Chapter 1: Introduction
1.4 Popular Matrices for DOA Analysis
1.4.3 Hair Matrix for DOA Analysis
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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).
29 violates an individual's privacy); easy transportation and storage (a solid and durable structure assures long stability); negligible risk of infection; and assessment of retrospective and cumulative drug exposure from months to years. Hair analysis also allows for assessment of the length of time an individual has been exposed to a substance (because drugs incorporating into hair have a large window of detection). Additionally, it is possible to evaluate chronic drug use through segmental analysis, in which the hair is cut into smaller pieces and then analyzed separately (Kintz, 2018; Kuwayama et al., 2021; Wada, Ikeda, Kuroda, & Nakashima, 2010). Additionally, it is possible to locate and characterize drugs on a single hair sample by using matrix-assisted laser
desorption/ionization (MALDI) combined with imaging mass spectrometry (Kamata et al., 2020; Kernalléguen et al., 2018). It is possible that a hair sample is the only
biological matrix that can be analyzed postmortem and for those investigations that take place a marked amount of time after the person has passed away. These types of
investigations are called postmortem toxicology analyses (de Campos et al., 2022).
The hair test has its own set of disadvantages as well. It is not possible to make a determination regarding recent drug use (within the past week). Methods that are
accurate and sensitive are required in order to detect extremely low drug concentrations, and the cost of analysis is significantly higher for drug samples than it is for other types of biological samples (Cuypers & Flanagan, 2018; Ferreira, Paulino, & Quintas, 2019).
1.4.3.1 Mechanisms of Drug Incorporation Into Hair
Still unknown are the precise mechanisms involved in the incorporation of drugs into hair. The model that is widely accepted postulates that drugs and their metabolites become incorporated in hair through a process known as passive diffusion. This process occurs when blood capillaries connect to the cells of the growing matrix, which are located at the base of the hair follicle. As the cells continue to divide and age, they eventually die and coalesce to form the hair fibre, which is responsible for transporting the drug that is incorporated into the matrix. Other possible transmission mechanisms include the diffusion of bacteria from sweat (sweat glands) or sebum into the hair, as well as the contamination of the environment (smoke, dust, or the physical transfer of bacteria from contaminated hands) (de Campos et al., 2022).
30
It is generally believed that drugs can enter the body through one of two
mechanisms: first, they can be absorbed from the surrounding environment, and second, they can be incorporated into the growing hair shaft by the blood that is supplying the hair follicle. Exposure to chemicals found in aerosols, smoke, or the secretions produced by sweat glands and sebaceous glands can all cause the hair to become contaminated with drugs. It is well known that sweat can contain the same drugs that are found in blood. Because hair is extremely porous and can add up to 18% to its weight simply by absorbing liquids, it is possible for drugs to easily be transferred into hair through perspiration. Lastly, chemicals that are present in the air (such as smoke and vapors) have the potential to become deposited onto hair (Figure 10) (Kintz, 2004).
At least three different pathways appear to be responsible for the incorporation of drugs into the hair: the first is through the blood during the process of hair formation, the second is through sweat and sebum, and the third is through the external environment. In this model it describes several experimental findings, compared to the passive model (transfer from blood into the growing cells of the hair follicle). Such findings include:
the drug and metabolite(s) ratios in blood are quite different from those found in hair;
and the drug and metabolite(s) concentrations in hair differ significantly in individuals receiving the same dose. Evidence that the drug was transferred through sweat and sebum, can be supported as the drugs and metabolites are present there at high
concentrations and continue to be present in these secretions for a longer period of time than in blood (Ferreira et al., 2019; Usman et al., 2019; Wada et al., 2010).
Figure 10: Possible model of drug incorporation, reproduced with permission from Elsevier (Kintz, 2004)
31 It is not known what the precise mechanism is that causes chemicals to become bound in hair. It has been hypothesized that a drug's ability to bind to intracellular
components of the hair cells, such as the melanin found in the hair, may increase the rate at which it diffuses passively through the hair. For instance, the amount of melanin in the hair determines the amount of codeine that remains in the hair after oral administration.
In spite of this, it is highly unlikely that this is the only mechanism given that drugs can become lodged in the hair of albino animals due to the absence of melanin. The
interaction of medications with the sulphydryl-containing amino acids that are present in hair is yet another proposed mechanism. In hair, there is a high concentration of amino acids such as cysteine, which help to maintain the integrity of the network of protein fibres by forming cross-linking S–S bonds. Through this mechanism, drugs that diffuse into hair cells could be bound (Khajuria et al., 2018; Madry, Poetzsch, Steuer, Kraemer,
& Baumgartner, 2021).
