conditions for specific analyte panels in different matrices. For a broad multi-class drug analysis, developing a one-size-to-fits-all method using SPE can be challenging.

Part IV. Properties of analytes

The compounds evaluated for this white paper and their log P and pKa values are listed in Table 1. The log P and pKa were sourced from chemicalize.com or the Drug Bank database.

Table 1. Analytes evaluated in the whole blood drug panel.

Part V. Methodologies

For this study, 100 µL whole blood was spiked with drug analytes at two concentrations, 5 ng/mL a representing ‘low’ level and 50 ng/mL representing a ‘high’ level. Whole blood samples were extracted using ISOLUTE® PPT+, PLD+, SLE+, and EVOLUTE® EXPRESS ABN, WCX, and CX 96-well plates, respectively according to the protocols described in Table 2. The extraction protocols were automated on the Biotage® Extrahera™ Sample Preparation workstation. Subsequent evaporation was performed using the TurboVap® 96 Dual. All extraction methodologies were compared to manually prepared protein- precipitated extracts. LC-MS/MS analysis was performed on a Nexera X2 HPLC system coupled to a SCIEX 5500 mass spectrometer using a Restek Raptor Biphenyl HPLC column (2.7 µm, 100 x 2.1 mm). Retention time and the MRM ion transitions for each analyte are listed in Table 1. All samples were analyzed using the same LC-MS method.

The performance of each extraction technique was evaluated based on extraction
recovery, matrix effects, and extrac- tion reproducibility using pre-extraction spiked samples (pre-spiked), post-extraction spiked samples (post-spiked), and neat solutions. Pre-spiked samples (n=5) were prepared by spiking whole blood samples at 5 ng/mL and 50 ng/mL and extracted following the described extraction protocol, while post-spiked samples (n=5) were spiked after extraction at the same concentrations immediately before reconstitution. Neat solutions (n=5) were prepared by spiking reconstitution solvent aliquots at 5 ng/mL and 50 ng/mL. All sample groups were analyzed using the same LC-MS method, and the data was processed using the same data processing method.

• Extraction recovery: calculated as the ratio ( %) of the analyte’s peak areas between the pre- and post-spiked samples.
• Matrix effect: determined as the ratio of the analyte’s peak areas between the post-spiked and neat samples. Ideally. This ratio is equal to or close to 1. A matrix effect is <1 indicates ion suppression, while a matrix effect >1 indicates signal enhancement caused by the matrix.
• Extraction reproducibility: measured as the relative standard deviation (RSD, %) of the pre-spiked sample replicates (n=5), reflecting the overall workflow variability.
• Extraction efficiency: evaluated as the ratio ( %) of the analyte’s peak areas between pre-spiked and neat samples, assesses the performance of extraction conditions (e.g., pretreatment pH, elution solvent.). Extraction efficiency and recovery are similar when the matrix effect is close to 1.

Table 2. Recommended sample preparation procedures for drugs of abuse analysis in whole blood by technique

Note: The listed pressure (psi) is according to the positive pressure manifold (PPM). The corresponding pressure of different extraction technologies suggested for Biotage® Extrahera™ Classic is listed in Table 4.

Part VI. Results and discussion by extraction technique

Figure 4. A) Recovery, B) Matrix effect, and C) Reproducibility (RSD) of DoA analytes in WB processed by manual protein crash, ISOLUTE® PPT+, PLD+, SLE+, and EVOLUTE® EXPRESS ABN, CX, and WCX.

Drug analytes were spiked in the WB samples (n=5) and evalu- ated at 5 ng/ml and 50 ng/ml. Analyte information and sample- preparation protocols are described in Tables 1 and 2. Dots in the boxplot indicate the results of individual analytes. All sample extraction and cleanup were processed on the Biotage® Extrahera™ workstation except for the manual protein crash.

The phospholipid profile was monitored by the MRM transition Q1 184 Da/ Q3 184 Da. (A) Phospholipid profiles of WB samples extracted by different sample extraction techniques. (B-F) Comparison of phospholipid removal between PPT and SLE (B), PLD (C), ABN (D), CX (E), and WCX (F).
Figure 5. Phospholipid removal by ISOLUTE® PPT+, ISOLUTE®PLD+, ISOLUTE® SLE+, EVOLUTE® EXPRESS ABN, EVOLUTE® EXPRESS CX, and EVOLUTE® EXPRESS WCX.

