175

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

TOXICOLOGICAL ANALYSIS OF HAIR IN FORENSIC TOXICOLOGY:

APPLICATIONS, LIMITATIONS, AND CLINICAL AND EXPERT

PERSPECTIVES

Sara Luciana de Andrade1

Abstract: Hair toxicological analysis has become an important tool in clinical and forensic toxicolo-

gy due to its ability to detect long-term exposure to drugs of abuse, medications, and other chemical

substances. Unlike conventional biological matrices such as blood, urine, and saliva, hair provides a

wide detection window, allowing the reconstruction of an individual’s history of substance use over

weeks or months. This study aimed to analyze the applications and limitations of hair toxicological

analysis in clinical and forensic investigations, highlighting its relevance for detecting psychoactive

substance use, the analytical methods employed, and the challenges associated with result interpreta-

tion. This is a qualitative, descriptive bibliographic review based on the analysis of scientiﬁ c articles,

books, international guidelines, and technical documents retrieved from national and international

databases. The ﬁ ndings demonstrated that techniques such as Gas Chromatography–Mass Spectro-

metry (GC-MS) and Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) provide

high sensitivity and speciﬁ city, contributing to the reliability of toxicological examinations. However,

factors such as external contamination, hair pigmentation, cosmetic treatments, hair growth rate, and

the lack of universal standardization in result interpretation remain signiﬁ cant limitations to their

application. It is concluded that hair toxicological analysis is a valuable scientiﬁ c tool for clinical and

forensic investigations, provided that results are interpreted comprehensively by considering labora-

tory, clinical, and forensic aspects, together with validated analytical protocols and internationally

recognized technical guidelines.

1 Mestranda pela Universidade Ivy Enber Christian University – E-mail: drasaraandrade@

yahoo.com - ORCID iD: https://orcid.org/0009-0004-6094-502X

176

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

Keywords: Forensic investigation; Forensic toxicology; Hair toxicological analysis; LC-MS/MS; Fo-

rensic sciences.

INTRODUCTION

Forensic toxicology is a multidisciplinary area of Health Sciences that plays a fundamental

role in the investigation of events related to the use of psychoactive substances, medications, poisons

and other chemical agents capable of causing physiological changes or contributing to the occurrence

of crimes, accidents and deaths.

Its work integrates knowledge from toxicology, pharmacology, analytical chemistry, forensic

medicine and molecular biology, allowing the identiﬁ cation, quantiﬁ cation and interpretation of the

presence of substances in different biological matrices.

In this context, toxicological tests represent important tools to support clinical, forensic and

judicial decisions, providing scientiﬁ c evidence that helps in the reconstruction of facts and in the

determination of exposure to chemical compounds (Levine, 2020).

Traditionally, blood, urine and saliva samples are widely used in toxicological investigation

due to the ease of collection and the ability to detect recent drug and medication use. However, these

matrices have limitations related to the short detection period of the substances, making it difﬁ cult to

identify exposures that occurred weeks or months before collection.

In view of this limitation, toxicological analysis in hair has been gaining prominence in

clinical and forensic toxicology because it allows a signiﬁ cantly wider detection window, allowing the

evaluation of patterns of chronic or repetitive consumption of drugs of abuse, medications, and other

chemical substances over time (Cooper; Kronstrand; Kintz, 2019).

Hair has biological characteristics that make it a differentiated matrix for retrospective

investigations. During its growth, substances present in the bloodstream can be incorporated into

the hair structure, remaining stable for months or even years, depending on the length of the sample

177

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

analyzed.

Considering that the average growth of human hair is approximately one centimeter per

month, the segmentation of the strands makes it possible to establish an approximate chronology of

exposure to substances, a characteristic that is especially relevant in criminal investigations, labor

forensics, therapeutic monitoring, abstinence programs, child custody processes, monitoring of drug

users and evaluations related to professional aptitude (Kintz, 2018).

The technological advances observed in recent decades have contributed signiﬁ cantly to the

improvement of capillary toxicological analysis. Highly sensitive and speciﬁ c instrumental techniques,

such as gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography

coupled to tandem mass spectrometry (LC-MS/MS), allow the identiﬁ cation of extremely low

concentrations of different chemical compounds with high analytical precision.

These methods have signiﬁ cantly reduced the occurrence of false-positive and false-negative

results, increasing the reliability of the tests and strengthening their use as scientiﬁ c evidence in court

proceedings and forensic investigations (UNODC, 2023).