Numerous studies have shown that after receiving the same dosage, black hair incorporates a significantly greater quantity of drugs than blond hair does. As a consequence of this, conversations have ensued about the possibility of genetic
variability in drug deposition in hair, which is still subject to investigation (Hegstad et al., 2008; Kintz, 2004; Zakrzewska et al., 2020).
1.4.3.2 Hair Sample Collection Protocol
There is no universally accepted procedure for the collection of a hair sample from a victim or a patient suffering from addiction, for the purposes of forensic
investigation. The strands of hair samples are taken at random from various locations on the body. In the majority of studies that have been published, the samples are taken from various parts of the scalp at random. The vertex posterior, which is located at the back of the head, is the optimal location from which to harvest hair. When compared to other regions of the head, this region has less variation in the rate at which hair grows, the number of hairs that are in the growing phase is more consistent, and the hair is less susceptible to the effects of age and sex. Strands of hair are cut as close to the scalp as is practically possible, and their precise locations on the scalp must be documented. After being collected, hair samples can be kept at room temperature in aluminum foil, an envelope, or a plastic tube, depending on the container chosen. The amount of the
32
sample that is taken can vary quite a bit from one laboratory to the next. It can also depend on the substance that is being analyzed and the testing methodology. The sample sizes that are reported in the literature range from a single hair all the way up to 200 mg, and they are taken from as close to the scalp as possible. The following procedures are included in the preparation and examination of hair (Kintz, 2004; Usman et al., 2019).
1.4.3.2.1 Hair Sample Decontamination
Washing the hair sample is necessary for achieving precision and accuracy in hair testing as well as improving analytical performance. This is because the residue of hair care products, dust, oils and lipids, sweat, and other impurities must be removed before the sample can be analyzed. It is possible for drugs to bind to the hair or the hair matrix as a result of passive exposure to the environment; however, this will depend on the porosity of the hair. The use of decontamination procedures allows for the removal of these drugs, which are only loosely bound, and the prevention of false-positive results.
As part of the decontamination process, the hair is washed with methanol, acetone, sodium dodecyl sulphate, dichloromethane, and various other organic solvents, in addition to detergents and phosphate buffers (Khajuria et al., 2018; Kintz, 2004, 2018).
1.4.3.2.2 Digestion or Extraction from The Hair Sample
Extraction of bound drugs from the hair matrix can be accomplished using one of three distinct methods: 1) alkaline digestion, which entails the prolonged incubation of samples in alkaline solutions such as sodium hydroxide, which is followed by extraction procedures such as solid phase extraction (SPE) and liquid–liquid extraction (LLE); 2) acid extraction, which is a technique that requires samples to be incubated for an extended period of time in acidic solutions such as sulphuric acid that is then followed by extraction processes such as SPE and LLE, and 3) the drug can be extracted from the hair through a process known as enzymatic digestion, which involves the utilization of enzymes such as b-glucoronidase/arylsulfatase.
The process of analyzing hair has been sped up and made more effective by some of the more recently developed methods, such as those that use ultrasonic or microwave energy to assist in the extraction process. Miniaturized techniques, such as headspace solid-phase micro extraction, hollow-fiber liquid-phase micro extraction, and micro-
33 extraction by packed sorbent, markedly cut down the amount of organic solvents used and the amount of toxic residue generated during the cleaning processes (Khajuria et al., 2018; Kintz, 2004, 2018, 2019). Studies identifying various drugs in hair with the use of different extraction methods is described in Table 6.
