Current status of keratinized matrices in Toxicology: Comparison of hair and nails

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REVIEW ARTICLE
Current status of keratinized matrices in Toxicology:
Comparison of hair and nails
M. Cobo-Golpe | A. de-Castro-Ríos | E. Lendoiro
Toxicology Service, Institute of Forensic
Sciences, Santiago de Compostela, Spain
Correspondence
M. Cobo-Golpe, Toxicology Service, Institute
of Forensic Sciences, C/San Francisco, s/n,
15782 Santiago de Compostela, Spain.
Email: m.cobo@usc.es
Abstract
Nails are a keratinized matrix that has been proposed as an alternative to hair to eval-
uate long-term and retrospective consumption of drugs of abuse and pharmaceuti-
cals. This matrix has been gaining interest in recent years, with new studies focusing
on the analysis of fingernails and/or toenails for different substances. However, nails
and hair present differences in structure, growth, and incorporation pathways that
may affect drug incorporation and analysis and complicate the interpretation of the
results. To better understand the results in nail samples, a comparison of concentra-
tions found in hair, fingernails, and toenails has been described in the literature for
some drugs. This review unifies the results found in the literature, with special inter-
est on studies that report paired samples from the same individuals. Differences
between fingernail and toenail samples, as well as proposed cut-offs in nails, are also
discussed. Definite conclusions can be reached for some drugs, but, in general, more
standardized studies are needed to better understand nail results.
KEYWORDS
alternative matrix, hair, nails, review, toxicology
1|INTRODUCTION
Hair and nails are keratinized structures that can be used as biologi-
cal matrices for the detection of endogenous and exogenous sub-
stances. Unlike conventional matrices like blood or urine, the hair
and nails can incorporate and accumulate substances over a long
period of time (weeks to months), offering a wide window of detec-
tion that theoretically makes them useful for many applications in
the context of Clinical and Forensic Toxicology.
1
Hair analysis has
been routinely applied in Toxicology for decades, for the chronologi-
cal study of drug consumption, for driving license regranting,
2
in
drug facilitated crimes,
3
to determine adherence to treatment,
4
or to
detect drug consumption during pregnancy.
5
Nails, however, have
only been studied in a handful of occasions in the past, although in
the last years there has been a growing interest in this matrix.
Despite both being keratinized matrices, hair and nails present some
structural differences that can affect the results of the analysis and
their interpretation.
1.1 |Hair and nail structure
Hair is originated in the hair follicles, where rapidly proliferating matrix
cells (keratinocytes) in the hair bulb produce the hair shaft. The
matrix cells differentiate through a process of keratinization, the for-
mation of disulfide bonds and cellular dehydration, move upwards,
and are compressed into their final shape.
The hair shaft is composed of three concentric layers of cells, the
medulla, cortex, and cuticle. The medulla is the innermost layer and
determines the final diameter of the hair; the cells are keratinized but
loosely bound, with air in between. The cortex is bound to the
medulla and constitutes the bulk of the hair shaft. It is composed of
Received: 25 April 2024 Revised: 28 May 2024 Accepted: 29 May 2024
DOI: 10.1002/dta.3748
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© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.
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long keratinized cells bound into long fibers by the tegument.
Between the cells in the cortex, there are very small air spaces called
fusi. Interspersed among the matrix cells are the melanocytes, which
produce the pigment melanin. The outermost layer is the cuticle. It
covers the other two and is strongly bound to the cortex. It consists
of five to seven layers of long, overlapping cells that anchor the hair
to the follicle and protect the internal fibers. However, the cuticle can
be penetrated by aqueous solutions and be damaged by chemical
products, heat, light, or mechanical injury.
Nails are originated in the nail matrix and attached to the nail
bed. The nail plate is formed as matrix cells become larger and paler,
and eventually the nucleus disintegrates. Melanocytes are also pre-
sent in the nail matrix
6
in two compartments, the first has quiescent
melanocytes unable to synthesize melanin under normal conditions,
and the second is of functionally differentiated melanocytes. Never-
theless, their density is low (217 mm
2
) compared with that of the
epidermis.
7
In Caucasian nails, the matrix melanocytes contain pre-
melanosomes and melanosomes I and II with little or no active syn-
thesis of mature melanosomes.
6
The melanocytes in Japanese
people contain all gradation of maturing melanosomes, and in Black
subjects, most of the melanosomes are mature.
7
When the normally
dormant matrix melanocytes are activated, melanin is deposited in
the growing nail plate resulting in a pigmented band.
8
This is known
as longitudinal melanonychia. Longitudinal melanonychia is a benign
phenomenon found particularly in Afro-Caribbeans
9,10
and Japanese
populations,
11
while in Caucasians, it has a higher chance of being
malignant.
12
The nail plate gains thickness and density as it grows distally. The
length of the matrix seems to influence the thickness of the nail plate,
along with other factors such as the linear rate of nail growth,
13
vas-
cular supply, subungual hyperkeratosis, and the use of drugs.
Three regions of the nail plate have been defined. The dorsal
plate is physically hard and has little acid phosphatase activity. It has a
high calcium, phospholipid, and sulfhydryl group content. The inter-
mediate nail plate has a high acid phosphatase activity, a high number
of disulfide bonds, and a low content of bound sulfhydryl groups,
phospholipid, and calcium. The existence of a ventral plate is more
controversial. Jarrett and Spearman
14
define the ventral plate as a
layer one or two cells thick, with the same high content in calcium,
phospholipid, and sulfhydryl groups as the dorsal plate, and high acid
phosphatase activity and disulfide bonds as the intermediate plate,
while Jemec and Serup
15
suggest the nail plate being a bilamellar
structure without a ventral layer. The nail can be compared in some
aspects to a hair follicle, sectioned longitudinally, and laid on its side.
The hair bulb is considered analogous to the intermediate nail matrix
and the cortex to the nail plate.