Despite the numerous advantages, the interpretation of the results obtained through hair

analysis remains one of the greatest challenges of contemporary forensic toxicology. Several factors

can interfere with the incorporation and permanence of substances in the hair, including individual

characteristics, hair color and type, cosmetic treatments, frequency of exposure, metabolism, route of

drug administration and, especially, the possibility of external contamination.

The environmental deposition of drugs by smoke, dust, or physical contact can lead to

results that require careful laboratory evaluation, making it essential to adopt strict protocols for

decontaminating samples before performing analyses (Society of Hair Testing, 2023).

In addition to the limitations related to external contamination, there are still challenges

regarding the international standardization of reference values, analytical cutoff points, and

interpretative criteria used by different laboratories. These differences can directly inﬂ uence the

clinical and expert interpretation of the results, especially in situations involving criminal liability,

178

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

investigation of suspicious deaths, occupational monitoring, or assessment of abstinence in chemical

dependency treatment programs.

Thus, the interpretation of the tests must consider not only the laboratory results, but also the

clinical context, the individual’s history, the sample’s chain of custody, and other available evidence.

In view of the growing use of toxicological analysis in hair in clinical and forensic practice,

it is necessary to critically discuss its potentialities, limitations and applications.

Thus, the present study aims to analyze, through a review of the scientiﬁ c literature, the

main applications of toxicological analysis in hair in clinical and forensic investigation, addressing its

detection window, the analytical methods employed, especially LC-MS/MS and GC-MS, the factors

that can interfere in the results, including external contamination, as well as the challenges related to

the interpretation of toxicological tests.

It is hoped that this review will contribute to broadening the understanding of this important

diagnostic and forensic tool, highlighting its relevance for the Health Sciences and for the production

of reliable scientiﬁ c evidence in the ﬁ eld of forensic toxicology.

OBJECTIVES

General Objective

To analyze the applications and limitations of toxicological analysis in hair in clinical and

forensic investigation, highlighting its importance as a tool for detecting the use of psychoactive

substances, the analytical methods employed and the challenges related to the interpretation of the

results.

Speciﬁ c Objectives

Describe the principles of toxicological analysis in hair and its use in clinical and forensic

179

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

toxicology.

Discuss the detection window provided by capillary analysis compared to other biological

matrices.

To present the main analytical techniques used in the identiﬁ cation of substances, with

emphasis on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography

coupled to tandem mass spectrometry (LC-MS/MS).

To analyze the limitations of toxicological analysis in hair, especially the risks of external

contamination and the factors that interfere in the incorporation of substances into the hair.

Discuss the challenges related to the interpretation of toxicological results and their application

in clinical practice and forensic investigation.

MATERIALS AND METHODS

The present study is characterized as a bibliographic research, with a qualitative approach

and descriptive nature, developed with the objective of analyzing the applications and limitations of

toxicological analysis in hair in clinical and forensic investigation.

Bibliographic research allows us to gather, organize and interpret scientiﬁ c knowledge

already published, providing a broad understanding of the current state of the investigated theme.

According to Gil (2019), this type of research is based on the systematic analysis of materials already

prepared, enabling the construction of critical discussions based on scientiﬁ c evidence.

To carry out the research, national and international databases recognized in the area of

Health Sciences were consulted, including Scientiﬁ c Electronic Library Online (SciELO), PubMed/

MEDLINE, Virtual Health Library (VHL), Scopus and Google Scholar. Technical documents and

guidelines published by reference organizations, such as the United Nations Ofﬁ ce on Drugs and

Crime (UNODC), the Society of Hair Testing (SoHT) and the European Workplace Drug Testing

Society (EWDTS) were also used, considering their relevance for the standardization of toxicological

180

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

tests and for the interpretation of results in different clinical and expert contexts.

The search strategy was carried out by combining descriptors in Portuguese and English,

using the Boolean operators AND and OR to expand the retrieval of pertinent studies. Among the

main descriptors used are: “forensic toxicology”, “toxicological analysis in hair”, “hair analysis”,

“hair testing”, “forensic toxicology”, “LC-MS/MS”, “GC-MS”, “detection window”, “external

contamination”, “toxicological interpretation” and “biological samples”.

The use of these terms made it possible to identify studies related to the clinical and forensic

applications of hair analysis, the analytical methods employed and the factors that interfere in the

interpretation of toxicological tests.

Inclusion and exclusion criteria were established to ensure the quality and timeliness of

the information analyzed. Scientiﬁ c articles published preferably between 2018 and 2025, literature

reviews, experimental studies, technical guidelines, and ofﬁ cial documents related to forensic

toxicology, toxicological examinations of hair, and instrumental chromatography techniques coupled

to mass spectrometry were included.

