Chapter 1: Introduction
1.4 Popular Matrices for DOA Analysis
1.4.2 Blood Matrix for DOA analysis
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lemon juice, eyedrops, and products that are commercially available via the internet (Dasgupta, 2007; Fu, 2016).
Table 4: Dilution, adulteration, substitution of urine specimen Urine States Description
Diluted Urine creatinine ≥ 2 mg/dL but < 20 mg/dL
Specific gravity > 1.001 but < 1.003 Substituted Urine creatinine < 2 mg/dL
Specific gravity < 1.001 or > 1.020 Adulterated pH < 3 or > 11
Nitrite concentration > 500 mcg/mL Chromium concentration > 50 mcg/mL Presence of: Halogen (bleach, iodine, fluoride), glutaraldehyde, pyridine, surfactant
23 samples generally detect alcohol and parent drug compounds themselves rather than their metabolites (Flanagan & Maurer, 2020).
Blood is probably the only medium with the potential to indicate whether an individual is under the influence of BZDs, or not, at the time of collection. It is
considered as an essential element in the control of drug abuse in the workplace (Qriouet et al., 2019). Blood tests can be performed to quantify the levels of certain BZDs and their metabolites but are more rarely practiced because of their invasive procedure. The detection window in the blood is narrower than that in urine, and the concentrations are lower. Therefore, a sensitive and a very specific confirmation technique is mandatory for the detection of BZDs and their metabolites in the blood (Perez et al., 2016). Studies reported the development of a sensitive and specific method for the detection of
nitrazepam (LOQ 50 ng/mL), clonazepam (LOQ 100 ng/mL) and lorazepam (LOQ 100 ng/mL) in blood samples with the use of LC-MS/MS (Moretti et al., 2019). Furthermore, a study presented a validated method for the simultaneous determination of morphine, codeine, and 6-monoacetymorphine with LOD 5 ng/ml (Prata et al., 2019).
Nevertheless, the developments in sample preparation, chromatography, and detection techniques have made it possible to use blood as a screening matrix. Within a single matrix, it is possible to perform both identification and quantification. A further significant benefit is that drugs can be identified shortly after consumption, ahead of the processes of metabolism and/or filtration (Mali et al., 2011).
1.4.2.1 Mechanisms of Drug Incorporation Into Blood
After administration (e.g., oral, intravenous, inhalation) of a drug, absorption into the bloodstream occurs at the site. The rate of oral absorption depends on factors such as the presence of food in the intestine, the particle size of the drug preparation, and the acidity of intestinal contents; this step usually lasts 1 to 6 h. Intravenous administration of a drug can result in its effects within a few seconds, making this a useful method for emergency treatment. Subcutaneous or intramuscular injection usually produces effects within a few minutes, depending largely on the local blood flow at the site of the
injection. Inhalation of volatile or gaseous agents also produces effects in a matter of minutes (Buxton & Benet, 2015).
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Absorption of drugs into the bloodstream can be done either through passive (simple) diffusion or carrier-mediated membrane transporters (Figure 8) (Sakai, 2008).
The most common mechanism of absorption for drugs is passive diffusion. This process simply occurs when the drug molecule moves according to the concentration gradient, from a higher drug concentration to a lower concentration until equilibrium is reached.
Numerous specialized carrier-mediated membrane transport systems are present in the body to transport ions and nutrients. Such systems include active and facilitated
diffusion. Active diffusion is an energy-consuming system essential for absorption (GM Cooper, 2000).
Figure 8: The variety of ways drugs move across cellular barriers in their passage throughout the body, reproduced with permission from The McGraw-Hill (Sakai, 2008) 1.4.2.2 Blood Sample Collection Protocol
Blood samples are best obtained in antemortem cases from the cephalic vein. For post-mortem cases venous and/or arterial femoral blood should be collected as it is relatively isolated from the internal organs of the chest and abdomen and therefore less influenced by the post-mortem redistribution phenomenon. Blood samples volumes collected should at least 3–5 mL (Dinis-Oliveira et al., 2016).