The phospholipid profile was monitored by the MRM transition Q1 184 Da/ Q3 184 Da. (A) Phospholipid profiles of WB samples extracted by different sample extraction techniques. (B-F) Comparison of phospholipid removal between PPT and SLE (B), PLD (C), ABN (D), CX (E), and WCX (F).

ISOLUTE® PPT+ and PLD+

The PPT and PLD extraction techniques both yielded 70 % recoveries or better across all analytes (Figure 4A). The two automated techniques demonstrated better recovery and matrix effects than samples extracted by the manual protein crash procedure (Figure 4A and 4B). Method development involved optimization of sample and crash solvent ratio to achieve efficient precipitation of proteins, phospholipid removal, and maximum analyte recovery. Multiple sample-to-crash solvent ratios were investigated. Pre-mixing whole blood samples with water (1:1, v/v, for 100 µL whole blood) and precipitating with MeCN in a 1:8 ratio demonstrated improved recoveries for both PPT and PLD techniques (Figure 6).

PPT and PLD can be easily automated in 96-well formats. When automating the extraction procedures for these techniques on the Biotage® Extrahera™ Classic automated sample preparation system, aspiration and dispensing flow rates must be adjusted properly to ensure accurate aliquoting of whole blood samples. Higher aspiration and dispensing flow rates achieved better pipetting of samples.

ISOLUTE® PLD+ and PPT techniques are based on the same familiar protein, condenser approach, however, the scavenging adsorbent in ISOLUTE® PLD+ provides an additional level of sample clean up. Matrix cleanup with both techniques was evaluated by monitoring the phospholipid profile of sample extracts. ISOLUTE® PLD+ yielded highly cleaner extracts in comparison to PPT and effectively removed >99 % of phospho- lipids (Figure 5B). Removal of phospholipids is extremely important in LC-MS/MS analysis of biological fluids, particularly blood-based samples, as these compounds can elute at various points throughout the LC chromatogram causing ion suppression.
Figure 6. Impact of sample/solvent mixing ratio on extraction performance of drug panel in WB using PPT and PLD.

Drug analytes were spiked (5 ng/ml) in WB samples (n=5) and extracted under 3 conditions: sample/ACN ratio at 1:6, sample/ ACN ratio at 1:8, and sample/ACN ratio at 1:8 (WB pre-mixed with water). Recovery (A), matrix effect (B), and reproduc- ibility RSD (C) were evaluated under each condition. Dots in the boxplot indicate the results of individual analytes. All sample extraction and cleanup were processed on the Biotage® Extrahera™ workstation.

ISOLUTE® SLE+

Whole blood samples extracted using the SLE extraction method described in Table 2, yielded 60 %-80 % extraction recoveries for most analytes (Figure 4A) with exception of amphoteric analytes, such as ritalinic acid, pregabalin, and gabapentin. SLE effectively removed >99 % phospholipids (Figure 5A&C) producing cleaner extracts with low matrix effects (0.9-1.2) across all analytes (Figures 6C).

Sample pretreatment and PH

SLE is a technique analogous to traditional liquid-liquid extrac- tion (LLE) utilizing the same principle of analytes portioning between aqueous and water immiscible organic solvents. Unlike LLE where solvents are in direct contact, in SLE the extraction interface takes place on the diatomaceous earth sorbent. Effective partitioning of analytes relies on the same parameters as in LLE; analyte functionality (LogP and pKa) and analyte solubility in the water immiscible extraction solvent. Neutralizing target analytes increases their degree of hydropho- bicity and improves their partitioning into the water immiscible organic phase. Given that most drug analytes in this study are weakly basic to basic, we examined pretreatment solutions with pH levels ranging from 7.0 to 13 (Figure 7A). The transition from water (pH 7.0) to ammonium acetate (pH 8.5) significantly enhanced the extraction efficiency for most analytes. However, pretreating the sample with the more basic 0.5M NH4OH did not result in further improvement. Hydrophobic analytes generally showed good extraction efficiencies regardless of pretreatment pH, as they tend to favor the water-immiscible organic solvent over aqueous (Figure 7A). In contrast, polar analytes, such as oxymorphone, EME, morphine, 6-AM, zopiclone, oxycodone, and codeine, were more impacted by the pH of the pretreatment solution. This demonstrates that pH control for polar analytes is crucial due to their poor solubility in water-immiscible solvents when charged. Due to their amphoteric nature, gabapentin and pregabalin are difficult to extract in a large panel using SLE. In this study, milder pH conditions (ammonium acetate, pH 8.5) were applied to avoid analyte degradation.
Figure 7. Impact of different pretreatment buffers (A) and elution solvents (B) on extraction efficiency of drug analytes in WB samples using SLE.

