Chapter 1: Introduction
1.4 Popular Matrices for DOA Analysis
1.4.1 Urine Matrix for DOA Analysis
As a result of the high concentration of drugs in urine, it was traditionally the sample of choice for the screening and identification of unknown drugs. Urine tests are more common because they are noninvasive, fast, offer the advantage of a large sample volume, and are able to qualitatively detect a wide range of substances including BZDs, OPI, and CAD (Sanabria et al., 2021). However, for an adolescent patient, its collection is somewhat invasive because it requires either a sophisticated collection protocol, which is not readily available in medical offices, or direct observation (e.g., by a clinician or a parent) to prevent tampering. Compounding this, many pediatricians are unfamiliar with proper collection procedures and with the limitations of urine drug screening (Hadland &
Levy, 2016).
Urine is used as the biological specimen in a notable number of different research projects. Some authors have developed LC-MS methods, mostly to study the metabolism of specific BZD drugs in the urine matrix, including phenazepam (Kriikku et al., 2012), clonazolam, meclonazepam, and nifoxipam (Meyer, Bergstrand, Helander, & Beck, 2016), pyrazolam (Moosmann et al., 2013); flubromazepam (Kintz et al., 2017);
diclazepam (Moosmann, Bisel, & Auwärter, 2014); and deschloroetizolam,
flubromazolam, and meclonazepam (Huppertz et al., 2015). In addition, morphine and
17 codeine were detected in urine samples at concentrations higher than 6-acetyl morphine (6-AM) (Dinis-Oliveira, 2019).
1.4.1.1 Mechanisms of Drugs Incorporation Into Urine
Drug excretion is the final step in the Absorption, Distribution, Metabolism, and Excretion (ADME) process and consists of a series of pathways that remove an
administered drug and/or its metabolites from the body (Barreto, Larson, & Koubek, 2021). Most drugs, particularly water-soluble drugs and their metabolites, are eliminated largely by the kidneys. The hydrophobic drugs to be excreted, must undergo metabolic modification, making them more polar. Drugs are excreted mainly by renal excretion, a process in which the drug passes through the kidney to the bladder and ultimately into the urine (AZ, SB, & R, 2022; Shargel, Wu-Pong, & Yu, 2012). A general view of the urinary system is shown in Figure 7 (Shargel et al., 2012).
Figure 7: The general organizational plan of the urinary system, the outer zone of the kidney is called the cortex, and the inner region is called the medulla, reproduced with permission from The McGraw-Hill (Shargel et al., 2012)
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1.4.1.2 Urine Sample Collection Protocol
In the event of an antemortem scenario, a plastic container with screw cap
(without preservative) is used in order to collect a urine specimen. Preferably at least 10 mL of urine should be taken for this drug analysis (Beresford, 2018). Information
including the date of collection, donor/s name and/or identification number as well as the collector’s initials should be placed on the bottle label (S. Y. Lin, Lee, Lee, & Chen, 2018).
Moreover, one should ensure that the sample comes from the person being examined to guarantee the integrity of the sample. It may be necessary to take
precautions in certain situations to tamper proof the sample. This is because adulteration of the sample can occur in a number of different ways. In the event of a post-mortem examination, a hypodermic syringe will be inserted into the bladder in order to collect a sample of the deceased person's urine. The process of acquiring urine on the side of the road is just as challenging. However, determining whether or not someone has used a drug in the past can be aided by looking at the concentration of that drug in their urine (Dinis-Oliveira, Vieira, & Magalhãaes, 2016). All specimens should be stored at 4 oC before transporting them to the laboratory. Each specimen bottle should be securely sealed to prevent leakage (Beresford, 2018).
1.4.1.3 Extraction Methods In Urinalysis
There are numerous extraction techniques that range from simple to rigorous, which prepare urine samples for drug analysis. These include dilutions (e.g., dilute and shoot); phospholipid depletion; liquid–liquid extraction (LLE); supported liquid, protein precipitation (PP); and solid phase extractions (SPE). Even within each type of
extraction, there is variability in the complexity and number of steps involved, as well as the amenability to automation. These techniques can be scaled to accommodate the matrix type and sample volume. Furthermore, as the complexity of the matrix increases from simple fluids (e.g., urine) to solids (e.g., tissue or bone), the process can be adjusted accordingly to disrupt the matrix so that the target analytes are released and can be
recovered (S. Jones et al., 2022; J. F. Wilson et al., 2001).