Classics widely recognized in the literature were also considered when indispensable for the

conceptual foundation of the theme. Duplicate studies, publications without full text available, studies

that did not directly address toxicological analysis in hair, and documents whose methodology had

limitations that compromised the reliability of the results were excluded.

After selecting the studies, an exploratory reading was carried out, followed by an analytical

and interpretative reading of the included publications. The extracted data were organized in a

thematic way, covering aspects related to the applications of toxicological analysis in hair, the detection

window of the substances, the analytical techniques used, especially gas chromatography coupled

to mass spectrometry (GC-MS) and liquid chromatography coupled to tandem mass spectrometry

(LC-MS/MS), as well as the limitations arising from external contamination, the biological factors

that inﬂ uence the incorporation of substances into the threads and the challenges involved in the

interpretation of laboratory results.

181

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

The data analysis was conducted in a descriptive and critical manner, seeking to compare the

evidence available in the scientiﬁ c literature and identify consensuses, divergences and gaps related

to the use of toxicological analysis in hair in clinical and forensic investigation.

RESULTS AND DISCUSSION

Toxicological analysis in hair and its use in clinical and forensic toxicology

Hair toxicology analysis has established itself as an important tool in clinical and forensic

toxicology because it enables the identiﬁ cation of prolonged exposure to drugs of abuse, medications,

alcohol and other chemical substances. Unlike blood, urine, and saliva, this biological matrix allows

you to evaluate the history of consumption for weeks or months, making it especially useful in forensic

investigations, therapeutic monitoring, and clinical follow-up.

According to Kintz (2018), the main advantage of hair analysis is the wide detection window,

which makes it possible to reconstruct the temporal proﬁ le of exposure to substances and identify

chronic patterns of consumption or withdrawal.

The incorporation of substances into the hair occurs mainly during the anagen phase of hair

growth, when compounds present in the bloodstream reach the hair follicle and remain retained in the

keratin structure. Cooper, Kronstrand and Kintz (2019) explain that this process occurs predominantly

by blood diffusion, although mechanisms related to sweat and sebum can also contribute to the

deposition of analytes.

For Levine (2020), this characteristic differentiates hair from other biological matrices, as it

allows obtaining retrospective information that would be difﬁ cult to identify by blood or urine tests

performed days after exposure.

In clinical practice, capillary analysis has been widely used in the follow-up of patients

undergoing treatment for chemical dependency and in the monitoring of treatment adherence.

Kintz (2023) highlights that the technique makes it possible to identify relapses and monitor the

182

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

evolution of the treatment through segmental analysis of the hair. Musshoff and Madea (2021) add

that this approach allows for the estimation of different periods of consumption, offering important

chronological information for clinical evaluation and therapeutic planning.

In the ﬁ eld of forensic toxicology, hair analysis has high probative value, being used in

investigations of suspicious deaths, trafﬁ c accidents, domestic violence, sexual crimes, legal disputes

related to child custody and monitoring of individuals subjected to legal measures.

According to the United Nations Ofﬁ ce on Drugs and Crime (UNODC, 2023), the chemical

stability of the sample, the ease of storage, and the lower susceptibility to adulteration make this matrix

especially useful in the production of scientiﬁ c evidence. Similarly, the European Workplace Drug

Testing Society (EWDTS, 2022) recognizes its importance in monitoring professionals who perform

risky activities, as long as the results are interpreted in conjunction with clinical and occupational

assessment.

Despite its numerous advantages, the literature highlights that toxicological analysis in

hair has limitations that require careful interpretation. Pragst and Balikova (2006) demonstrated

that factors such as hair pigmentation, cosmetic treatments and environmental contamination can

inﬂ uence the incorporation of substances into the hair.

Corroborating this perspective, Kintz (2023) states that the mere detection of a drug in the

hair does not conﬁ rm, in isolation, the frequency or intensity of consumption, and it is essential to

consider the clinical context, laboratory data, and other evidence of the investigation.

Thus, there is a consensus among the main authors regarding the relevance of toxicological

analysis in hair for clinical and forensic toxicology. As highlighted by Cooper, Kronstrand and Kintz

(2019) and Levine (2020), the reliability of this methodology depends on the use of validated analytical

techniques, the adoption of standardized protocols and the specialized interpretation of the results.

Thus, when used judiciously, capillary analysis is a tool of high diagnostic and expert value,

contributing signiﬁ cantly to the production of scientiﬁ c evidence in the Health Sciences and in forensic

investigation.