1.2 |Hair and nail growth
Hair growth is not continuous but asynchronous; that is, it grows in
cycles. Moreover, human hair grows in a mosaic pattern; that is, each
individual follicle follows a growth cycle independently of the
surrounding hair follicles.
16
The growth cycle of a hair follicle consists
of four phases: a growing phase (anagen), a regression phase (cata-
gen), a resting phase (telogen), and a shedding phase (exogen).
17
Hair
grows at a different rate depending on the body site. In head hair, a
range of 0.6 to 3.36 cm/month has been observed, although a mean
of 1 cm/month is accepted at the posterior vertex zone (Figure S1).
Unlike hair, nails grow continuously and in two directions: 80% of
the growth is longitudinal, from the nail matrix, and 20% is growth in
thickness from the nail bed upwards. There are no significant growth
rate differences between right and left fingernail/toenails,
18,19
although growth rates vary from finger to finger, with longer fingers
showing a faster rate. In general, an average fingernail growth rate of
3 mm/month is accepted, about twice that of toenails
(1.5 mm/month).
20
Thickening is constant but slow, with a mean value
of 0.027 mm/month
20
(Figure S2).
1.3 |Drug incorporation into hair and nails
Substances are incorporated into the hair following a multicompart-
ment model. The principal mechanism of incorporation is through pas-
sive diffusion of the substances from the blood irrigating the dermal
papilla into the cells of the hair follicle, remaining trapped after kerato-
genesis, but there is also incorporation from sweat and sebum after
hair formation, and from the external environment after the hair has
emerged from the skin.
Passive diffusion is influenced by two factors. One is the liposolu-
bility of the molecules: the more lipophilic, the easier the entrance
through the membranes to the inside of the cells. The other is the pKa
of the molecules and the cellular pH: nonionized molecules diffuse
more easily through the membrane, and because the pH in the inside
of melanocytes and keratinocytes is more acidic than the plasma
(pH =3–6 in the cells versus pH =7.3 in the plasma), basic molecules
can enter the cells more easily. It has been observed that drug incor-
poration might be augmented by binding to components in the hair
cells, especially to melanin,
21,22
although this is not the only mecha-
nism, because in albino animals there is also some incorporation of
drugs to hair.
23
Another augmenting mechanism might be the binding
to sulfhydryl-containing amino acids present in hair, like cysteine,
especially for divalent cations that can form covalent bonds.
24
The
model of passive diffusion assumes that the amount of drug incorpo-
rated depends on blood concentrations, which depend on the dose
ingested. This model is also the basis for segmental analysis. Because
hair is assumed to grow at a constant rate, drug disposition along the
hair shaft can be correlated with the moment the drug was present in
the blood flow.
On the other hand, the porous nature of the hair allows it to
absorb liquids, increasing its weight and incorporating substances pre-
sent in the sweat and sebum that are secreted as the hair grows. The
free and nonionized drugs diffuse from blood to sweat. The pH differ-
ence between sweat (pH =5.8) and blood (pH =7.3) facilitates the
passive diffusion of basic molecules from blood to sweat, where they
are ionized, thus blocking their return to the blood.
25
Sweat and
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sebum incorporation is especially relevant in the case of drugs of
abuse, which are found in these secretions at high concentrations,
26
and the high interindividual variability in their secretion can explain
differences in concentrations in people receiving the same dose, as
well as the incorporation of drugs along the hair shaft, not corre-
sponding to the times of administration.
26
These secretions are bound
less tightly, because they occur after hair formation, so they should be
easier to remove by washing the hair before the analysis. External
contamination from air, water, or dust has been proposed as an incor-
poration path for trace elements found in hair, and it is potentially
important in the case of smoked drugs such as marijuana, cocaine, and
heroin.
27
If the hair is exposed to high concentrations of these con-
taminants, the molecules can be incorporated to the external layers of
the hair. This exposition is not indicative of an active consumption
of the drugs, and it should be removed by a washing procedure before
the analysis to avoid false positive results.
Likewise, drugs are primarily incorporated into nails through diffu-
sion from the blood flow, but unlike hair, nails grow in two different
directions, and substances can be incorporated both from the germi-
nal matrix and from the nail bed. Because 80% of the nail is formed in
the germinal matrix, most of the incorporation occurs longitudinally,
but there is also some incorporation throughout the nail bed and from
sweat. Two studies about the incorporation of zolpidem in nails have
found evidence of these three incorporation paths.
28,29
As with hair,
incorporation into nails through external contamination, especially in
fingernails due to drug handling, should be considered as a possible
incorporation pathway.
The differences in the mechanism of drug incorporation in hair
and nails must be taken into account for the interpretation of the
results.
20
Because in the posterior vertex zone of the head hair
growth is more constant, long hair samples can be divided into sec-
tions where each centimeter corresponds to 1 month of hair
growth.
26
Segmentation has been studied in whole nails
30
and in
one case with nail clippings,
31
but very sensitive instrumentation is
needed to detect the very low concentrations present in these seg-
ments. Moreover, even if nails could be reliably divided into seg-
ments, the interpretation of the results is complex due to the double
path of substance incorporation. As for structural differences, one is
the presence of melanin in hair, known to influence the incorpora-
tion of certain substances depending on their physicochemical prop-
erties.
32
In Caucasians, melanin is not present in nails under normal
circumstances, avoiding the bias due to pigmentation.
33
In other
populations, however, the presence of melanin is more common, and
its influence in the incorporation of substances has not been studied
yet. The other difference is the absence of a cuticle layer in nails that
can act as a barrier to substance incorporation and extraction. This
means that nail samples are more susceptible to contamination from
drug handling and to wash-out effects due to frequent
handwashing.
34
All these differences suggest that concentrations in hair and nail
samples from the same individual could be very different, and cut-off
concentrations established for hair to determine chronic consumption
might not be appropriate for nails.
