Atypical Asphysia

http://www.jfsmonline.com/article.asp?issn=2349-5014;year=2018;volume=4;issue=4;spage=233;epage=237;aulast=Cao

Forensic investigation of atypical asphysia

Zhe Cao¹, Zhiyuan An², Xiaoning Hou¹, Dong Zhao^3
1 Anshan Public Security Bureau, Anshan, China
² Key Laboratory of Evidence Science (China University of Political Science and Law), Ministry of Education, China, Collaborative Innovation Center of Judicial Civilization, China
³ Key Laboratory of Evidence Science (China University of Political Science and Law), Ministry of Education, China, Collaborative Innovation Center of Judicial Civilization; Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, Beijing, China

Correspondence Address:
Dr. Dong Zhao
25 Xitucheng Road, Haidian, Beijing 100088 
China

Abstract

Smothering, choking, confined spaces, traumatic asphyxia, positional asphyxia, and other kinds of atypical mechanical asphyxia are not rare in forensic practice. However, these are not commonly well demonstrated in forensic monographs worldwide. The authors researched related works and literatures and summarized these with a view to contribute to the existing teaching resources and provide help to forensic practitioners who are involved in scene investigation and identification of such deaths.

Keywords: Asphyxia, forensic pathology, forensic medicine

Introduction

Death caused by compression of the neck, such as from hanging, strangling, or throttling, is termed "mechanical asphyxia" and usually has obvious physical findings. However, asphyxias that result from no direct pressure on the neck vessels or trachea, lack typical morphological changes, or result in minimal damage are called "subtle asphyxias"^[1] or "atypical mechanical asphyxias," used in this article. Atypical mechanical asphyxias include smothering, choking, environmental hypoxia, traumatic asphyxia, and positional asphyxia, among others.

Smothering

Smothering is a form of asphyxia death caused by obstructing the mouth and nose with hands, airtight papers, soft textiles, or the weight of one's own head.^[2]

Smothering can be seen in homicidal or suicidal cases. Homicidal smothering is common in infants, older adults, and people who are unconscious or have restricted motion due to fabric bundling, disease, poisoning, or intoxication. Homicidal smothering can also result when there are significant physical power differences between a perpetrator and victim.^[3],[4],[5] Suicidal smothering is common in psychiatric patients; an example includes wrapping tape around one's mouth, nose, or the entire face.^[6] Smothering can also occur accidentally. For example, adults who are unconscious or paralyzed because of drunkenness, epilepsy, drug overdose, or having another disease might accidentally asphyxiate themselves. Similarly, for an infant lying face down on an airtight mattress or pillow, the weight of the infant's head might obstruct, distort, and occlude his or her mouth and nose, leading to suffocation. In a third example, sleeping infants with clothes or bedding covering their faces are at an increased risk of suffocation.^[1],[2]

In general, it is difficult to identify a case of smothering during forensic scene examination because physical findings are nonspecific.^[7],[8] If smothering is suspected, there may be local signs of pressure on the face.^[2],[3] In adults, with even slight resistance, signs include skin exfoliation from fingernails; contusions on the nose, cheeks, or chin from fingers; bleeding and skin tears corresponding to the teeth in the oral mucosa; and intramuscular bleeding at the mandibular margin. Nasal deformation is also considered a sign of smothering, but can be caused by emergency tracheal intubation.^[3],[5],[7] In infants and adults who are unable to physically resist during asphyxiation, physical damage is difficult to detect.^[3] Of note, a body in the prone position concentrates pressure on the face, preventing accumulation of blood into the compressed skin around the mouth and nose, leading to the formation of distinct pale areas caused by the absence of pooled blood. It is, therefore, important not to assume that pale areas such as these have resulted from indentation by smothering.^[2]

Without positive physical findings in smothering cases, scene investigation plays a decisive role. Pillows and bedding should be examined for blood or lipstick.^[5],[9] For suspected cases of smothering, even if postmortem changes are obvious, suspicious skin lesions should be biopsied for histological examination.^[5] In cases of smothering by textiles, the mouth, nasal cavity, and airways should be examined for inhaled fabric fibers. Fibers in the trachea indicate that a patient may have been alive during smothering.^[8]

Gagging generally involves placing fabric in a victim's mouth to prevent yelling; the fabric gradually becomes soaked with saliva, and if airtight, will lead to suffocation. Another form of gagging involves placing tape over the mouth or nose, which results in trapped mucus production that eventually leads to suffocation. Obstruction of the nasopharynx by objects in the oral cavity may also lead to gagging and subsequent death.^[2] Usually, suspected gagging is confirmed when blocking objects are found, not by any specific physical signs of asphyxia.^[3]

Choking

Choking refers to upper respiratory tract blockage by a foreign body leading to suffocation. The foreign body is usually lodged between the larynx and trachea.^[10],[11] Death may result from simple hypoxia; however, many deaths occur quickly before the onset of hypoxia. Studies have found that, even in cases in which the airway is not completely blocked, death often occurs, likely from neurogenic-induced cardiac arrest.^{[2],[9],[11],[12]}