183

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

Detection window of capillary analysis and comparison with other biological matrices

The choice of the biological matrix is an essential step in toxicological investigation, as each

type of sample has speciﬁ c characteristics related to the period of detection of the substances, ease

of collection, and clinical or forensic applicability. Among the most used matrices are blood, urine,

saliva and hair, the latter being recognized for its wide detection window, capable of identifying the

consumption of drugs and medications weeks or months after exposure (Levine, 2020).

The detection window corresponds to the period in which a substance remains detectable in

a given biological matrix. According to Kintz (2018), this interval depends on the pharmacokinetic

characteristics of the drug, the frequency of use, the dose administered, the individual metabolism,

and the type of sample analyzed. Thus, the selection of the matrix should consider the purpose of the

examination and the time elapsed since exposure.

Blood is the most suitable matrix to evaluate acute intoxication and the immediate effects

of psychoactive substances. Musshoff and Madea (2021) state that blood concentrations reﬂ ect the

individual’s state at the time of collection, and are widely used in trafﬁ c accidents and medico-

legal investigations. However, Levine (2020) points out that its short detection window limits the

identiﬁ cation of exposures that occurred several days ago.

Urine remains one of the most used matrices in screening and monitoring programs for

drug use. According to the European Workplace Drug Testing Society (EWDTS, 2022), it allows

metabolites to be identiﬁ ed for a period that generally varies between one and seven days. However,

Cooper, Kronstrand and Kintz (2019) point out that this test only conﬁ rms previous exposure, without

indicating the exact moment of consumption or the degree of impairment of the individual.

Saliva, in turn, has a simple and non-invasive collection, and is indicated to detect very

recent consumption. According to the United Nations Ofﬁ ce on Drugs and Crime (UNODC, 2023),

this matrix mainly reﬂ ects the free fraction of substances present in the bloodstream, which is why it

184

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

is often used in trafﬁ c inspections. However, its reduced detection window restricts its application in

retrospective investigations.

In contrast, toxicological analysis on hair allows you to reconstruct the history of consumption

over long periods. Kintz (2023) explains that, considering the average growth of approximately one

centimeter per month, a six-centimeter sample can represent about six months of exposure. In addition,

Cooper, Kronstrand and Kintz (2019) point out that the segmental analysis of the strands makes it

possible to identify different periods of consumption, making this technique especially useful in

therapeutic follow-up and forensic investigations.

Despite these advantages, hair analysis has limitations. Levine (2020) observes that there

is an interval of approximately seven to fourteen days between the consumption of the substance

and its incorporation into the visible segment of the hair, making it impossible to detect very recent

exposures.

In addition, factors such as hair pigmentation, hair growth speed, and cosmetic treatments

can interfere with the concentrations detected. Pragst and Balikova (2006) demonstrated that more

pigmented hair tends to incorporate greater amounts of certain drugs, evidencing the need for careful

interpretation of the results.

According to the Society of Hair Testing (SoHT, 2023), no biological matrix should be

considered superior to the others, as each one meets speciﬁ c objectives. While blood and saliva are

more suitable for identifying recent consumption, urine is widely used in monitoring programs,

and hair stands out for its ability to document prolonged exposure. Thus, the scientiﬁ c literature

demonstrates that capillary analysis does not replace the other matrices, but complements them,

increasing diagnostic accuracy and strengthening clinical and forensic investigation when the results

are interpreted in an integrated manner.

185

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

Analytical methods used: GC-MS and LC-MS/MS in toxicological analysis of hair

Advances in analytical techniques have signiﬁ cantly strengthened forensic toxicology,

allowing the identiﬁ cation of chemical substances in increasingly smaller concentrations and with

high reliability. In toxicological analysis of hair, instrumental methods are fundamental due to the

low concentration of drugs incorporated into the capillary matrix. Among the main techniques used,

gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled

to tandem mass spectrometry (LC-MS/MS) stand out, considered reference methods for analytical

conﬁ rmation in clinical and forensic laboratories (UNODC, 2023).

Before instrumental analysis, the sample undergoes rigorous laboratory preparation.

Cooper, Kronstrand and Kintz (2019) describe that this process involves standardized collection,

decontamination, drying, fragmentation and extraction of the analytes present in the hair. According

to the authors, these steps are essential to ensure the reliability of the results, since environmental

residues can remain adhered to the threads and compromise toxicological interpretation.

In this context, the Society of Hair Testing (SoHT, 2023) recommends speciﬁ c washing

protocols to remove external contaminants without signiﬁ cantly altering the substances incorporated

into the interior of the hair ﬁ ber.