Nowadays, there are still few published methods for drug detec-
tion in nail samples, and even fewer publications have tackled the
direct comparison of both matrices. Two reviews summarize the pub-
lished methodologies for nail analysis
35
and the usefulness of nail
analysis in Forensic Toxicology.
20
The present review will focus on
the studies that analyze and compare drug concentrations in nail and
hair samples, with special focus on paired samples, to try to elucidate
the relationship between both keratinized matrices. Moreover, given
the differences in growth rate and contamination pathways of finger-
nail and toenail samples, studies assessing the comparison between
fingernails and toenails will also be reviewed.
2|MATERIALS AND METHODS
A literature search (1980–2022) was performed using PubMed and
Web of Science using combinations of the search terms nail, hair,
drug, abuse, pharmaceutical, toxicology, and detection. The identified
articles were reviewed and publications including the detection of
substances in both nail and hair samples were selected. Only articles
written in English were included.
3|RESULTS
3.1 |Comparison of concentrations in nail and hair
samples
A total of 29 publications were found that compare concentrations
between nail and hair samples (Table 1). The results are separated by
compound class and described in detail in the following sections.
3.1.1 | Amphetamine derivatives
Amphetamine derivatives have been studied in six publications
36–41
with varying results. In the first study, amphetamine and methamphet-
amine were analyzed in samples from nine suspects of drug abuse,
finding similar mean concentrations in hair and nails; moreover, in
paired samples, the matrix with the highest concentration was vari-
able.
36
Another study
37
analyzed 12 paired hair and nail samples from
regular users and found the same variability for these drugs. Cirimele
et al.
38
analyzed hair and fingernails from one drug user and found
concentrations of amphetamine, MDA and MDMA slightly higher in
fingernails than in hair. Lin et al.
39
analyzed fingernails from 97 drug
users, finding 62 positive cases for amphetamine and/or methamphet-
amine. From six of the positive subjects, they collected paired nail and
hair samples and found concentrations in nail clippings lower than in
the corresponding 1.5 cm hair samples for both drugs. Madry et al.
40
analyzed hair and nail samples from 15 subjects after the administra-
tion of two doses of MDMA. Concentrations for MDMA and MDA
were higher in hair in most samples, and similar or lower in two and
three, respectively. Finally, Cappelle et al.
41
analyzed hair, fingernails,
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TABLE 1 Summary of published methods comparing concentrations in hair and nail samples.
Reference Analyte Samples Paired Comparison
Amphetamines
Suzuki 1984
36
AMP, MAMP Hair (n=7)
Fingernail (n=8)
Toenail (n=4)
Yes Hair nails
Suzuki 1989
37
AMP, MAMP Hair (n=15)
Nail (n=20)
Yes Hair > nails (n=4/9) for MAMP
Hair > nails (n=2/3) for AMP
Cirimele
1995
38
AMP, MDA, MDMA Hair (n=1)
Fingernail (n=1)
Yes Fingernails > Hair
Lin 2004
39
AMP, MAMP Hair (n=6)
Fingernail (n=97)
Yes Hair > fingernails
Madry 2016
40
MDA
MDMA
Hair (n=13)
Fingernail (n=15)
Toenail (n=3)
Yes Hair (5 cm) > nail
Hair nail in n=2
Cappelle
2018
41
AMP, MAMP, MDMA, MDEA Hair (n=26)
Fingernail (n=24)
Toenail (n=18)
Yes Nails > hair
Antidepressants and benzodiazepines
Irving 2007
42
ALP, CLOB, CLON, DIA, MID,
OXA, TEM, TRIAZ, ZOP
Hair (n=21)
Nail (n=21)
Yes Hair nails
Hang 2013
28
ZOL Hair (n=7)
Fingernail (n=7)
Yes Hair > nails
Madry
2014a
29
ZOL Hair (n=9)
Fingernail (n=9)
Yes Hair > nails
Cobo-Golpe
2021a
34
VENL, TRAZ, CITA, PARO, FLUO, SERT,
ZOL, OXA, ALP, LORA, NDIA, DIA
Hair (n=16)
Fingernail (n=16)
Toenail (n=17)
Yes Hair > nails if active treatment,
except for DIA
Antipsychotics
Uematsu
1989
43
HALO Hair (n=40)
Nail (n=20)
Yes Hair > nails
Uematsu
1990
23
HALO Hair (n=20)
Nail (n=20)
Yes Hair > nails
Chen 2012
44
CLOZ Hair (n=16)
Nail (n=16)
Yes Hair > nails
Chen 2014
45
CLOZ Hair, fingernails, toenails from
cadaver case (n=1)
Yes Hair > nails
Cobo-Golpe
2020
46
QUET, HALO, LMZ, CLOZ, OLAN Hair (n=13)
Fingernail (n=13)
Toenail (n=13)
Yes Hair > nails, except for OLAN
Cannabis
Jones 2013
47
THCCOOH Hair (n=60)
Nail (n=60)
Yes Nails > hair
Cobo-Golpe
2021b
48
CBN, CBD, THC Hair (n=17)
Fingernail (n=22)
Toenail (n=19)
Yes Fingernails > hair
Hair > toenails
Cocaine and opioids
Ropero-Miller
2000
49
AEME, BE, COC, CE, EEE, EME, NBE,
NCOC, 6-AM, COD, MOR, NCOD, NMOR
Hair (n=8)
Fingernail (n=8)
Yes Hair > nails
Cingolani
2004
50
MOR, 6-AM, COC Hair (n=18)
Toenail (n=18)
Yes Toenail > hair for COC, MOR
Toenail hair for 6-AM
Shen 2014
51
6-AM, MOR, COD, AC, heroin Hair (n=18)
Fingernail (n=18)
Yes Hair > nails for 6-AM, AC, COD
Nail > hair for MOR
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and toenails from 26 patients in treatment for drug abuse and found
higher concentrations of amphetamine, MDMA, and MDEA in nails
than in hair.
3.1.2 | Cannabinoids
Two publications detected cannabinoids in paired hair and nail sam-
ples.