The containers used for the samples may vary depending on the analysis to be performed, and it is vital that the correct types are used. The preferred collection tube for blood samples is an EDTA tube. If ethanol is suspected, it is best to use a
fluoride/oxalate tube. In contrast, the glass or plastic tube is used if carbon monoxide or other volatiles are suspected (Flanagan & Maurer, 2020). Samples should be stored in
25 tightly sealed containers at 4 °C (short-term) or at −20 °C; however preferably at −80 °C (long-term) (Dinis-Oliveira et al., 2016).
1.4.2.3 Extraction Methods in Blood
Different sample pre-treatments and extraction techniques have been applied for the determination of drugs in whole blood plasma as well as urine as discussed earlier (Figure 9) (Casas, Hansen, Krogh, Styrishave, & Björklund, 2014). For the whole blood, extraction (LLE or SPE) can be done either directly on blood (S. Jones et al., 2022) or by the dried blood spot (DBS) technique (Minzi, Rais, Svensson, Gustafsson, & Ericsson, 2003; Wilhelm, den Burger, & Swart, 2014). The plasma can be directly extracted by SPE (Lindegardh et al., 2009) or LLE (Wiesner, Govender, Meredith, Norman, & Smith, 2011) and further extracted after a protein precipitation (Lindegårdh et al., 2005). It should be emphasized that nearly all methods published for the three matrices mentioned include either LLE or SPE as part of the sample preparation strategy (Casas et al., 2014).
Various studies identified a diversity of drugs in the blood samples with the use of different extraction methods, illustrated in Table 5.
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Figure 9: Sample preparation strategies for drugs analysis in blood, plasma and urine, reproduced with permission from Elsevier (Casas et al., 2014)
27 Table 5: Chromatography-based methods for drug determination from blood samples
Drugs Extraction protocol Instrument Linear range References
Prazepam LLE LC-MS/MS 1–200 ng/mL (Banaszkiewicz
et al., 2020)
Tetrazepam LLE LC-MS/MS 1–200 ng/mL (Banaszkiewicz
et al., 2020)
Pyrazolam SPE LC-MS/MS 1–500 ng/mL (Mei,
Concheiro, Pardi, &
Cooper, 2019) Morphine Protein precipitation GC–MS/MS 2.5–1000 ng/mL (Jinlei et al.,
2021)
Hydromorphone Protein precipitation GC–MS/MS 2.5–1000 ng/mL (Simão et al., 2022)
Buprenorphine LLE LC-MS/MS 1–100 ng/mL (Phillips,
Oliveto, Mancino, &
Hendrickson, 2021) Pregabalin Protein precipitation LC-MS/MS 1-100 mg/L (Antunovic,
Dzudovic, Kilibarda, Vucinic, &
Djordjevic, 2023)
Gabapentin LLE LC-MS/MS 50 – 10 000
ng/mL
(Phillips et al., 2021)
Carisoprodol Protein precipitation LC-MS/MS 1-35 mg/L (Essler, Bruns, Frontz, &
McCutcheon, 2012)
1.4.2.4 Factors Affecting Blood Analysis
Some blood specimen containers can interfere with certain drug assays. For the analysis of volatile poisons (for example solvents and fuel gases) from acutely poisoned
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patients and autopsy subjects, plastic tubes are unsuitable. The principle underlying this is that the solvents present in the specimen will be removed by dissolution into the plastic wall of the container or into the rubber or plastic cap liner of a glass tube (Kapur
& Aleksa, 2020). Gross contamination by toluene, 1-butanol, ethylbenzene, and xylene has also been reported from blood collection tubes containing gel separators. In addition, plastic or rubber materials in contact with the specimen may introduce contaminants into the specimen and thereby produce spurious additional peaks in the chromatogram. These problems can be avoided if the rubber cap liner is wrapped in aluminum foil. Blood collected into the wrong anticoagulant tube will thus be contaminated with the wrong anticoagulant (Richardson, 2000).
The site of sampling of blood from a patient is generally critical and needs to follow the normal rules for common clinical chemistry assays. In addition, It will usually be necessary to note the time of sampling with respect to time of ingestion as dictated by the rules of pharmacology (Dinis-Oliveira et al., 2016).
Alcohol swabs may also contaminate blood samples being collected from
patients. Such swabs, however, usually contain propan-2-ol, which is readily identified by any GC method for alcohols and volatiles (Beresford, 2018).