After decontamination, the substances present in the keratin are extracted. Levine (2020) points

out that different digestion and extraction methods can be used, depending on the physicochemical

properties of the analyte and the analytical technique used. Musshoff and Madea (2021) add that

efﬁ cient extraction increases the recovery of compounds and improves the sensitivity of the exams,

making the results more reliable for clinical and forensic applications.

GC-MS remains one of the most consolidated techniques in forensic toxicology. Levine

(2020) explains that this method separates volatile compounds by gas chromatography and performs

their molecular identiﬁ cation by mass spectrometry, presenting high speciﬁ city and reducing the

occurrence of false-positive results. However, Cooper, Kronstrand and Kintz (2019) observe that some

186

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

substances require chemical derivatization before analysis, making the procedure more complex and

limiting its use to certain thermolabile compounds.

On the other hand, LC-MS/MS has expanded its use due to its ability to analyze polar and

thermally unstable molecules without the need for derivatization. According to Kintz (2023), this

technique has high sensitivity, speed, and versatility, allowing the simultaneous detection of different

drugs and their metabolites in a single analysis. Levine (2020) reinforces that this characteristic is

especially important in investigations involving polyuse of psychoactive substances, while UNODC

(2023) highlights its wide applicability in both clinical practice and forensic investigation.

When comparing the two methods, Cooper, Kronstrand and Kintz (2019) state that both have

high performance when correctly validated. However, LC-MS/MS has been gaining more space in

laboratories due to the possibility of multi-residue analyses with less sample preparation and higher

productivity, while GC-MS continues to be considered a standard of excellence for several classes of

drugs.

To ensure the reliability of the results, SoHT (2023) recommends that all methods be

previously validated for selectivity, precision, linearity, limits of detection, and quantiﬁ cation. In

addition, UNODC (2023) emphasizes the importance of laboratories’ participation in quality control

programs and the adoption of international standards, such as ISO/IEC 17025.

Thus, there is a consensus between Levine (2020), Cooper, Kronstrand and Kintz (2019),

Musshoff and Madea (2021), Kintz (2023), SoHT (2023) and UNODC (2023) that GC-MS and LC-

MS/MS constitute the main technologies of modern forensic toxicology. When associated with

standardized laboratory protocols and qualiﬁ ed professionals, these techniques offer high sensitivity,

speciﬁ city, and reliability, contributing to the production of consistent scientiﬁ c evidence and the

safety of clinical and judicial decisions.

187

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

Limitations of toxicological analysis in hair: external contamination and factors that interfere

in the interpretation of the results

Although toxicological analysis in hair represents an important advance in clinical and

forensic toxicology, its use still has limitations that require careful interpretation of the results.

The scientiﬁ c literature demonstrates that several biological, environmental and

methodological factors can inﬂ uence both the incorporation and the permanence of substances in

the hair structure, making any conclusion based exclusively on the qualitative detection of an analyte

inadequate. In this context, the interpretation of the exams must consider not only the laboratory

aspects, but also the individual characteristics of the examinee, the clinical history, the context of the

investigation, and the protocols used during the analysis (Kintz, 2023).

Among the main limitations of hair analysis is the possibility of external contamination.

This phenomenon occurs when particles containing drugs or medications adhere to the surface of the

hair without the consumption of the substance by the individual. According to Cooper, Kronstrand

and Kintz (2019), environments contaminated by illicit drug smoke, industrial dust, aerosols or

direct contact with certain substances can favor this type of surface deposition. Thus, the simple

identiﬁ cation of a chemical compound in hair does not constitute, in isolation, deﬁ nitive evidence of

ingestion or use of the substance.

The concern with environmental contamination has motivated the elaboration of international

protocols aimed at the standardization of toxicological tests. The Society of Hair Testing (SoHT, 2023)

recommends that all samples undergo rigorous decontamination procedures prior to instrumental

analysis, using standardized washing sequences capable of removing external contaminants without

compromising the substances effectively incorporated into the interior of the hair ﬁ ber.

However, Kintz (2023) notes that there is still no universal protocol capable of completely

eliminating all possibilities of contamination, which is why laboratory results should always be

interpreted in an integrated manner.

188

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

In addition to external contamination, another widely discussed factor refers to the biological

characteristics of the hair. Pragst and Balikova (2006), in a study considered a reference in the area,

demonstrated that melanin exerts an important inﬂ uence on the incorporation of several basic drugs,

especially cocaine, amphetamines and opioids. According to the authors, darker hair, because it has a

higher concentration of melanin, tends to retain higher amounts of these substances when compared

to light hair.

This observation continues to be corroborated by more recent studies. Kintz (2023) highlights

that differences related to hair pigmentation can signiﬁ cantly inﬂ uence the concentrations found,

making it necessary to carefully interpret the results obtained.