Choking is almost always accidental, with cases of homicide and suicide relatively rare.^[1],[11] For infants, accidental choking most often occurs with foreign body ingestion; for adults, choking most often occurs with food.^[1],[11] Victims in homicidal choking cases are most likely to be older adults, infants, young children, people who are unconscious, or persons debilitated by illness or intoxication. Suicidal choking most often occurs in patients with psychosis or prisoners in jail.^[1]

Evidence of coughing helps eliminate choking as a cause of death because it signifies that the respiratory tract was open during upper respiratory blockage.^[3] Computed tomography imaging can provide information before an autopsy on the location of a foreign body and can help inform an autopsy plan.^[13] Few physical findings are generally seen in choking deaths, so the discovery of a foreign body in the airway, a detailed clinical history, descriptions of the death environment and any resuscitation attempts, and exclusion of other causes of death are critical when forming a conclusion.^{[1],[9],[11],[12]} If the foreign body shifts during resuscitation or otherwise is moved, clinical history might be the only evidence.^[3],[13]

Foreign bodies blocking the airway leading to choking generally belong to the following categories.^[2]

Foreign objects

Attackers may put a towel or sock into the victim's mouth to prevent shouting; this can cause choking and gagging.^[3] In another example, people may inhale sand, piles of gravel, or piles of soil when they fall on them, causing respiratory blockage and resulting in choking death. This scenario may occur accidentally at a construction site, during a traffic accident, or in children playing in or eating sand.^[3],[14]

Acute obstruction

Acute allergy, steam stimulation, heat inhalation, and acute inflammation may cause swelling of the throat organs, including the epiglottis, tonsils, or glottis, leading to choking. Trauma in the anterior or lateral cervical neck structures can also result in severe swelling of the respiratory tract from bleeding and edema.^[1],[2],[7] Tumors, polyps, or cysts can also block respiration, leading to choking.^{[1],[10],[11]}

Foods

The most common foreign bodies causing choking death in adults are foods.^[10] Susceptible factors include old age, neuromuscular disease, poor dentition leading to chewing problems, consumption of alcohol or other central nervous system depressants weakening the gag reflex, or other neurological or mental illness (of which poor dentition is an important risk factor).^{[1],[11],[12],[13]} Of patients with mental illness, those with schizophrenia are most likely to choke on food, possibly from a propensity to swallow incompletely chewed food.^[11] The majority of adult choking cases occur at patients' homes, nursing homes, or mental hospitals, and often take place suddenly during meals.^[1]

When a sudden death occurs while eating or soon after, the possibility of choking must be considered. A search for a blocked airway should be initiated, but in addition, the investigator should also consider factors that could have aggravated the choking episode. Therefore, quality and number of teeth, food debris in the esophagus – which can cause tracheal obstruction from the external oppression – and exclusion of neurological diseases and intoxication are all important when evaluating sudden death during a meal.^{[1],[9],[11],[12]}

It is typical for gastric contents to be present in the throat, trachea, and bronchi after death, caused by reflux or shifting of contents. This is a common postmortem phenomenon, found in 20%–25% of routine examinations. As a result, if a small amount of gastric content is found in the respiratory tract, this does not mean that choking had occurred; however, if the throat or airway is completely blocked by gastric contents, choking can be concluded.^[2],[3],[13] The inhalation of gastric contents is more common in people who are unconscious.^[1]Importantly, there is no reliable way to distinguish natural food reflux early in the dying process from true inhalation while alive, unless the inhalation occurred during a clinical procedure or another person witnessed the event. In most cases, in the absence of hard evidence, it is unreasonable for forensic officers to conclude that the inhalation of gastric contents is secondary to choking death.^[2]

Environmental Hypoxia

Environmental asphyxiation is usually caused by a lack of oxygen in the local environment,^[1],[2],[3] and is almost always accidental. Oxygen deficiency can occur secondary to breathing exercises, microbial consumption, activities related to industrial work (such as welding), environmental chemical reactions (such as rust), absorption by chemical substances (such as activated carbon), and presence of toxic gases (such as propane, nitrogen, and methane).^[1],[2],[3] An atmospheric oxygen concentration below 5%–10% will cause death in a few minutes, and a concentration of carbon dioxide higher than 10% is lethal.^[1] In some cases, death occurs before the onset of hypoxia, and is secondary to overexcitement of the body's chemical sensing system, which causes parasympathetic nervous system-mediated cardiac arrest.^[2]

In hypoxia-asphyxia deaths caused by low atmospheric oxygen levels, physical findings are usually absent,^[2] making elucidation of the specific cause of death difficult. Investigators must carefully analyze the environment and exclude other causes of death to conclude environmental hypoxia-asphyxia.^[3] Measurements of toxic gases and oxygen concentrations in the air, as well as postmortem analysis of blood and tissues, should be performed; in addition, scene simulations may be required.^[1]