47,48
Cobo-Golpe et al.
48
analyzed paired fingernail, toenail, and
hair samples from 23 chronic cannabis users and found THC, CBN,
and CBD concentrations 4.9 to 21.2 times higher in fingernails than in
hair. Conversely, concentrations in toenails were lower than in hair.
Jones et al.
47
analyzed paired samples from 60 subjects and found
THCCOOH concentrations in fingernails 4.3 times higher than in hair.
Because THCCOOH is an endogenous metabolite of THC, the higher
concentrations in fingernails cannot be attributed to external contami-
nation but rather to a greater accumulation in nails over time, consid-
ering the slower growth of nails compared with hair.
3.1.3 | Cocaine
For cocaine, four publications compare concentrations in hair and
nails.
33,41,49,50
Three of them found higher concentrations in hair than
nails,
31,41,49
and one
50
found higher concentrations in nails. Ropero-
Miller et al.
49
analyzed hair and fingernail scrapings from eight partici-
pants in an in-patient study. Participants received alternating low
doses of cocaine and codeine for 6 days, placebo for 2 weeks and a
high dose for another week. The analytes were detected in the nail
washing solution, but not in the washed nail scrapings. Considering
the total drug detected (sum of sample concentration and washes
concentration), the maximum concentrations were five to 30 times
higher in hair than nails. Madry et al.
33
analyzed paired hair (1.5–6 cm)
and toenail samples from cocaine users. Cocaine concentrations in
hair were two to 42 times higher than toenail clipping concentrations
(median =16 times); however, the sum of metabolites (benzoylecgo-
nine [BE], norcocaine, and cocaethylene) hair concentrations were
only 0.6 to nine times higher than toenails (median =2.5 times).
TABLE 1 (Continued)
Reference Analyte Samples Paired Comparison
Madry
2014b
33
COC, BE, NCOC, CE Hair (n=20)
Toenail (n=20)
Yes Hair > toenails
Cappelle
2018
41
COD, MOR, 6-AM, MTD, EDDP,
COC, BE, EME
Hair (n=26)
Fingernail (n=24)
Toenail (n=18)
Yes Nails > hair
Hair > nails for COC
Tzatzarakis
2015
52
BUP, NBUP Hair (n=46)
Fingernail (n=46)
Yes Nails > hair for BUP
Hair nails for NBUP
EtG
Jones 2012
53
EtG Hair (n=570)
Fingernail (n=561)
Yes Nails > hair
Cappelle
2017
54
EtG Hair (n=45)
Fingernail (n=41)
Toenail (n=13)
Yes Nails > hair
Fosen 2017
55
EtG Hair(n=40) Fingernail (n=40) Yes Nails > hair
Paul 2019
56
EtG Hair (n=50)
Fingernail (n=50)
Yes Nails > hair in paired positive samples
Hair nails considering all samples
Other
Faergemann
1993
57
Terbinafine Hair (n=12)
Nail (n=12)
Yes Hair > nails
Kim 2020
58
COT, 3-HCOT Hair (n=26)
Nail (n=26)
Yes Hair > nails
Krumbiegel
2016
30
76 drugs Hair (n=7)
Nail (n=7)
Postmortem cases
Yes Concentrations not comparable
Segmental analysis of nails showed
different drug concentration per
segment
Abbreviations: AC, acetyl codeine; AEME, anhydroecgonine methyl ester; ALP, alprazolam; AMP, amphetamine; BE, benzoylecgonine; BUP,
buprenorphine; CBD, cannabidiol; CBN, cannabinol; CE, cocaethylene; CITA, citalopram; CLOB, clobazam; CLON, clonazepam; CLOZ, clozapine; Cmax,
maximum concentration; COC, cocaine; COT, cotinine; DIA, diazepam; EDDP: 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EEE, ecgonine ethyl
ester; EME, ecgonine methyl ester; EtG, ethylglucuronide; HALO, haloperidol; LMZ, levomepromazine; LORA, lorazepam; MDA,
3,4-methylendioxiamphetamine; MDEA, 3,4-methylenedioxyethylamphetamine; MDMA, 3,4-methylendioximethamphetamine; MID, midazolam; MOR,
morphine; MTD, methadone; NAMP, noramphetamine; NBE, norbenzoylecgonine; NBUP, norbuprenorphine; NCOC, norcocaine; NCOD, norcodeine;
NDIA, nordiazepam; NMOR, normorphine; OLAN, olanzapine; OXA, oxazepam; QUET, quetiapine; TEM, temazepam; THCCOOH, tetrahydrocannabinolic
acid; THC, tetrahydrocannabinol; TRIAZ, triazolam; ZOL, zolpidem; ZOP, zopiclone; 3-HCOT, 3-hydroxycotinine; 6-AM, 6-acetylmorphine.
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Higher concentrations in hair were attributed to melanin binding and
external contamination in the case of the parent compound. Cappelle
et al.
41
found higher concentrations in hair for cocaine, but concentra-
tions for its metabolites BE and ecgonine methyl ester (EME) were
higher in nails (n=26). Cingolani et al.
50
detected cocaine in hair and
toenail samples from 18 autopsies of drug abusers. Entire nails were
removed from the big toe, and the proximal portion of both nails and
hair was used for the analysis. Mean concentrations of cocaine in toe-
nails were twice those in hair.
3.1.4 | Cotinine
Kim et al.
58
analyzed hair and nail samples from 26 infants to deter-
mine second-hand tobacco exposure. They detected the nicotine
metabolites cotinine and hydroxycotinine in eight and two paired
samples, respectively. Concentrations of both analytes were higher in
hair than in nails.
3.1.5 | Ethylglucuronide
Ethylglucuronide (EtG) concentrations were compared in four
publications.
53–56
All of them found higher concentrations in nails (1.5
to five times higher, depending on the study). Jones et al.