Table 6: Chromatography-based methods for drug determination in hair samples
Drugs Amount
of hair
Decontamination Extraction protocol
Instrument Linear range
References
Codeine 4 - 6 cm 20 mg
DCM MeOH LC-MS/MS 100–
3000 pg/mg
(Huang, Liu, Huang, &
Chien, 2009) Codeine 20 mg (<1
mm)
DCM, deionized water, MeOH
Methanol, SPE, derivatization
MSTFA
GC–MS 0.05-50 ng/mg
(M. Barroso, Dias, Vieira,
López- Rivadulla, &
Queiroz, 2010) Codeine 10 mg (1–
3 mm)
SDS, distilled water, MeOH
Cation- selective exhaustive injection sweeping
micellar electrokinetic chromatography
(CSEI-Sweep- MEKC)
LC–MS 0.3–30 ng/mg
(Y. H. Lin, Lee, Lee, Ko,
& Wu, 2007)
Codeine 10mg (1-4 cm)
MeOH SPME,
derivatization BSTFA + 1%TMCS
GC–MS 0.005–
5.0 ng/mg
(Moller, Aleksa, Walasek, Karaskov, &
Koren, 2010) Codeine 50 mg (2
mm)
DCM SPE LC-MS/MS 0.05- 10
ng/mg
(Imbert et al., 2018) Morphine 4 - 6 cm
20 mg
DCM MeOH LC-MS/MS 100–
3000 pg/mg
(Huang et al., 2009)
Morphine 20 mg (<1 mm)
DCM, deionized water, MeOH
Methanol, SPE, derivatization
MSTFA
GC–MS 0.05-50 ng/mg
(M. Barroso et al., 2010)
Morphine 10 mg (1–
3 mm)
SDS, distilled water, MeOH
Cation- selective exhaustive injection sweeping
micellar electrokinetic chromatography
(CSEI-Sweep- MEKC)
LC–MS 0.3–30 ng/mg
(Y. H. Lin et al., 2007)
Morphine 10mg (1-4 cm)
MeOH SPME,
derivatization BSTFA + 1%TMCS
GC–MS 0.01–5.0 ng/mg
(Moller et al., 2010)
34
Table 6: Chromatography-based methods for drug determination in hair samples (Continued)
Drugs Amount
of hair
Decontamination Extraction protocol
Instrument Linear range
References
Morphine 50 mg (2 mm)
DCM SPE LC-MS/MS 0.05- 10
ng/mg
(Imbert et al., 2018) 6-acetylmorphine
(6-AM)
4 - 6 cm 20 mg
DCM MeOH LC-MS/MS 100–
3000 pg/mg
(Huang et al., 2009)
6-acetylmorphine (6-AM)
20 mg (<1 mm)
DCM, deionized water, MeOH
Methanol, SPE, derivatization
MSTFA
GC–MS 0.05-50 ng/mg
(M. Barroso et al., 2010)
6-acetylmorphine (6-AM)
10mg (1-4 cm)
MeOH SPME,
derivatization BSTFA + 1%TMCS
GC–MS 0.01–5.0 ng/mg
(Moller et al., 2010)
6-acetylmorphine (6-AM)
50 mg (2 mm)
DCM SPE LC-MS/MS 0.05- 10
ng/mg
(Imbert et al., 2018) Heroin 50 mg (2
mm)
DCM SPE LC-MS/MS 0.05- 10
ng/mg
(Imbert et al., 2018) Tramadol 20 mg (<1
mm)
DCM, deionized water, MeOH
Methanol, SPE, derivatization
MSTFA
GC–MS 0.05-40 ng/mg
(M. Barroso et al., 2010)
Pregabalin 20 mg (2–
3 mm)
MeOH Addition of
methaqualone.
UPLC–
MS/MS
50- 1600 pg/mg LOD of
0.76 pg/mg and an LLOQ of 2.5 pg/mg
(Pauly, Yegles, &
Schneider, 2013)
Pregabalin 25 mg (<1 mm)
DCM Ultrasound bath. UPLC–
MS/MS
10 to 2000 pg/mg
(Kintz, Ameline, &
Raul, 2018)
benzoylecgonine (BE)
4 - 6 cm 20 mg
DCM MeOH LC-MS/MS 100–
3000 pg/mg
(Huang et al., 2009)
benzoylecgonine (BE)
50 mg (2 mm)
DCM SPE LC-MS/MS 0.05- 10
ng/mg
(Imbert et al., 2018)
35 Table 7 provides a summary of the confirmation of the cut-off and expected
concentrations for various drugs of abuse found in the hair. These concentrations were recommended by the Society of Hair Testing (SoHT) (G. A. A. Cooper et al., 2012) to prevent false-positive results from external contamination, particularly during workplace drug testing. Sometimes, in the forensic investigations involving a single drug exposure, such as those involving child abuse, crimes that were facilitated by drugs, or doping offences, these cut-offs should be applied. It is important to keep in mind that the hair cut-offs for many different drugs have not yet been determined. Some examples of these drugs include GHB, BZDs, PGB, carisoprodol, new psychoactive substances (NPS), and anabolic steroids (Kintz, 2018).