As a type of environmental hypoxia-asphyxia, plastic bag suffocation is often used as a suicide technique in Western countries. This method is common in young men and elderly women.^[15] Some people even use the propane, ether, or helium gas along with the plastic bag. Plastic bag suffocation deaths can also occur accidentally or unexpectedly, such as during sexual asphyxia, children playing with plastic bags, and other occurrences.^[1] It is very rare for the use of plastic bags to result in death; however, it is more likely in cases in which the victim is unconscious, or when there is a large difference in strength between the perpetrator and victim.^[16]

Plastic bag suffocation often occurs rapidly with few physical signs;^[1],[2] however, in a small number of cases, marks on the neck are present corresponding to the areas of bag bundling (such as from a rubber band), or there may be signs of prior injury, such as wrist cutting or abuse.^[1],[2] It is a common misconception that the postmortem presence of moisture in the plastic bag confirms that the bag was placed on a breathing human; water droplets form as gas evaporates from the skin, nose, and mouth even if the person was previously deceased.^[2]

Because there are usually no specific physical findings, it is difficult to identify cases of plastic bag suffocation unless the bag is over the head at the time of scene investigation or autopsy.^[2] If the plastic bag is removed before forensic workers see the corpse, they will not be able to determine the cause of death through forensic examination, and may even conclude that a natural death occurred. Therefore, to identify such cases, forensic workers must pay careful attention during scene exploration and investigation.^{[1],[3],[9],[16]} If necessary, forensic workers can conduct simulations under close monitoring in a protected environment, which can help to pinpoint a cause of death through analysis of time measurements.^[4],[6],[17] Specimens collected from the blood, lungs, liver, or other organs for poison analysis should be extracted and stored in a sealed empty bottle along with a plastic bag,^[2],[7],[16] frozen, and delivered promptly.^[1]

Traumatic Asphyxia

Traumatic asphyxia refers to the compression of the chest or abdomen by massive mechanical forces resulting in thoracic fixation – expansion of thoracic and lower phrenic muscles – leading to respiratory disturbance and death by asphyxiation.^[2]

Traumatic asphyxia is common in the following types of accidents: motor vehicle compression or extrusion during traffic accidents; pinning from building collapse, falling rocks, or other objects; trampling by a crowd; compression while standing in a crowded population from sudden external forces; compression by fallen tools or furniture; and compression of infants and children while sleeping with parents (overlaying asphyxia).^[1],[2],[18] There are also reports of homicide resulting from a perpetrator kneeling or sitting on the chest of a victim.^[19]

The pathological features of traumatic asphyxia are usually quite specific. These include prominent facial and nuchal hyperemia and swelling; numerous petechial hemorrhages on the face or conjunctiva; subconjunctival hemorrhage and edema; and nasal bleeding. In general, a person who dies from traumatic asphyxiation often appears strangled with features extending down to the neck, with no signs of local damage.^{[2],[20],[21]}

However, physical features such as these are not always visible. Studies have shown that, in up to 10% of cases, no petechial hemorrhages are seen on the face or conjunctiva. The reason for this is unclear, but may be related to rapidness of death, lack of obvious chest compression or vagus nerve stimulation, lack of occlusion of the epiglottis, or concurrence of both left heart and right heart impairment at the time of chest compression.^{[1],[18],[20],[21]} On gross examination, lungs may have a purplish red color, congestion, or subserous bleeding with or without obvious expansion of the right heart or superior vena cava; sometimes, there is no evidence of trauma despite severe direct external compression on the chest and abdomen.^{[1],[2],[3],[9]}

Traumatic asphyxia is a diagnosis of exclusion. In addition to supporting evidence from a scene investigation, suffocation death should only be considered after excluding fatal injuries and poisoning.^[1],[9],[21]

Overlaying asphyxia is a special form of traumatic asphyxia, often secondary to nasal compression. Physical examination findings are usually absent, so overlaying can be difficult to determine unless the same-bed sleeper admits to crushing the infant or child. Overlaying asphyxia is sometimes attributed to sudden infant death syndrome, so it is important to examine adults' and children's clothes and bedding carefully as well as the scene.^[1],[3],[22]

Positional Asphyxia

Positional asphyxia refers occurrences in which respiration is compromised from splinting of the chest or diaphragm preventing normal respiration, or occlusion of the upper airway due to abnormal positioning of the body.^[23] Positional asphyxia is almost always an accident, during which the victim cannot extract himself or herself from a specific position or small space. The victim may be further impaired by alcohol or drug intoxication, weakness, neurological disease, or fabric bundling. Common examples of positional asphyxia include limbs tied behind the back while in a prone position (may be performed for restraint by police or psychiatrists for suspects or patients); head-down position (inversion of the body, or head hanging down off the edge of a bathtub); jack-knife position (upper body significantly curved from the waist down); bundled thoracic or abdominal horizontal sling (e.g., a young girl wearing a belt hanging by the abdomen on a swing); excessive flexion or extension of the neck (e.g., during a motor vehicle accident); lack of chest wall expansion in a restricted space (wedging); and a person sandwiched between the wall and the mattress after falling off the bed.^{[1],[2],[3],[4],[5],[6],[7],[24]} A typical case of postural asphyxia involves a drunken person who collapses into a narrow space, excessively distorting the neck and hindering breathing, leading to death.^[9]