53
analyzed
paired hair and fingernails of 529 participants. EtG concentrations were
higher in fingernails, with mean concentration three times higher than
in hair. Cappelle et al.
54
analyzed samples from 45 patients in treatment
for alcohol dependency. Of these, 41 had paired fingernail-hair samples
and 13 had paired toenail-hair samples. Median concentrations in nail
samples were higher than in hair (1.5 and 4.2 times higher in fingernails
and toenails, respectively). Fosen et al.
55
analyzed paired hair and nails
from 40 patients of an alcohol rehabilitation clinic. Median concentra-
tions of EtG were five times higher in nails than hair. Finally, Paul
et al.
56
analyzed paired hair and fingernails from 50 participants. The
mean EtG concentrations in hair and fingernails were similar when con-
sidering all cases, but for those positive in both matrices (n=21), EtG
concentrations were 1.5 times higher in nail samples.
3.1.6 | Opioids
There are five publications comparing opioid compounds in both matri-
ces.
41,49–52
Morphine was detected in three of them, all at higher con-
centrations in nails
41,50,51
; codeine was detected in higher
concentrations in hair in two publications,
49,51
and lower in another
41
;
and 6-acetylmorphine (6-AM) was detected in similar concentrations in
both matrices in one study,
50
higher in hair in another,
51
and lower
in the last one.
41
Specifically, Shen et al.
51
analyzed fingernail and hair
samples from 18 subjects whose urine had tested positive for mor-
phine. Morphine concentrations were higher in nails, while 6-AM,
codeine, and acetylcodeine were higher in hair. Cingolani et al.
50
found
mean concentrations of morphine 1.6 times higher in toenails than hair,
but mean concentrations of 6-AM were similar in both matrices. Cap-
pelle et al.
41
detected morphine, 6-AM, codeine, methadone, and its
metabolite EDDP (2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine)
and found higher concentrations in nails (fingernails and toenails) than
hair. Buprenorphine was studied only by Tzatzarakis et al.,
52
who ana-
lyzed samples from 46 patients and found higher median concentra-
tions of buprenorphine in fingernails than hair, but similar
concentrations in both matrices for its metabolite norbuprenorphine.
3.1.7 | Antidepressants and benzodiazepines
There are five publications comparing antidepressants or benzodiaze-
pines in nails and hair. Two of them focused on the detection of zolpi-
dem after the administration of a single dose.
28,29
Madry et al.
29
investigated concentrations in paired hair and fingernail samples from
nine volunteers. Comparing concentrations in the segments corre-
sponding to incorporation at the moment of administration, they found
concentrations two to 10 times higher in hair than nails. Hang et al.
28
studied zolpidem concentrations in paired fingernails and hair from
seven subjects and found that hair concentrations were around 1000
times higher. The other three publications included multiple drugs.
Krumbiegel et al.
30
investigated 76 substances in hair and nails from
seven postmortem cases. The nail and hair samples were segmented
differently in each case, making it difficult to compare concentrations in
the two matrices. The analytes of interest detected in these cases were
zolpidem, diazepam, nordiazepam, and oxazepam; the first three were
found in higher concentrations in hair, whereas for oxazepam, results
varied. Other drugs of abuse and pharmaceuticals were detected in the
samples, with high variability in the results between sample segments
and individual cases. Irving and Dickson
42
detected alprazolam, cloba-
zam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazo-
lam, zopiclone, and some metabolites in hair, fingernails, and toenails of
21 patients that had been prescribed any of the drugs. In general, con-
centrations were similar in hair and nail samples, but for each drug, a
pattern could usually be observed. For example, for N-desmethyl cloba-
zam, diazepam, nordiazepam, and triazolam, concentrations were higher
in nails, while for midazolam, temazepam, and zopiclone, concentrations
were higher in hair. Cobo-Golpe et al. described hair and nail concentra-
tions for 12 drugs, namely, venlafaxine, trazodone, citalopram, paroxe-
tine, fluoxetine, sertraline, zolpidem, oxazepam, alprazolam, lorazepam,
nordiazepam, and diazepam,
34
although comparison between the two
matrices was only possible for some of them. Hair concentrations were
higher than nail concentrations in patients under active treatment with
trazodone, sertraline, alprazolam, zolpidem, venlafaxine, paroxetine, and
fluoxetine. Diazepam was the only exception, with higher concentra-
tions in nails in the only case available.
3.1.8 | Antipsychotics
Five publications show antipsychotic drugs concentrations in nail and
hair samples. Uematsu et al.
23,43
found much higher concentrations of
6COBO-GOLPE ET AL.
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TABLE 2 Summary of published methods comparing concentrations in fingernail and toenail samples.