Drug analytes spiked (5 ng/ml) WB samples (n=3) were premixed with 4 different buffers (water, ammonia acetate at pH 8.5, 1 % NH4OH, and 0.5 M NH4OH), loaded onto the SLE plate, and eluted by 95:5 DCM-IPA to compare the pretreatment buffers (A). The same spiked WB samples were pretreated with ammonium acetate (pH 8.5), loaded onto the SLE plate, and then eluted by EtOAc and DCM-IPA (95:5, v/v) to compare the elution solvents (B). The extraction efficiency was evaluated as the ratio ( %) of peak areas between spiked WB sample and spiked neat solution.

Loading

To improve extract cleanliness when extracting whole blood, it is recommended to decrease the loading volume (use ¾ of maximum recommended volume). For example, load 300 µL pre-treated sample onto a 400 µL capacity plate or column.

Elution solvent

SLE requires eluting with water-immiscible organic solvents, such as hexane, MTBE, DCM, DCM/IPA, and EtOAc. This study compared the extraction efficiency of two more polar elution solvent combinations: 95:5 (v/v) DCM/IPA and EtOAc (Figure 7B). Overall, both solvents demonstrated comparable perfor- mance for most analytes. However, for certain analytes, such as opioids and cocaine metabolites (norhydrocodone, norfentanyl, benzoylecgonine, and EME), the 95:5 (v/v) DCM/IPA elution solvent yielded significantly higher extraction efficiency than EtOAc (Figure 7).

Evaporation

Some analytes are prone to loss during evaporation, which can reduce their extraction recovery. These include basic analytes, such as cocaine, EME, benzoylecgonine, amphetamine, and mephedrone. Evaporation loss of these analytes can be mitigated by adding 100 mM HCl (50 µL/sample) which converts them to their more stable, non-volatile salt form (Figure 8).Figure 8. Acid reduces drug analyte loss during the evaporation of non-polar solvents.

Analyte-spiked WB samples (n=3) were extracted by SLE or PLD methods and eluted by (A) EtOAc or (B) ACN. Recoveries were compared with and without the addition of 0.38 % HCl-methanol to the eluted fraction. The addition of 0.38 % HCl-methanol significantly reduces the drug analyte loss when eluting with EtOAc but not with ACN.

EVOLUTE® EXPRESS SPE

Most drug analytes are weakly basic to basic small molecules. They are best suited for the reverse-phase ABN and mixed-mode CX and WCX sorbents.Figure 9. Structure backbones of EVOLUTE® EXPRESS polymeric SPE chemistries: (A) ABN, (B) CX, and (C) WCX.

Interaction sites based on the reverse-phase mechanism are circled in orange, and interaction sites based on ion exchange are circled in blue.

EVOLUTE® EXPRESS ABN

EVOLUTE® EXPRESS ABN is a polymeric sorbent based on a reverse-phase retention mechanism and is widely used to extract a range of small molecules from biological fluids.

EVOLUTE® EXPRESS ABN demonstrated good extraction recoveries for most analytes (>60 % at 5ng/mL, >80 % at 50 ng/mL) and improved matrix effects in comparison with traditional protein crash (Figure 4A & 4B). The polymeric nature of EVOLUTE® EXPRESS ABN sorbent makes it water-wettable without conditioning. However, due to the high viscosity of whole blood, conditioning and equilibrating the sorbent was crucial. This process reduces the surface tension and allows the whole blood to quickly permeate the sorbent preventing potential clogging and enhancing method reproducibility. Prior to loading onto the EVOLUTE® EXPRESS ABN 96-well plate, whole blood samples were diluted with water (1:3, v/v) to increase sample fluidity. Sample loading is followed by an aqueous wash and a subsequent weak organic wash, H2O:MeOH 95:5, to further aid cleanliness without losing polar analytes, such as morphine, 6-AM, and ritalinic acid. Analyte polarity plays a significant role in their retention mechanism with ABN’s reverse-phase sorbent and subsequent disruption of that retention in the elution step. Considering the variability in polarity of the drug analytes in this panel, we examined the effect of varying the eluotropic strength of the elution solvent on analytes recovery and matrix effects. Elution solvent mixtures containing DCM and EtOAc yielded good extraction recoveries (>70 %) for most analytes (Figure 9). Eluting with EtOAc:MeOH (95:5) yielded good extraction recoveries (>70 %) and low matrix effects (between 0.6 and 1) for most analytes (Figure 4A, 4B) .