Another relevant aspect involves the cosmetic treatments performed on the hair. Procedures

such as bleaching, coloring, chemical straightening, perm and frequent use of high temperatures

can alter the structure of keratin and favor the partial loss of previously incorporated substances.

Levine (2020) explains that these structural changes can reduce the concentration of analytes detected

during laboratory tests, increasing the possibility of false-negative results or underestimation of the

intensity of consumption. Cooper, Kronstrand and Kintz (2019) add that the individual’s cosmetic

history should be part of the forensic evaluation, as it directly interferes with the reliability of the

quantitative analysis.

Hair growth also represents an important variable in the chronological interpretation of the

results. Although it is widely accepted that human hair has an average growth of approximately one

centimeter per month, Musshoff and Madea (2021) point out that this speed is inﬂ uenced by genetic

factors, age, sex, nutritional status, systemic diseases, and hormonal conditions.

Consequently, the time estimate obtained by the segmental analysis should be considered

approximate, and it is not possible to establish exact consumption dates solely based on the position

of the substance along the wire.

Another challenge often mentioned in the literature refers to the individual variability

of metabolism. According to Levine (2020), differences related to the absorption, distribution,

189

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

metabolization, and elimination of drugs directly inﬂ uence the amount of substance available for

incorporation into the hair. Thus, individuals subjected to the same consumption pattern may present

different concentrations in the laboratory analysis. This observation is reinforced by Kintz (2023), who

highlights the impossibility of establishing a linear relationship between the concentration detected in

the hair and the amount actually consumed by the individual.

The quantitative interpretation of the exams also deserves special attention. The United

Nations Ofﬁ ce on Drugs and Crime (UNODC, 2023) emphasizes that high concentrations do not

necessarily mean heavier consumption, just as reduced concentrations do not exclude the occurrence

of repeated exposure. Factors related to the type of drug, frequency of use, chemical afﬁ nity for

melanin, hair growth, and laboratory procedures can signiﬁ cantly modify the quantitative results.

Thus, the values obtained must be analyzed together with clinical information, toxicological

history and other available expert evidence.

Another aspect discussed refers to the lack of universal standardization of cut-off values. SoHT

(2023) establishes recommendations for several substances, but different countries and laboratories

still adopt their own interpretation criteria. According to Cooper, Kronstrand and Kintz (2019), this

lack of uniformity can generate divergences between reports issued by different institutions, especially

in legal proceedings in which small analytical differences can inﬂ uence relevant decisions.

The chain of custody of the sample is also an essential factor to ensure the reliability of

toxicological tests. Levine (2020) highlights that failures in identiﬁ cation, collection, packaging,

transportation, or storage can compromise the integrity of the sample and reduce its evidential value.

In this sense, UNODC (2023) recommends that all stages of processing be rigorously documented,

ensuring complete traceability from collection to the issuance of the expert report.

Therefore, it is observed that the limitations of toxicological analysis in hair do not reduce

its importance in clinical and forensic investigation, but highlight the need for multidisciplinary

interpretation of the results. The studies by Levine (2020), Cooper, Kronstrand and Kintz (2019),

Musshoff and Madea (2021), Kintz (2023), as well as the recommendations of SoHT (2023) and

190

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

UNODC (2023), converge in stating that hair examination should be understood as a complementary

tool within the investigative process.

When associated with validated laboratory protocols, high-precision analytical methods,

and careful evaluation of the clinical and forensic context, this test has high potential to contribute

to diagnoses, therapeutic monitoring, and the production of reliable scientiﬁ c evidence in forensic

toxicology.

Challenges in interpreting toxicological results and future prospects

The interpretation of the results obtained through toxicological analysis in hair is one of

the most complex steps of clinical and forensic toxicology. Although technological advances have

provided highly sensitive and speciﬁ c analytical methods, the simple detection of a substance in

the capillary matrix does not allow a conclusion on the frequency, intensity or circumstances of

consumption in isolation.

Thus, the interpretation of the exams requires a multidisciplinary approach, integrating

knowledge of toxicology, pharmacology, forensic medicine, analytical chemistry, and forensic

sciences, in addition to a careful analysis of the clinical and investigative context (Levine, 2020).

One of the main challenges refers to the differentiation between passive exposure and

effective consumption of the substance. According to Kintz (2023), certain drugs can be deposited

on the surface of the hair through environmental smoke, physical contact, or direct manipulation of

the substance, without systemic absorption by the individual. This possibility reinforces the need to

interpret the laboratory results together with clinical information, the history of the examinee, the

circumstances of the collection and other elements of the forensic investigation.