Cause of death from positional asphyxia often results from reverse suspension of the body such that the movement of the chest wall is restricted by intra-abdominal organs compressing the diaphragm. This prolongs inspiration, and eventually results in respiratory muscle fatigue, leading to slowed movement of the chest wall and subsequent hypoxia. Venous return is effectively limited, and blood flow to the brain is shifted, decreasing blood flow and further aggravating respiratory muscle fatigue; eventually, the heart stops.^[1] Positional asphyxia does not require reversal of the entire body; fatal asphyxia may result from the reversal of torso position, excessive flexion of the neck, or pressure on one's face, such as in an intoxicated person whose face is pressed to the floor.^[25] The difference between traumatic asphyxia and positional asphyxia is whether the chest and abdomen are compressed by external forces. If chest compression is from an external source, he or she should have been died from traumatic asphyxia. If a deceased person is found in a specific position or restricted space that limits chest activity, the person should have been died from positional asphyxia.^[1],[23]

Positional asphyxia can be identified by the following criteria: The body position is consistent with restricted or disordered respiration; scene investigation or historical investigation identifies that an accident had occurred; the deceased person cannot change his or her position for some reason; and other obvious natural or violent causes of death are excluded. A diagnosis of accidental positional asphyxia mainly depends on the evidence obtained from the scene environment.^[24],[25] Some forensic investigators believe that, if another disease is present, then either the cause of death is not associated with positional asphyxia, or the onset of the disease makes the deceased patient prone to positional asphyxia.^[23] It should be noted that alcohol consumed by a patient with positional asphyxia may be metabolized. Thus, even if the concentration of alcohol in the blood or urine is very low or negative, the possibility of positional asphyxia cannot be ignored.^[24]

Wedging is a special form of positional asphyxia, commonly seen in infants and young children whose body or head are compressed in a narrow space. The chest wall is fixed, resulting in airway obstruction that results in asphyxia. Wedging usually occurs between a mattress and wall or mattress and furniture or baby crib. It is most common in infants aged 3–6 months, intoxicated adults, or comatose patients who accidentally fall between a mattress and wall, leading to death. Physical findings of wedging are usually absent.^[1],[22]

Acknowledgments

This study was supported by the Open Project of Key Laboratory of Forensic Genetics, Ministry of Public Security (2017FGKFKT05), Program for Young Innovative Research Team from China University of Political Science and Law (2016CXTD05), and Project of Interdisciplinary Science Construction-Forensic Psychology from China University of Political Science and Law.  

Determination in hair samples by gas chromatography/mass spectrometry : 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), methadone (1,1-diphenyl-1-(2-dimethylaminopropyl)-2-butanone), cannabinol (6, 6, 9-trimethyl-3-pentyl-6H-dibenzo[b, d]pyran-1-ol), cannabidiol (2-[(1R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol), Δ9-tetrahydrocannabinol (THC), UR144 (1-pentyl-1H-indol-3-yl)(2, 2, 3, 3-tetramethylcyclopropyl)methanon), CP47497 (2-[(1R,3S)-3-hydroxycyclohexyl]-5-(2-methyl-2-octanyl)phenol) and its homolog CP47497-C7, 1-([5-fluoropentyl]-1H-indol-3-yl)-(naphthalen-1-yl)methanone (AM2201), (1-hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-019), (4-methoxy-1-naphthalenyl) (1-pentyl-1H-indol-3-yl)methanone (JWH-081), (4-methyl-1-naphthalenyl) (1-pentyl-1H-indol-3-yl)methanone (JWH-122), 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), tetrahydrocannabinol-D3 (THC-D3), EDDP-D3, and methadone-D3

http://www.jfsmonline.com/article.asp?issn=2349-5014;year=2018;volume=4;issue=4;spage=184;epage=191;aulast=Anzillotti

ORIGINAL ARTICLE

Year : 2018  |  Volume : 4  |  Issue : 4  |  Page : 184-191

Determination of methadone and eight new psychoactive substances in hair samples by gas chromatography/mass spectrometry

Luca Anzillotti, Luca Calò, Marianna Giacalone, Antonio Banchini, Rossana Cecchi
Department of Medicine and Surgery, Institute of Legal Medicine, University of Parma, Parma, Italy

Date of Web Publication

27-Dec-2018

Correspondence Address:
Dr. Luca Anzillotti
Institute of Legal Medicine, University of Parma, Via A. Gramsci 14, Parma 43126 
Italy