Reference Analyte Samples Paired Comparison
Amphetamines
Suzuki
1984
36
AMP, MAMP Fingernail (n=8)
Toenail (n=4)
Yes Toenails > fingernails
Madry
2016
40
MDA
MDMA
Fingernail (n=15)
Toenail (n=3)
Yes Fingernails toenails
Cappelle
2018
41
AMP, MAMP, MDMA, MDEA Fingernail (n=24)
Toenail (n=18)
Yes Toenails > fingernails for AMP, MDMA
Shu 2015
59
AMP, MAMP >10,000 cases No Fingernails toenails
(Median concentrations)
Antidepressants and benzodiazepines
Hang 2013
28
ZOL Fingernail (n=7)
Toenail (n=4)
Yes Toenails > fingernails
Cobo-Golpe
2021a
34
VENL, TRAZ, CITA, PARO, FLUO, SERT,
ZOL, OXA, ALP, LORA, NDIA, DIA
Fingernail (n=16)
Toenail (n=17)
Yes Toenails fingernails for ZOL, VENL
Toenails > or fingernails for ALP,
TRAZ
Toenails > fingernails for FLUO, SERT
Shu 2015
59
ALP, DIA, NDIA >10,000 cases No Fingernails toenails
(Median concentrations)
Antipsychotics
Chen 2014
45
CLOZ Fingernails, toenails from
cadaver case (n=1)
Yes Fingernails > toenails
Cobo-Golpe
2020
46
QUET, HALO, LMZ, CLOZ, OLAN Fingernail (n=13)
Toenail (n=12)
Yes Toenails > or fingernails depending
on the participant
Cannabis
Cobo-Golpe
2021b
48
CBD, CBN, THC Fingernail (n=22)
Toenail (n=19)
Yes Fingernails > toenails
(Median concentrations)
Shu 2015
59
THCCOOH >10,000 cases No Fingernails toenails
(Median concentrations)
Cocaine and opioids
Garside
1998
60
COC, BE Fingernail (n=14)
Toenail (n=14) Postmortem
cases
Yes Fingernails > toenails for COC
Fingernails > or toenails for BE
Engelhart
2002
61
COC, BE, EME, NCOC, CE, MOR, 6-AM,
COD
Fingernail (n=17)
Toenail (n=17) Postmortem
cases
Yes Fingernails > toenails
Fingernails toenails for COD
Cappelle
2018
41
COC, BE, EME, COD, MOR, 6-AM, MTD,
EDDP
Fingernail (n=24)
Toenail (n=18)
Yes Fingernails > toenails
Toenails > fingernails for COD, MTD
Shu 2015
59
COC, BE, NCOC, CE, 6-AM, MOR, COD,
HCOD, HYM, MTD, EDDP, OXC, OXM
>10,000 cases No Fingernails > toenails
EtG
Cappelle
2017
54
EtG Fingernail (n=41)
Toenail (n=13)
Yes Toenails > fingernails
(Mean concentrations)
Shu 2015
59
EtG >10,000 cases No Fingernails > toenails
(Mean and median concentrations)
Other
Jenkins
2006
62
PCP Fingernail (n=4)
Toenail (n=4) Postmortem
cases
Yes Fingernails > toenails
Abbreviations: ALP, alprazolam; AMP, amphetamine; BE, benzoylecgonine; CBD, cannabidiol; CBN, cannabinol; CE, cocaethylene; CITA, citalopram; CLOZ,
clozapine; Cmax, maximum concentration; COD, codeine; COC, cocaine; DIA, diazepam; EDDP, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine; EME,
ecgonine methyl ester; EtG, ethylglucuronide; HALO, haloperidol; HCOD, hydrocodone; HYM, hydromorphone; LMZ, levomepromazine; LORA,
lorazepam; MDA, 3,4-methylendioxiamphetamine; MDEA, 3,4-methylenedioxyethylamphetamine; MDMA, 3,4-methylendioximethamphetamine; MOR,
morphine; MTD, methadone; NCOC, norcocaine; NDIA, nordiazepam; OLAN, olanzapine; OXA, oxazepam; OXC, oxycodone; OXM, oxymorphone; PCP,
phencyclidine; QUET, quetiapine; THCCOOH, tetrahydrocannabinolic acid; THC, tetrahydrocannabinol; ZOL, zolpidem; 6-AM, 6-acetylmorphine.
COBO-GOLPE ET AL.7
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haloperidol in hair than nails (concentrations in nails about 3%–4%
those in hair) in two different studies when comparing paired samples
from 20 patients after receiving a fixed daily dose for more than a
month. Chen et al. found nail concentrations about 5%–10% those in
hair, after analyzing hair and fingernail samples from 16 psychiatric
patients
44
and in one case of a drowned cadaver.
45
More recently,
Cobo-Golpe et al.
46
published an article detecting five antipsychotic
drugs (quetiapine, haloperidol, levomepromazine, clozapine, and olan-
zapine) in hair and nail samples from psychiatric patients under
chronic treatment. Hair concentrations of quetiapine, haloperidol,
levomepromazine, and clozapine were higher than nail concentrations,
whereas results for olanzapine were very variable. In general concen-
trations of antipsychotics were higher in hair, most likely due to mela-
nin binding in hair samples.
3.1.9 | Terbinafine
Faergemann et al.
57
measured terbinafine concentrations in paired
hair and toenails from 12 participants during and after ingesting a
daily dose for 28 days. Mean concentrations of terbinafine were 2.3
to 9.6 times higher in hair than nails during the study.
3.2 |Comparison of concentrations in fingernail
and toenail samples
Fingernail and toenail samples differ in growth rates and possible con-
tamination pathways. This could lead to different concentrations in
both samples and should be taken into account when choosing the
type of nail sample to analyze. Thirteen publications describe
the comparison between fingernail and toenail concentrations
(Table 2), and the results are detailed in the following sections.
3.2.1 | Amphetamine derivatives
Amphetamine derivatives were compared in fingernail and toenail in
four studies,
36,40,41,59
finding discrepant results. Madry et al.
40
found
similar MDA and MDMA concentrations in three toenail samples
compared with those in 13 fingernail samples, with one outlier that
was attributed to a past consumption of MDMA more than 2 months
before the study. Shu et al.
59
analyzed nail samples from more than
10,000 high-risk cases, detecting 52 different drugs of abuse. The
median concentrations for amphetamines were also similar in both
nail samples. However, Suzuki et al.
36
found higher concentrations for
amphetamine and methamphetamine in toenails than fingernails in
nine subjects. Similarly, Cappelle et al.
41
found concentrations of
amphetamine and MDMA higher in toenails than fingernails in
26 patients in treatment for drug abuse.
3.2.2 | Cannabinoids
Only two studies compared concentrations in fingernail and toenail
samples. Cobo-Golpe et al.
48
analyzed paired fingernail and
toenail samples from 23 chronic cannabis consumers and found much
higher concentrations in fingernails than in toenails (eight to 29 times
higher) for THC, CBD, and CBN. Shu et al.
59
found similar THCCOOH
concentrations in fingernails and in toenails, although due to the high
number of samples, the authors did not compare paired samples but
the range and median of concentrations.