In this sense, Cooper, Kronstrand and Kintz (2019) state that toxicological interpretation

should not be limited to the quantitative data provided by the laboratory. The authors point out that

similar concentrations can represent completely different situations, depending on the frequency

191

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

of consumption, the interval between exposure and collection, the physiological characteristics of

the individual and the factors related to hair growth. Levine (2020) shares this understanding by

highlighting that the laboratory result should be understood as part of a body of evidence and never

as an isolated element to support clinical or judicial decisions.

Another aspect widely discussed in the literature concerns the cut-off values used to interpret

the tests. These values correspond to the minimum concentrations established for a sample to be

considered positive for a given substance. The Society of Hair Testing (SoHT, 2023) recommends

speciﬁ c values for several classes of drugs, seeking to reduce the inﬂ uence of external contamination

and increase the speciﬁ city of exams. However, Kintz (2023) notes that there are still differences

between protocols adopted by different countries and laboratories, which can result in different

interpretations for the same sample.

The United Nations Ofﬁ ce on Drugs and Crime (UNODC, 2023) also points out that the

absence of complete international standardization represents a challenge for the harmonization of

toxicological reports. According to the institution, although several technical recommendations have

been published in recent years, there are still differences related to washing methods, extraction,

analytical validation, detection limits and interpretative criteria. This variability reinforces the need

for continuous development of international standards capable of standardizing laboratory procedures.

Another important challenge refers to the quantitative interpretation of the concentrations

found. Musshoff and Madea (2021) state that there is no direct proportional relationship between the

amount of drug detected in the hair and the degree of clinical impairment presented by the individual.

This is because factors such as metabolism, frequency of use, hair pigmentation, cosmetic treatments,

and hair growth speed can signiﬁ cantly modify the concentrations observed. Thus, high levels do not

necessarily indicate heavy consumption, just as low concentrations do not exclude repetitive use of

the substance.

In addition to biological aspects, technical factors related to laboratory processing can also

interfere in the interpretation of the results. Levine (2020) highlights that failures in collection, storage,

192

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

sample identiﬁ cation, chain of custody, or methodological validation can compromise the reliability of

the tests. For this reason, it is recommended that toxicology laboratories operate in accordance with

international quality assurance systems, performing internal controls, proﬁ ciency tests and periodic

validation of the analytical methods employed.

Technological evolution represents, on the other hand, a promising perspective to minimize

many of the limitations that currently exist. Kintz (2023) points out that the development of high-

resolution mass spectrometry equipment, new chromatographic techniques, and advanced data

processing software has signiﬁ cantly increased the ability to simultaneously identify different

substances present in the capillary matrix. These innovations make it possible to detect compounds

in increasingly smaller concentrations, reducing analytical interference and increasing the robustness

of the results.

Cooper, Kronstrand and Kintz (2019) add that advances in metabolomics, proteomics and

molecular analysis technologies tend to expand the diagnostic potential of hair toxicology in the

coming years. According to the authors, new approaches may make it possible not only to identify the

presence of substances, but also to understand metabolic changes related to the pattern of consumption,

offering more complete information for clinical and forensic applications.

Another important perspective involves the use of artiﬁ cial intelligence and computational

tools to interpret laboratory results. Recent studies indicate that algorithms capable of integrating

clinical, laboratory, and toxicological information can help standardize the interpretation of tests,

reducing the currently existing subjectivity and increasing diagnostic accuracy (UNODC, 2023).

Although this application is still in the development phase, its potential to strengthen forensic

toxicology has been widely recognized by the scientiﬁ c community.

It is observed, therefore, that toxicological analysis in hair represents a consolidated

methodology of high scientiﬁ c value, but its correct use depends on the integration between technology,

technical knowledge and specialized interpretation. The studies by Levine (2020), Cooper, Kronstrand

and Kintz (2019), Musshoff and Madea (2021), Kintz (2023), as well as the recommendations of the

193

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

Society of Hair Testing (2023) and the United Nations Ofﬁ ce on Drugs and Crime (2023), demonstrate

consensus on the need to interpret the tests in a contextualized way, considering biological, analytical

and legal factors.

Thus, the evolution of laboratory techniques, combined with the international standardization

of procedures and the continuous improvement of professionals in the area, tends to further strengthen

the reliability of toxicological analysis in hair, expanding its contribution to clinical investigation,

forensic medicine and the production of scientiﬁ c evidence in the ﬁ eld of forensic toxicology.

FINAL CONSIDERATIONS

Hair toxicology analysis has established itself as an important tool in clinical and forensic

toxicology due to its ability to identify the prolonged consumption of psychoactive substances,

medications and other chemical agents.