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jfsm.jfsm_22_18

Abstract

Many new psychoactive substances (NPSs) with different chemical structures have emerged in the illicit drug market in the last decade. The present work was aimed at the development of a simple method in gas chromatography/mass spectrometry (MS) for the determination of NPS of different classes, the use of cannabinoids, and, at the same time, the evaluation of methadone therapy in hair matrix, within our routine analysis control for methadone treatment or from autopsy cases. The determination of synthetic cannabinoids and methadone therapy used an extraction method based on incubation in concentrated sodium hydroxide (NaOH) solution, providing a dissolution of the keratin matrix. The described method was applied on 15 authentic specimens from our cases: five showed the presence of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). The described method can be useful not only in the forensic investigation of NPS-related addiction histories but also in epidemiological and retrospective studies on the spread of NPS among specific safety-sensitive social workers. The GC instrument was an Agilent 7820A (Agilent Technologies, Santa Clara, CA, USA), and the detection system was an Agilent 5977B single quadrupole MS operating in selective ion monitoring mode. Validation parameters such as limit of detections (LODs), limit of quantifications (LOQs), repeatability, accuracy, and linearity were satisfactory for its application on real specimens. LODs, LOQs, R%CV, standard deviation, and the mean concentration for the analyzed compounds are reported in Table 1b. Accuracy and repeatability were acceptable for all the analytes at their respective LOQs. Recovery experiments varied from 58.3% to 103.0%, thus allowing the application on authentic specimens. The described method can be useful not only in the forensic investigation of NPS-related addiction histories but also in epidemiological and retrospective studies on the spread of NPS among specific safety-sensitive social workers, such as drivers.

Keywords: Drugs of abuse, forensic, gas chromatography/mass spectrometry, hair, new psychoactive substance, toxicology

How to cite this article: Anzillotti L, Calò L, Giacalone M, Banchini A, Cecchi R. Determination of methadone and eight new psychoactive substances in hair samples by gas chromatography/mass spectrometry. J Forensic Sci Med 2018;4:184-91

How to cite this URL: Anzillotti L, Calò L, Giacalone M, Banchini A, Cecchi R. Determination of methadone and eight new psychoactive substances in hair samples by gas chromatography/mass spectrometry. J Forensic Sci Med [serial online] 2018 [cited 2018 Dec 29];4:184-91. Available from: http://www.jfsmonline.com/text.asp?2018/4/4/184/248696

Introduction

Many new psychoactive substances (NPSs) with different chemical structures have emerged in the illicit drug market in the last decade. NPSs are different chemical compounds sold online through the e-commerce and the deep web as legal substitutes for classical drugs of abuse, including synthetic cannabinoids, synthetic cathinones, phenethylamines, piperazines, or substances not relating to any of these groups and plant-based materials.^[1] The ease of NPS distribution favored their quick spreading worldwide through different channels. However, as soon as NPSs are scheduled, new derivatives appear on the market; therefore, the number of NPS reported by the European Monitoring Centre for Drugs and Drug Addiction increases each year.^[2] This rapid increase of NPS sets new challenges not only in drug prevention and legislation but also in clinical and forensic toxicology, as the acute and chronic toxicity of many of these compounds is still partially unknown. Hence, the identification in biological samples is of great concern for forensic and clinical toxicologists, to evaluate the spread of NPS among population. According to the 2016 European Early Warning System Report, the largest substance categories monitored are the synthetic cannabinoids (over 160 substances, including 11 new cannabinoids reported in 2016), followed by the synthetic cathinones (over 100 substances, 14 reported for the first time in 2016).^[3] Even within the same class (i.e., synthetic cannabinoids), as soon as legislation is passed banning their use, different compounds show up in the next wave. These substances show different function group chemistry that dictates a sample extraction procedure that will capture the various chemical functionalities. Some NPSs are extremely potent in terms of dosage, so that they may only be present at trace levels. Hence, the necessity and the ability to analyze the complex chromatographic data in the presence of large amounts of coextractant material. These materials are also structurally similar in terms of chromatographic retention time (RT) and mass spectral appearance. Data analyses need to be able to identify the subtle differences in these species and be able to detect such substances in complex mixtures.^[3],[4]

Many analytical methods were developed for NPS determination in biological fluids, such as oral fluid,^[4],[5],[6] blood,^[7],[8],[9] or urine.^[10],[11]To date from a recent search in literature, only few studies deal with the determination of NPS in hair;^{[12],[13],[14],[15],[16],[17]} to the best of our knowledge, no paperwork mentioned the determination of these classes of substances together. The present pilot study was aimed at the development of a simple method in gas chromatography/mass spectrometry (GC/MS) for the determination of eight NPSs of different classes (mainly synthetic cannabinoids), the use of cannabinoids, and, at the same time, the evaluation of methadone therapy in hair matrix, within our routine analyses control for patients with methadone treatment or from autopsy cases. The development of the method involved an extraction technique based on incubation in concentrated sodium hydroxide (NaOH) solution, providing a dissolution of the keratin matrix.^[12],[13] Hair sampling is easy to perform, not invasive, and relatively stable, moreover less affected by adulterants.^[18] Hair samples allow a retrospective determination of the drug use history depending basically on hair length due to their accumulation in keratin, taking into account that head hair grows at an average rate of 1 cm circa each month,^[19] and being able to confirm long-term exposure: it is, therefore, a reliable and valuable tool to assess chronic use of drugs in a specific population.^[18] Moreover, parent drugs prevalently accumulate in hair and keratinized matrices in general with respect to unmetabolized drugs,^[20] avoiding hydrolysis steps for the determination of metabolites.