3.2.3 | Cocaine and opioids
Cocaine
59–61
and opioids
41,59,61
concentrations in nail samples were
compared in three studies each.
For cocaine and opioids, fingernails tend to have higher concen-
trations than toenails, but with some exceptions. Specifically, Gar-
side et al.
60
analyzed paired fingernail and toenail samples from
14 postmortem cases of suspected cocaine users. Cocaine and BE
were the analytes predominantly detected; cocaine concentrations
were higher in fingernails than toenails, with some samples being
positive in fingernails while negative in toenails. BE was also higher
in fingernails in some cases, but similar concentrations were found in
others. Engelhart and Jenkins
61
analyzed cocaine analytes and opi-
ates in paired fingernails and toenails from 17 postmortem cases.
For cocaine and its metabolites (BE, EME, norcocaine, cocaethylene),
concentrations in fingernails were seven to 20 times higher than in
toenails. The opioids majorly detected were morphine, 6-AM, and
codeine; concentrations of morphine and 6-AM were 30 times
greater in fingernails, while codeine concentrations did not differ
significantly between both matrices. Shu et al.
59
found mean and
median concentrations of cocaine and opioids higher in fingernail
samples. Cappelle et al.
41
found cocaine, BE, EME, morphine, 6-AM,
and EDDP higher in fingernails and codeine and methadone higher
in toenails (n=26).
3.2.4 | EtG
EtG was only studied by Shu et al.,
59
who found mean and median
concentrations higher in fingernail samples.
3.2.5 | PCP
PCP was investigated by Jenkins and Engelhart,
62
who analyzed nail
samples from four postmortem cases that tested positive for PCP in
urine. Paired fingernails and toenails were available in three individ-
uals, with PCP concentrations higher in fingernails.
8COBO-GOLPE ET AL.
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3.2.6 | Antidepressants and benzodiazepines
Three authors compared concentrations in both nail samples for these
drugs. Hang et al.
28
studied zolpidem concentrations in paired finger-
nails and toenails from seven subjects after the administration of a sin-
gle dose. Concentrations in toenails were higher than in fingernails
(up to three times). Cobo-Golpe et al.
34
studied different antidepressant
and benzodiazepine drugs in 21 patients and found similar concentra-
tions in fingernail and toenail samples when considering patients under
active treatment. Specifically, concentrations for zolpidem, venlafaxine,
and two samples containing trazodone were similar in both nail sam-
ples, and in one case, trazodone concentrations were five times higher
in toenails than fingernails. For alprazolam, one case showed similar
concentrations in both samples, while another was positive in toenails
but negative in fingernails. Fluoxetine and sertraline concentrations
were higher in toenails (n=1) (1.75 and 1.5 times higher, respectively).
Shu et al.
59
analyzed nail samples from more than 10,000 high-risk
cases, detecting 52 different drugs of abuse. The range of concentra-
tions for alprazolam, diazepam, and nordiazepam was similar.
3.2.7 | Antipsychotics
Fingernail and toenail concentrations for antipsychotic drugs were only
compared in two studies. Cobo-Golpe et al.
46
compared samples from
13 patients and found similar or higher concentrations in toenails for all
analytes except olanzapine and quetiapine, for which one sample and
four samples, respectively, showed higher concentrations in fingernails.
Chen et al.
45
found mean concentrations of clozapine in fingernails
slightly higher (1.3 times) than in toenails from a bloated cadaver case.
3.3 |Cut-offs in nail samples
Due to the differences in concentrations between hair, fingernails,
and toenails, the cut-off concentrations established for hair are not
valid to interpret the concentrations found in nails. To date, only five
studies have proposed preliminary cut-off concentrations in nail sam-
ples for EtG, THC, amphetamine, cocaine, BE, and EME (Table 3).
Regarding EtG, Berger et al.
63
analyzed 547 hair samples and
506 fingernail samples, paired when both samples were available.
Concentrations of EtG were always higher in fingernails than in hair,
and the cut-off for excessive alcohol consumption proposed in finger-
nails was 56 pg/mg. Cappelle et al.
54
analyzed 45 hair, 41 fingernail,
and 13 toenail samples (paired when possible) from patients treated
for alcohol use disorders. Higher concentrations were present in fin-
gernails and toenails compared with hair. The study proposes prelimi-
nary cut-off values for EtG concentrations in fingernails: >123 pg/mg
for chronic excessive alcohol consumption, 59–123 pg/mg for moder-
ate alcohol consumption, and <59 pg/mg for alcohol abstinence.
Fosen et al.
55
also detected higher EtG concentrations in fingernails
than in hair and proposed that the cut-off in fingernails should be
higher than in hair, but considered that the number of samples
(n=40) was not enough to calculate a cut-off. More recently, a study
by Vermeulen et al.
64
analyzed hair and fingernails from 111 teeto-
talers and proposed a cut-off for alcohol abstinence based on the
measured EtG concentrations in fingernails in their study. EtG concen-
trations were lower than the LOQ of 2.1 pg/mg in most samples, and
the highest concentration was 23 pg/mg. The authors proposed the
97.5% percentile of 7.6 pg/mg as a cut-off for alcohol abstinence.
Cobo-Golpe et al.
46
analyzed 22 fingernail, 19 toenail, and 16 hair
samples from chronic cannabis users, finding a better correlation of hair
concentrations with toenails, and proposed a THC cut-off in toenails of
16.5 pg/mg.
Finally, Cappelle et al.
41
analyzed hair and nail samples from in-
patients engaged in a treatment program for substance use disorders.
Twenty-six hair samples, 24 fingernail samples, and 18 toenail samples
were collected. Concentrations were higher in nails for BE, EME, and
amphetamine, while for cocaine, concentrations were higher in hair.
Cut-off values were proposed in fingernails and toenails for cocaine
(440 and 150 pg/mg), BE (175 and 105 pg/mg), EME (80 and 40 pg/
mg), and amphetamine (485 and 505 pg/mg, respectively).