EVOLUTE® EXPRESS CX

EVOLUTE® EXPRESS CX is a polymeric sorbent with both reverse phase and strong cation exchange retention mechanisms. The CX sorbent is based on the same ABN chemistry with a sulfonic acid moiety designed to retain cationic species (Figure 9B). To facilitate the mixed-mode retention mechanism, samples were pretreated with an acidic buffer to maximize ionization of basic functional groups. However, due to whole blood reacting with strong acidic buffers, samples were pretreated with 50 mM NH4OAc. Wash 1 (50 mM NH4OAc) maintained analyte charge while removing water-soluble interferences such as salts, small proteins and some phospholipids. The subsequent organic wash (40:60 H2O:MeOH) removed remaining phospholipids along with neutral and acidic interferences while retaining cation exchange interactions. For some analytes, an additional wash with strong acid (4 % H3PO4) improved their extraction recovery (Figure 10). Additionally, for unionized analytes at a low pKa, such as secobarbital, phenobarbital, lorazepam, phenytoin, desalkylflurazepam, a weak organic wash (MeOH:H2O 60:40) improved their extraction recoveries. While a weak organic wash compromised extract cleanliness, it improved the overall extraction recovery of analytes. Analytes elution was optimized with DCM:MeOH:NH3OH (78:20:2) which broke up hydrophobic interactions and eliminated analyte charge. The described CX extraction protocol in Table 2 achieved excellent recoveries (>80 %) for most analytes in comparison with other techniques (Figure 4).Figure 10. Strong acid (4 % H3PO4) wash/rinsing enhances the recovery of weakly basic to basic analytes with EVOLUTE® EXPRESS CX.

Drug analytes were spiked (5ng/mL) in the whole blood samples (n=5). Sample loading was followed by ammonia acetate (pH 6) wash; wash 2 with and without 4 % H3PO4; methanol-water (60:40) wash; and elution with EtOAc-IPA-ammonia (78:20:2).

EVOLUTE® EXPRESS WCX

EVOLUTE® EXPRESS WCX combines reverse-phase and weak cation exchange functionalities, thereby selectively retaining strongly basic, or cationic species, from aqueous samples. It shares the same polymeric backbone as ABN but is modified with a negatively charged group carboxylic acid group that promotes strong retention of positively charged cationic analytes. Given the variability in pKa among analytes, a weak organic wash (H2O:MeOH 40:60) yielded better extraction recoveries for analytes with a lower pKa that otherwise could not get fully ionized after the aqueous wash. Weakly basic analytes that are not fully ionized in water (e.g., 7-aminofl- unitrazepam, 7-AC, ketamine) and amphoteric analytes (e.g., ritalinic acid, gabapentin, pregabalin) showed low recoveries due to poor retention on the WCX sorbent. While WCX yields overall lower recoveries for most analytes, the ability to elute under acidic conditions is advantageous due to LC-MS/MS compatibility.

Part VII. Results and discussion by drug class

Opioids and opioids agonists

Fentanyl, norfentanyl, hydrocodone, norhydrocodone, 6-AM, codeine,
hydromorphone, oxycodone, oxymorphone, morphine, methadone, naloxone, buprenorphine, desmethyltramadol

Opioids are drugs that bind to opioid receptors producing morphine-like effects. Most opioids contain primary, secondary, or tertiary amines and are classified as weakly basic to basic. Both the PLD and SLE methods demonstrated good recovery (>70 %) and matrix effects (0.85-1.10) for opioids, with some exceptions (Figure 15 A1 and A2). Opioids with a secondary amine group, such as norhydrocodone and norfentanyl, showed improved extraction recovery with SLE when treated with a base and eluted with DCM-IPA (Figure 7). All opioid analytes showed good recoveries (63-82 %) using ABN when eluted with DCM:MeOH:NH4OH (95:5). Extraction by CX resulted in excel- lent recoveries (>85 %), when eluted with DCM-MeOH-NH4OH (78:20:2, v/v/v), for all opioids except buprenorphine (42 %) and 6-AM (77 %). Opiates, especially 6-AM and morphine, are unstable under extreme pH conditions, with 6-AM degrading if the reconstitution solution (0.1 % FA) is left overnight. To ensure analyte stability, minimize exposure to strong acids or bases and analyze samples immediately after reconstitution.