Subjects and Methods

Reagents and standards

Water, sodium dodecyl sulfate (SDS), acetone, acetonitrile, formic acid, phosphate buffer and methanol, chloroform and isopropanol, NaOH, hexane, and ethyl acetate were purchased from Sigma Aldrich, Milano, Italy. 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), methadone (1,1-diphenyl-1-(2-dimethylaminopropyl)-2-butanone), cannabinol (6, 6, 9-trimethyl-3-pentyl-6H-dibenzo[b, d]pyran-1-ol), cannabidiol (2-[(1R,6R)-3-methyl-6-(prop-1-en-2-yl)cyclohex-2-en-1-yl]-5-pentylbenzene-1,3-diol), Δ9-tetrahydrocannabinol (THC), UR144 (1-pentyl-1H-indol-3-yl)(2, 2, 3, 3-tetramethylcyclopropyl)methanon), CP47497 (2-[(1R,3S)-3-hydroxycyclohexyl]-5-(2-methyl-2-octanyl)phenol) and its homolog CP47497-C7, 1-([5-fluoropentyl]-1H-indol-3-yl)-(naphthalen-1-yl)methanone (AM2201), (1-hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone (JWH-019), (4-methoxy-1-naphthalenyl) (1-pentyl-1H-indol-3-yl)methanone (JWH-081), (4-methyl-1-naphthalenyl) (1-pentyl-1H-indol-3-yl)methanone (JWH-122), 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone (JWH-250), tetrahydrocannabinol-D3 (THC-D3), EDDP-D3, and methadone-D3 were supplied from LGC standards (Milan, Italy). Standard compounds were stored according to supplier recommendations until their use.

Calibration and sample preparation

Hair strands were collected either from routine analyses or from autopsies, cut from the posterior vertex region of the head, close to the scalp since this region is associated with least variation in growth rates (the amount required by SOHT guidelines is a pencil thickness.^[18]) Hair sample aliquots were washed with 3 mL × 3 of a solution of SDS 1%, rinsed twice with 3 mL of distilled water, and then twice with 1 mL of acetone. After drying, each sample was segmented in samples of 1 cm each circa, then each one shred and grinded into small pieces of 1 mm circa (30 mg in weight).

A working solution mixture of deuterated drugs of abuse (mix drugs-Deut) containing EDDP D3, methadone D3, and THC D3 at 10 μg/mL was prepared by proper dilution of the standard solutions and stored at −20°C until use.

Individual methanolic stock solutions were used to prepare a working solution at a concentration of 10 μg/mL. Calibration curves were prepared by addition of the appropriate amount of cannabinoids to 30 mg of blank hair sample (collected from three different drug-free subjects) to obtain the following concentrations: 0.1, 0.5, 1, 2, 5, 10, and 20 ng/mg.

Thirty milligrams of hair samples was put in a vial and 3 μL mix drugs-Deut plus 500 μL of NaOH were added to digest hair sample at 90°C for 30 min. Then, the sample was extracted with 1.5 mL of a mixture composed by hexane: ethyl acetate (9:1) by automated shaking for 10 min and centrifuged for 5 min at 2000 rpm. The supernatant was then transferred into another vial and evaporated under a gentle stream of nitrogen to dryness. After the evaporation step, the sample was reconstituted with 100 μL of MeOH and 2 μL was injected in the GC/MS equipment.

Gas chromatography/mass spectroscopy equipment

The GC instrument was an Agilent 7820A (Agilent Technologies, Santa Clara, CA, USA), and the parameters chosen for optimization were the following: the liner temperature was held at 270°C; helium was used as a carrier gas at a constant flow of 40 mL/min. The oven temperature started from 100°C, then by 20°C/min was held at 250°C for 10 min, then at 20°C/min to 280°C and was held for 5 min and finally to 320°C for 4 min.

The detection system was an Agilent 5977B single quadrupole MS operating in selective ion monitoring (SIM) mode. The column was a J and W DB-5 (5% phenylmethyl silicone) capillary column (30 mm × 0.25 mm. i.d., 0.25 μm film thickness, Agilent Technologies). Characteristic ion fragments of investigated compounds were chosen and optimized injecting the individual methanolic solutions in scan mode before developing the analytical method in SIM [as reported in [Table 1]a. After pretreatment of the sample, 2 μL was injected into the instrument.

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Method validation

The method was validated according to the Food and Drug Administration guidelines^[21] and was evaluated for linearity, limit of detection (LOD), limit of quantification (LOQ), lowest limit of quantitation (LLOQ), accuracy, and repeatability. The linearity of the assay was calculated by the method of least squares and expressed as coefficient of determination (R²). Calibration curves were prepared in triplicate in 3 different days by adding to blank hair samples a mixture of the commercially available standards at a concentration of 10 μg/mL in the proper amount to obtain the range of concentration and to determine the LOD and of quantification (LOQ). These parameters were studied using serial dilutions of the substances of interest in matrix in triplicate and analyzed in 5 different days. Repeatability and accuracy were assessed at three concentrations: low (quality control [QC] 1), medium (QC2), and high (QC3) injected in quintuplicate in 3 different days and were expressed, respectively, as CV% and E%. The parameters studied are listed below.