Table 7: Confirmation cut-off concentrations (when tested by chromatography) recommended by SoHT and expected concentrations for drugs of abuse in hair
Drug Confirmation Cut-off Concentrations
Expected Concentrations Heroin 0.2 ng/mg of 6-acetylmorphine,
morphine
0.5 – 70 ng/mg, in most cases < 30 ng/mg
Cocaine 0.5 ng/mg of cocaine and 0.05 ng/mg of benzoylecgonine and
cocaethylene
Presence of norcocaine and/or cocaethylene
0.5 – 100 ng/mg, in most cases < 50 ng/mg, in crack abusers > 300 ng/mg is
possible
Amphetamine, MDMA
0.2 ng/mg for each drug 0.5 – 50.0 ng/mg
Cannabis 0.05 ng/mg for THC 0.2 pg/mg for THC-COOH
THC: 0.05 – 10 ng/mg, in most cases <
2 ng/mg THC-COOH: 0.2 – 50 pg/mg, in most cases < 5 pg/mg
Methadone 0.2 ng/mg for methadone 0.05 ng/mg for EDDP
0.2 - 50 ng/mg Buprenorphine 0.01 ng/mg for buprenorphine 0.01
ng/mg for norbuprenorphine
< 1 ng/mg
36
1.4.3.3 Factors Affecting Hair Analysis
The applications of hair analysis include proving chronic intoxication in an individual, assisting in the resolution of drug-facilitated crimes and child custody cases, conducting post-mortem drug screening, and workplace drug testing; however, the scope of hair analysis is dependent on the detection of drugs and their metabolites followed by their quantification in the hair matrix. The accumulation of drugs in the hair can be influenced or affected by a number of different factors (Guthery, Bassindale, Bassindale, Pillinger, & Morgan, 2010; Khajuria et al., 2018).
1.4.3.3.1 Contamination
The fact that there is a large body of research available on hair analysis makes it a non-controversial biological sample; however, the most notable problem is the
possibility of obtaining false-positive results always reduces the benefits that it offers.
The effectiveness of the decontamination process is something that will never be proven beyond a reasonable doubt. It is not universally agreed upon that washing one's hair will completely remove any drugs that have been deposited on the outside of the body. The drugs that were found to be externally deposited on the hair were removed from hair samples by treating them with methanol and isopropanol/phosphate buffer and then washing them three to five times (Balíková & Habrdová, 2003; Cairns, Hill, Schaffer, &
Thistle, 2004; Schaffer, Wang, & Irving, 2002; Tsanaclis & Wicks, 2008; Yegles, Marson, & Wennig, 2000). According to the findings of a number of studies (Paulsen, Wilkins, Slawson, Shaw, & Rollins, 2001; Rollins et al., 2003; Tsanaclis & Wicks, 2008), the washing process can remove more than 90% of the drugs that have been deposited externally on hair. As a result, the criteria for the concentration ratio between the most recent wash and the sample of hair were established (Rollins et al., 2003;
Tsanaclis & Wicks, 2008).
The detection of the drugs' endogenous metabolites may also help reduce the number of false-positive test results. For a sample to be considered positive for cocaine, organizations such as SoHT and the Substance Abuse and Mental Health Services Administration have established metabolite cut-off levels such as the following: COC 500 pg/mg and at least one metabolite of cocaine 50 pg/mg (Bush, 2008; G. A. A.
37 Cooper et al., 2012). Additionally, drug markers were utilized in order to ascertain both active and passive exposure (Villain, Muller, & Kintz, 2010).
1.4.3.3.2 Hair Color
Several studies have shed light on how a drug binds to the pigments melanin and pheomelanin, as well as the mechanism underlying this process (Barbosa et al., 2013;
Mieczkowski & Kruger, 2007; Nosanchuk & Casadevall, 2006). When compared to lighter hair, darker hair contains a greater amount of melanin, which results in a greater accumulation of drugs. However, research is still being done to figure out how acid and base drugs of varying types bind to melanin so that they can be taken into the body.
Researchers have not completely dismissed the idea that heredity could play a significant part in determining melanin concentration, which in turn affects the way drugs are
incorporated into hair (Borges, Wilkins, & Rollins, 2001; Kintz P, 2008).
1.4.3.3.3 Cosmetic Treatments
Studies have shown that low concentrations of drugs may not be detected in hair that has undergone chemical treatment; however, studies have also reported that
chemical treatment damages the hair, making it more likely that the hair will be
contaminated with drugs from the outside. Therefore, the history of cosmetic treatment of the hair should always be taken into consideration when interpreting the results (Mário Barroso, Gallardo, Vieira, López-Rivadulla, & Queiroz, 2011; Kelly, Mieczkowski, Sweeney, & Bourland, 2000; Skopp, Pötsch, & Moeller, 1997).
1.4.3.3.4 Dose–Concentration Relationship
Even though some studies have found a tenuous connection between the dose of the drug and its concentration in the hair, it is of utmost importance to determine whether the quantity that is detected can accurately indicate the quantity that was consumed (Kronstrand, Roman, Hedman, Ahlner, & Dizdar, 2007; Ropero-Miller, Goldberger, Cone, & Joseph, 2000). The opiate dose and concentration in the suspectsts' hair, which was collected as part of a case-control study, did not provide any information that was statistically significant (Kintz, Bundeli, Brenneisen, & Ludes, 1998).