Table 3. Percent recovery and matrix effects of opioids extracted from whole blood using five different extraction techniques.

Benzodiazepines

Lorazepam, oxazepam, temazepam, desalkylflurazepam, bromazepam, clonazepam, nordiazepam, diazepam, 7-aminoflunitrazepam, 7-AC, triazolam, estazolam, alpha-hydroxyalprazolam, alprazolam, chlordiazepoxide, midazolam, and flurazepam

Benzodiazepines are a group of depressant drugs with a core chemical structure composed of a fused benzene and diazepine ring. They are considered weak bases with pKa values generally below physiological pH. All benzodiazepines had recoveries of 60 % or greater when extracted by ISOLUTE® PPT+, ISOLUTE® PLD+, ISOLUTE® SLE+ and EVOLUTE® EXPRESS CX (Figure 8, B1). For matrix effects, ISOLUTE® PPT+ and EVOLUTE® EXPRESS CX yielded more suppression than either ISOLUTE® SLE+ or ISOLUTE® PLD+ (Figure 8, B2). Benzodiazepines extraction recovery by EVOLUTE® EXPRESS CX was improved with a strong acid wash (4 % H3PO4) to ionize analytes followed by a weak organic wash (H2O:MeOH 40:60). When extracting benzodiaz- epines of low pKa, such as lorazepam, oxazepam, temazepam, and desalkylflurazepam, using CX a weak organic wash must be applied as they not well retained due to weak ionic interactions. EVOLUTE® EXPRESS WCX is less effective for the extraction of benzodiazepines as they are weakly basic and not retained well through ion exchange.

Table 4. Percent recovery and matrix effects of benzodiazepines extracted from whole blood using five different extraction techniques.

Stimulants and their metabolites

Cocaine, benzoylecgonine, EME, mephedrone, amphetamine, and ritalinic acid

Cocaine, phenethylamines, and their metabolites are classified as basic compounds, and some, like cocaine and amphetamine, are highly volatile. Extraction of these stimulants by EVOLUTE® EXPRESS CX yielded excellent recoveries (89-96 %), with lesser matrix effects (0.97-1.12). ISOLUTE® SLE+ provided 97 % recovery for EME, 77 % for cocaine, and 62 % for mephedrone, and <50 % for benzoylecgonine, amphetamine, and no recovery of ritalinic acid. Ritalinic acid, a metabolite of methylphenidate, is not recovered by SLE due to its amphoteric nature which doesn’t allow it to partition into the organic solvent. Recovery for EME and benzoylecgonine was improved, 97 % and 37 %, by changing the elution solvent to DCM-IPA (95:5, v/v) (Figure 7). Additionally, recoveries of benzoylecgonine, EME, cocaine, mephedrone, and amphetamine are improved by the addition of 0.38 % HCl-MeOH to the elution fraction. Acidification (with HCl or FA) helps prevent evaporative loss and improve analyte(s) stability (Figure 8).

Table 5. Percent recovery and matrix effects of stimulants and their metabolites extracted from whole blood using five different extraction techniques.

Barbiturates and Sedative-hypnotics

Phenobarbital, secobarbital, zolpidem, zopiclone, clonidine, xylazine

EVOLUTE® EXPRESS CX had >96 % recovery for zolpidem, zopiclone, and clonidine, 74 % for xylazine and <57 % for phenobarbital and secobarbital. Barbiturates are weakly acidic and cannot be fully ionized with acid pretreatment. Recovery of >70 % was achieved using ISOLUTE® SLE+ and >65 % using EVOLUTE® EXPRESS ABN.

Table 6. Percent recovery and matrix effects of barbiturates and other sedative drugs extracted from whole blood using five different extraction techniques.

Antidepressants, anticonvulsants, anesthetics and other drugs

Amitriptyline, nortriptyline, cyclobenzaprine, lamotrigine, phenytoin, pregabalin, gabapentin, risperidone, clozapine, trazodone, ketamine, norketamine, and phencyclidine (PCP)

EVOLUTE® EXPRESS CX yielded excellent recoveries of these analytes, >83 %, with matrix effects between 0.92-1.08, except for phenytoin as it could not be fully ionized. EVOLUTE® EXPRESS ABN and ISOLUTE® SLE+ produced >62 % recoveries for all analytes in this class except for nortriptyline, pregabalin and gabapentin. The amphoteric nature of pregabalin and gabapentin hinders their partitioning into the organic layer with SLE. Extraction by ISOLUTE® PLD+ resulted in >70 % recoveries all analytes listed in this class with matrix effects between 0.90-1.01.