Specificity

Ten negative hair samples from voluntary subjects were collected and analyzed to determine specificity and verify, therefore, the absence of interfering peaks that could hinder the analytes. Specificity was also assessed by analyzing samples spiked with a sample at a concentration of 500 pg/mg of compounds with most common illicit or therapeutic drugs (such as cocaine and metabolites, opiates, benzodiazepines, and various antipsychotic drugs). Hence, satisfactory specificity was established if no interfering signals were found in terms of characteristic fragments and RT related to endogenous or exogenous compounds.

Limit of detection and limit of quantification

The LOD was calculated at a concentration value giving an S/N >3 for at least three ion fragments for each substance while the LOQ was considered the concentration value giving an S/N >10 for three ion fragments and acceptable accuracy and precision (%CV and %E <20%). LLOQ was calculated at the concentration value giving an S/N ratio >5. These parameters were studied using scalar dilutions of the substances of interest in hair in quintupled.

Linearity

The linearity of the method for each compound was studied in the range from the LLOQ of each substance to 20 ng/mg, performing triplicate analyses for each level. Calibration curves were built by linear regression of the area ratio of each substance with their internal standard (IS) versus the concentration of analyte.

Accuracy and precision

QC samples were prepared at three concentration levels: low (QC1), medium (QC2), and high concentrations (QC3). Accuracy and precision were assessed by analyzing the QCs in quintuplicate in three different days and were expressed respectively as %error (%E) and standard deviation (STD).

Memory effect

Memory effect, intended as carryover of analytes from sample to sample, was evaluated: two blank samples were injected after each run of spiked samples at 50 and 100 ng/mg and analyzed after positive samples.

Identification criteria

The criteria to be fulfilled for the identification of analytes were RT, the presence of three ion fragments, and their relative ion intensities. For the identification of an analyte, RT should not vary more than ±2.5%; relative ion intensities should not vary more than ±20% for ions with relative intensities >50%, ±25% for ions with relative intensities between 10% and 50%, and ±50% for ions with relative intensities <10%, with respect to a spiked control sample.

Recoveries

Recovery experiments were performed by comparing the analytical results for extracted samples at three concentrations (low 1 ng/mg, medium 10 ng/mg, and high 20 ng/mg) with samples spiked with standards after the extraction procedure that represent 100% recovery.

Results

Specificity

The proposed method demonstrated its specificity for the detection and quantification of methadone and its main metabolite EDDP, including cannabinoids and the most common NPS in hair samples, verifying the absence of peaks that could interfere with the substances of interest.

Limit of detection and limit of quantification

All the analytes investigated were detectable in the range from 0.05 ng/mg to 0.5 ng/mg. The only exception was AM2201 which could be determined at 1 ng/mg [Table 1]b.

Linearity

From calibration curves, built by linear regression of the area ratio of each substance with their IS versus the concentration of analyte, the linearity of the assay was calculated by the method of least squares and expressed as coefficient of determination (R²). The method was linear in the range from LOQ to the highest concentration assessed with quadratic regression coefficients (R²) ranging from 0.9978 to 0.9997. R² is reported for each analyzed substance in [Table 1]b.

Accuracy, precision, and recoveries

Accuracy and precision were expressed, respectively, as %CV and STD, and the results are shown in [Table 1]b. CV% values are lower than 20% for low concentrations and lower than 15% for high concentrations; therefore, according to the guidelines, the method showed acceptable accuracy and precision values. As expected after liquid/liquid extraction, a low matrix effect was observed: recovery percentages were very high (around 100%) for almost all the monitored compounds, except for JWH 019 and JWH 122, as shown in [Table 1]b.

In summary, validation parameters such as LODs, LOQs, repeatability, accuracy, and linearity were satisfactory for its application on real specimens. LODs, LOQs, R%CV, standard deviation, and the mean concentration for the analyzed compounds are reported in [Table 1]b (nominal values of QCs 1, 2, and 3 were 1 ng/mg, 5 ng/mg, and 20 ng/mg, respectively). Accuracy and repeatability were acceptable for all the analytes at their respective LOQs. Recovery experiments varied from 58.3% to 103.0%. [Figure 1] shows an extracted ion chromatogram of the quantifier ion for all the substances investigated.

Figure 1: Extracted ion chromatogram of the quantifier ion for all the substances investigated at a concentration of 20 ng/mg Click here to view

Application to real samples

Since the recent application of the protocol in our laboratory, the described method was applied on 15 authentic specimens from our cases: five showed the presence of methadone and EDDP: for example, the first analyzed hair sample's segments were from a female subject found dead in her apartment from which we were able to collect 15 cm of hair strand. Nine segments (S) of 1 cm circa were prepared and analyzed and showed the following results: the root was positive for methadone at 4.31 ng/mg, 6.42 ng/mg (S-1), 5.47 ng/mg (S-2), 2.51 ng/mg (S-3), 53.19 ng/mg (S-4), 129.57 ng/mg (S-5), 361.08 ng/mg (S-6), 86.87 ng/mg (S-7), and 149.80 ng/mg (S-8), including its metabolite EDDP at 7.98 ng/mg (root), 7.22 ng/mg (S-1), 10.23 ng/mg (S-2), 7.65 ng/mg (S-3), 38.93 ng/mg (S-4), 72.38 ng/mg (S-5), 5 ng/mg (S-6) 23.47 ng/mg (S-7), and 118.47 ng/mg, respectively (S-8). In [Figure 2], chromatogram of authentic postmortem hair sample (S-5) positive for methadone and EDDP is shown at 129.57 ng/mg and 72.38 ng/mg, respectively. The subject had a well-known history of substance abuse and resulted positive to many xenobiotics in biological fluids as well. Results obtained demonstrated the proficiency of the developed method to determine, with a satisfactory sensitivity and sensibility, the drugs of abuse in hair samples involved in the study rapidly and with a simple sample pretreatment.