19 Specific compounds may exhibit a more effective extraction recovery with one technique than another. Certain techniques can be utilized to target diverse groups of compounds, while others will be more compound or class specific. Choosing the ideal sample preparation technique for the situation helps to ensure a successful analysis (S.
Jones et al., 2022). Various studies identified a diversity of drugs in urine with the use of different extraction methods, illustrated in Table 2. It showed that the extraction
technique of BZD drugs in urine varied from PP to supramolecular solvent (SUPRASs) extraction (Kang & Lin, 2018), while the listed opiate compounds had microextraction by the packed sorbent extraction method (Simão et al., 2022). Moreover, some of the detected CAD in urine such as PGB and GBP, were done through LLE (Almalki et al., 2021).
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Table 2: Chromatography-based methods for drug determination from urine samples
Drugs Extraction protocol Instrument Linear range (ng/ml)
References
Alprazolam Protein precipitation LC-MS/MS 5.0–1000 (Kang & Lin, 2018)
7-Aminoclonazepam Protein precipitation LC-MS/MS 25.0–1000 (Kang & Lin, 2018)
Nitrazepam Supramolecular solvent (SUPRASs) extraction
GC–MS 2-50 (Jinlei et al., 2021)
Estazolam Supramolecular solvent (SUPRASs) extraction
GC–MS 5-100 (Jinlei et al., 2021)
Tramadol Microextraction by packed sorbent
GC–
MS/MS
1–1000 (Simão et al., 2022)
6-
monoacetylmorphine
Microextraction by packed sorbent
GC–
MS/MS
1–1000 (Simão et al., 2022)
Pregabalin Liquid–liquid extraction LC-MS/MS 10–1000 (Almalki et al., 2021)
Gabapentin Liquid–liquid extraction LC-MS/MS 10–1000 (Almalki et al., 2021)
1.4.1.4 Factors Affecting Urinalysis
Numerous factors may affect the results of a drug analysis utilizing the urine matrix. The urine sample can be diluted, adulterated, or substituted (Kintz P, 2008).
Urine creatinine should range from 20–400 mg/dL in a healthy individual, and with a specific gravity (SG) ranging from 1.002–1.030 and a pH range from 4.5–8.0 as displayed in Table 3 (Flanagan & Maurer, 2020; Raouf, Bettinger, & Fudin, 2018).
21 Table 3: Characteristics of valid urine specimen
Characteristics Normal Range
Creatinine, mg/dL 20–400
Specific gravity 1.002–1.030
pH 4.5–8.0
The diluent urine specimen can have a creatinine range less than 20 mg/dL and a SG less than 1.003, except for a substituted specimen. A substituted urine specimen is a sample with the creatinine less than 2 mg/dL and relative density less than 1.002, or equal to or greater than 1.020. In contrast, an adulterated urine sample can be detected if the nitrite level is greater than or equal to 500 g/mL, or if the pH is less than 3, or greater than or equal to 11 (Gail Cooper & Negrusz, 2013). Table 4 indicates the parameters involved in the dilution, adulteration, and substitution of urine specimens (Flanagan & Maurer, 2020; Raouf et al., 2018).
Although low creatinine and SG values indicate dilute urine, a dilute sample does not necessarily indicate that the urine was intentionally diluted (Chaturvedi et al., 2013;
Holden & Guice, 2014). Dilution may occur pre-collection by consumption of excess amounts of fluids or post-collection by adding fluid to the specimen. Other causes of diluted urine should be considered, such as renal tubular dysfunction or diuretic use (Raouf et al., 2018).
Chemicals may be added to urine either in an attempt to mask a test result, or to destroy the analyte. The chief objective of adulteration is to produce a negative result, but even an invalid test may lead to a second collection, thereby ‘buying time’ for drug and/or metabolites to be eliminated before the second test. There is a large
‘underground’ sale of adulterants. Commonly used compounds include household chemicals, such as bleach, table salt, laundry detergent, toilet bowl cleaner, vinegar,
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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