Throughout this study, it was found that this biological matrix has signiﬁ cant advantages over

conventional matrices, mainly due to the wide detection window and the possibility of retrospective

reconstruction of the individual’s exposure history. These characteristics expand its applicability in

different contexts, including criminal investigations, therapeutic monitoring, abstinence programs,

labor forensics, and evaluations related to forensic medicine.

The results of the review showed that hair analysis is a complementary method to tests

performed on blood, urine and saliva. While these matrices make it possible to identify recent

exposures and assess immediate pharmacological effects, hair makes it possible to document patterns

of consumption over weeks or months. In this sense, there was a consensus among the authors

analyzed regarding the potential of toxicological analysis in hair to strengthen the production of

scientiﬁ c evidence and support more grounded clinical and judicial decisions.

It was also possible to verify that technological advances, especially with the use of gas

chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography coupled to

194

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

tandem mass spectrometry (LC-MS/MS), contributed signiﬁ cantly to the increase in sensitivity,

speciﬁ city and reliability of toxicological tests. These techniques make it possible to identify multiple

substances at extremely low concentrations, reducing analytical interference and strengthening the

credibility of the reports issued by specialized laboratories.

However, the research also demonstrated that the interpretation of the results remains one of

the greatest challenges of contemporary forensic toxicology. Factors such as external contamination,

individual biological characteristics, hair pigmentation, cosmetic treatments, hair growth speed and

metabolic differences can interfere with the incorporation of substances and modify the concentrations

detected. Thus, the mere presence of an analyte in the hair should not be considered isolated evidence

of consumption, and the integration between laboratory data, clinical history, investigative context

and other expert elements is indispensable.

Another relevant aspect identiﬁ ed refers to the need for greater international standardization

of laboratory procedures and the criteria used to interpret the tests. Although entities such as the Society

of Hair Testing (SoHT), the United Nations Ofﬁ ce on Drugs and Crime (UNODC) and the European

Workplace Drug Testing Society (EWDTS) have published important technical recommendations,

there are still methodological differences between laboratories and countries, which reinforces the

need for constant scientiﬁ c updating and harmonization of the protocols employed.

In view of this evidence, it is concluded that toxicological analysis in hair represents a tool of

high value for the Health Sciences and forensic investigation, as long as it is used judiciously, associated

with validated analytical methods and interpreted by qualiﬁ ed professionals. The continuous evolution

of analytical technologies, combined with the development of increasingly standardized international

protocols, tends to increase the reliability of this methodology and strengthen its application in

clinical, forensic and judicial practice. Finally, it is hoped that this study will contribute to expand

the knowledge about the potentialities and limitations of toxicological analysis in hair, encouraging

new research that promotes the improvement of laboratory techniques and the interpretation of

toxicological results.

195

ISSN: 2763-5724 / Vol. 06 - n 04 - ano 2026

REFERENCES

COOPER, G.; KRONSTRAND, R.; KINTZ, P. Society of Hair Testing guidelines for drug testing in

hair. Forensic Science International, v. 300, p. 34-43, 2019.

EUROPEAN WORKPLACE DRUG TESTING SOCIETY (EWDTS). European Laboratory

Guidelines for Workplace Drug Testing in Hair. London: EWDTS, 2022.

GIL, A. C. Methods and techniques of social research. 7. ed. São Paulo: Atlas, 2019.

KINTZ, P. Analytical and practical aspects of drug testing in hair. Boca Raton: CRC Press, 2018.

KINTZ, P. Hair analysis in clinical and forensic toxicology: an updated review. Forensic Sciences

Research, v. 8, n. 1, p. 1-18, 2023.

LEVINE, B. (Ed.). Principles of Forensic Toxicology. 5. ed. Cham: Springer, 2020.

MUSSHOFF, F.; MADEA, B. Handbook of Analytical Separations: Forensic Toxicology. Amsterdam:

Elsevier, 2021.

PRAGST, F.; BALÍKOVÁ, M. A. State of the art in hair analysis for detection of drug and alcohol

abuse. Clínica Chimica Acta, v. 370, n. 1-2, p. 17-49, 2006.

SOCIETY OF HAIR TESTING (SOHT). Consensus for hair testing in forensic toxicology. Version

2023. Available at: https://www.soht.org. Accessed on: 8 jul. 2026.

UNITED NATIONS OFFICE ON DRUGS AND CRIME (UNODC). Guidance for the validation

of analytical methodology and calibration of equipment used for testing of illicit drugs in seized

materials and biological specimens. Vienna: United Nations, 2023.