Figure 2: Chromatograms of an authentic postmortem hair sample positive for methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine at 129.57 ng/mg and 72.38 ng/mg, respectively Click here to view

Discussion

Although a few immunochemical rapid tests can detect few NPS, the gold standard for their detection is chromatography coupled to mass spectrometry. Drugs levels in hair are considerably lower than those found in matrices such as blood or urine; therefore, single or tandem mass spectrometry is employed for confirmation tests. Hair testing analysis provides a retrospective timeframe via segmental analysis, due to the larger detection window when compared with other specimens (up to months, depending on strands lengths); another advantage of hair sampling is low potential for donor manipulation. In the present work, we developed and validated an analytical method to employ either after autopsy or in a population of routine analysis for methadone treatment, showing a particularly high potential for the identification of NPS users. As our study confirms, the method is suitable for analyses of studied compounds; however, there are some limitations hereby discussed. The positivity rate obtained from the study is indeed influenced by the number of samples that we could be able to collect and analyze, although it has to be stressed that these are preliminary results and that more samplings and analyses will be performed; therefore, a significant greater number of authentic cases will be assayed.

In addition, since NPS comprehends a very wide range of substances with different chemical structures, it must be noticed that the application of our analytical procedure might not be suitable for other compounds (i.e., cathinones). In particular, heating the samples as a pretreatment procedure might affect and potentially have a destructive effect on the chemical stability of NPS with low boiling points or different structures, such as mephedrone or other synthetic derivatives.^[22] For example, ester analogs (e.g., PB-22) decompose (or participates in the ester-exchange reactions) in the injection port; another example is that cyclopropyl urinary metabolites (e.g., UR-144) undergo a thermal degradation mainly in GC column.^{[23],[24],[25]} To avoid such issues, when performing splitless injection, an injector temperature of 270°C and a surface deactivated injector liner without glass wool minimizes the degradation and enhances the sensitivity. These results indicate that special attention is required for GC-MS analysis of NPS.^[23] Similar mass spectra are sometimes obtained by GC–MS analyses due to regio- and ring-substituted analogs available on the market: the misidentification of these analogs arises when comparing data only with the available mass spectra. When tandem and high-resolution MS are used to identify the conformational isomers or regioisomers, such misidentification does not occur. Moreover, compounds that are thermally unstable might decompose in the GC injection port, especially those with polar groups (i.e., amino or hydroxyl groups) that can cause a polar interaction with the column stationary phase, leading to poor detection of the analyte. To overcome these problems, derivatization step should be added onto the extraction method for these compounds; however, this step would be time-consuming and potentially toxic. From our validation study, satisfactory results on our set of analytes can be obtained overcoming the derivatization step.

Indeed, synthetic cannabinoids are not ideal compounds for GC-MS analysis because they are neutral to weakly acidic compounds and have high molecular weight. However, concerning the substances taken into consideration in the validation of the presented method, no destructive effect was noticed; therefore, the method is applicable only for the relevant compounds included in this study and more experiments will be performed when other NPS will be included in the procedure. The phenomenon might interest some metabolites that, however, are not the focus of our study, because the parent compounds are the main targets in hair analyses.

Hair analysis for NPS is still at an early stage of development, particularly on the toxicological screening side. The proposed method allows for the identification of synthetic cannabinoids, cannabinoids, and methadone (including its main metabolite) in hair samples with a simple sample pretreatment.

Conclusions

The identification of NPS in biological samples is one of the emerging challenges for forensic laboratories due to the necessity of detection and confirmation of a very large class of substances, often not structurally correlated. Due to the reoccurring threat of synthetic cannabinoids to public health and their rapidly increasing abuse worldwide, it is necessary to develop reliable analytical methods for their detection in different biological matrices. SCs are constantly being modified and rapidly becoming widely available; therefore, laboratories should update their scope for detecting the most prevalent compounds at specific times.

Blood and urine are the first choice of sample for testing; however, hair is often used as an alternative matrix in repeated drug exposure. The method validation presented herein is a straightforward, selective, and accurate method for the determination of some drugs belonging to the CP and aminoalkylindole structural classes. The described method can be useful not only in the forensic investigation of NPS-related addiction histories but also in epidemiological and retrospective studies on the spread of NPS among specific safety-sensitive social workers, such as drivers.

This study has been approved by the local ethics committee and the consent was exempted.