Table 7. Percent recovery and matrix effects of antidepressants, anticonvulsants, and other drugs extracted from whole blood using five different extraction techniques.

PART VIII. General recommendations

We have presented six sample preparation techniques for extracting a diverse panel of 56 drugs and metabolites from whole blood. The choice of the final method depends on the drugs of interest and their properties.

ISOLUTE® PLD+ is recommended for the analysis of a broad panel of drugs and metabolites in whole blood. When using PLD, the crash solvent is dispensed first onto the PLD wells before loading pretreated blood samples and mixing as required. Pre-mixing whole blood samples and precipitating with MeCN in a 1:8 ratio is recommended to achieve optimal extraction recovery and reproducibility.

ISOLUTE® SLE+ is recommended for analyzing a range of drug classes and their metabolites, including opioids, benzodiaz- epines, antidepressants, anesthetics, anticonvulsants, and stimulants except for some amphoteric analytes such as ritalinic acid, gabapentin, and pregabalin. For elution, a solvent mixture of DCM-IPA (95:5, v/v) is recommended when the panel includes norhydrocodone, norfentanyl, benzoylecgonine, and EME.

EVOLUTE® EXPRESS ABN works well for most drugs classes including benzodiazepines, most opioids, barbiturates and other sedatives, cocaine, EME, antidepressants, anesthetics, and anticonvulsants except for pregabalin and gabapentin. For optimal overall results with a broad panel, a weak organic wash (5:95 methanol:water, v/v) is recommended to recover polar analytes.

EVOLUTE® EXPRESS CX is recommended for a drug panel comprising weakly basic to basic drugs, like opioids, benzodi- azepines, PCPs, stimulants, TCAs, and cocaine. While ISOLUTE® SLE+, PLD+ and EVOLUTE® EXPRESS ABN can also extract these analytes, the mixed-mode cation exchange sorbent of EVOLUTE® EXPRESS CX offers enhanced selectivity and better sample cleanliness.

When pretreating whole blood samples, avoid strong acids. Instead, use a weakly basic buffer (e.g. 0.05M ammonium acetate at pH 6.0) for pretreatment and the first wash. After sample loading and the initial wash, a strong acidic wash with 4 % H3PO4 is necessary to retain analytes on the sorbent through ion exchange.

For panels including weakly basic and basic drugs, a strong organic wash (e.g., methanol) is recommended. If the panel also includes cationic and non-cationic analytes, the strength of the organic wash needs to be adjusted based on the hydrophobicity of the non-cationic analyte(s). Alternatively, these analytes can be fractionated with a strong organic wash and extracted separately. For elution, both DCM-IPA-NH4OH (78:20:2, v/v) and DCM-MeOH-NH4OH (78:20:2, v/v) provide excellent extraction recoveries and matrix effects. If ritalinic acid, gabapentin, and pregabalin are included, elution with DCM-MeOH-NH4OH (78:20:2, v/v/v) is recommended for optimal results.

EVOLUTE® EXPRESS WCX is best suited for extracting basic and strongly basic analytes with high pKa values, such as norfentanyl, norhydrocodone, nortriptyline, amphetamine, and phencyclidine PCP. It is also effective for drug panels containing a mix of weak and strong bases, as weak bases can be eluted separately with an organic wash. However, partially charged or uncharged analytes like benzodiazepines, barbitu- rates, and zolpidem are retained only through reverse phase interactions, and their extraction recovery may be comprised, especially with strong organic washes.

Developing a single sample preparation method for a large panel of drugs and metabolites can be quite challenging. When analyzing a broad range of drug analytes with diverse structures and properties, compromises in recovery and sample cleanliness are inevitable. It is essential to utilize comprehensive information about sorbent chemistries, analyte properties, and starting conditions. Conducting a series of laboratory experiments will help to determine the performance of specific analytes and guide necessary optimizations. Sample preparation methods should be optimized for sufficient recovery and extract cleanliness that meet the overall analytical sensitivity, accuracy, reproducibility, and robustness requirements.

Literature number: PPS766