4|DISCUSSION
When comparing concentrations found in hair with those in nails,
some drugs showed a clear pattern. This is the case of
TABLE 3 Cut-off concentrations proposed for different drugs in nail samples.
Reference Drug Concentrations Cut-off Hair (pg/mg) Fingernail (pg/mg) Toenail (pg/mg)
Cappelle 2017
54
EtG Toenail > fingernail > hair Excessive consumption >30 >123 -
Abstinence <7 <59 -
Berger 2014
63
EtG Fingernail > hair Excessive consumption >30 >56 -
Vermeulen 2022
64
EtG - Abstinence <5 <7.6 -
Cappelle 2018
41
COC Hair > fingernail > toenail Chronic use 500 440 150
BE Fingernail > toenail > hair Chronic use 50 175 105
EME Fingernail > hair > toenail Chronic use 50 80 40
AMP Toenail > fingernail > hair Chronic use 200 485 505
Cobo-Golpe 2020
46
THC Fingernail > hair > toenail Chronic use 50 - 16.5
Abbreviations: AMP, amphetamine; BE, benzoylecgonine; COC, cocaine; EME, ecgonine methyl ester; EtG, ethyl glucuronide; THC, tetrahydrocannabinol.
COBO-GOLPE ET AL.9
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antidepressants, benzodiazepines, antipsychotics, terbinafine, and
cotinine, which have been found in higher concentrations in hair than
in nails, while EtG has always been found in higher concentrations in
nails. On the other hand, for most drugs of abuse, the distribution is
not so clear. Amphetamine derivatives, cocaine, and its metabolites
and opioids have been reported to be at higher concentrations in hair
in some studies, higher in nails in others, and even different across
participants from the same study. Furthermore, in the case of cannabi-
noids, while THCCOOH has been reported in higher concentrations in
nails, for THC, CBD, and CBN, concentrations in hair were higher than
in toenails, but much lower than in fingernails.
One of the main differences between these two matrices is the
lack of melanin in nails in most individuals. The different affinities of
each substance for melanin binding could explain not only differences
in concentrations in different hair colors but also the differences in
concentrations between hair and nails.
Studies of drug incorporation into hair depending on melanin con-
tent have been performed for different analytes. Lee et al.
65
studied
codeine and morphine concentrations in rat hair and found that in
dark gray hair concentrations were always higher than those in white
hair. Ramirez-Fernandez et al.
66
evaluated the effect of melanin in
antipsychotics concentrations in human hair and found that hair mela-
nin has a much higher affinity for these drugs than hair proteins.
Other authors measured the binding of the substances to melanin in
in vitro experiments. Joseph et al.
67
concluded that melanin was the
primary hair constituent responsible for both nonspecific and specific
binding of cocaine. Gautam et al.
68
determined that, in vitro, 32% of
amphetamine bound to eumelanin.
Melanin content in hair was quantified by two authors to better
search for its correlation with substance incorporation.
69,70
Scheid-
weiler et al.
69
found that BE concentrations correlated with melanin
content, and a positive linear relationship was found between total
hair melanin content and Cmax for codeine, cocaine, and metabolites
following high dosing. The authors suggest that the correlation with
melanin content could be better explained by the melanin role in drug
incorporation of these drugs inside the hair cell than because of the
direct union of melanin with these compounds. The melanin role in
drug incorporation was evaluated by in vitro studies, which suggest
that pigmented melanocytes contain a transport system responsible
for drug influx and efflux.
71
Polettini et al.
70
also found a strong corre-
lation between the melanin concentration in hair and the AUC and
Cmax of AMP and MAMP. Conversely, the incorporation of neutral
and acidic compounds in hair did not appear to be melanin-correlated,
as observed for THC and THCCOOH,
21,72
N-acetylamphetamine,
73
phenobarbital,
74
carbamazepine,
75
ethyl glucuronide,
76
and fatty acid
ethyl esters.
77
From the results available on nail and hair concentrations, we can
infer that the affinity of each analyte for melanin plays an important
role when comparing both samples. Taking this into account, basic
substances, such as cocaine, codeine, antipsychotics, terbinafine, and
cotinine, which have a high affinity for melanin, are found at higher
concentrations in hair. Conversely, compounds with no relevant affin-
ity for melanin, such as cannabinoids and EtG, are often detected at
higher concentrations in nails due to the slower growth of nails that
allows for the accumulation of the substances over time. An exception
to this is seen for morphine, 6-AM, and amphetamines. These are
basic substances that have been observed to accumulate in higher
concentrations in darker hair due to melanin binding.
73,78
However,
when comparing nail and hair concentrations, their distribution in both
matrices is equivocal; morphine has been detected in higher concen-
trations in nails in all studies,
41,49–51
and for 6-AM
41,49–51
and
amphetamines,
36–41
the matrix with higher concentrations fluctuates
between the different cases and studies.
Another important difference between hair and nails to consider
is the growth rate: While in head hair 1 cm/month is accepted, nails
grow slower (about 3 mm/month the fingernails and 1.5 mm/month
the toenails) and, therefore, substances can be incorporated over lon-
ger periods of time.
20
This means that hair, fingernail, and toenail sam-
ples collected at the same time represent different detection
windows. Furthermore, nail growth is constant, but in two directions,
which means that substance incorporation is different than in hair, as
previously described.
28,29
Hang et al.
28
found that zolpidem appeared
in the overhang of the nails just 1 week after a single dose was admin-
istrated, and the concentrations decreased over the next 6–12 weeks
until a peak of concentration appeared between the 10th–15th
weeks. Zolpidem stopped being detected at the 18th–30th weeks.
Madry et al.
29
found the highest peak concentration after 24 h,
another increase of concentrations after 2–3 weeks, and a final con-
centration peak after 10–18 weeks. A possible explanation for the
first peak concentration is the incorporation into nails from sweat
shortly after taking the dose of zolpidem. However, because the water
content of nails is 9%–10%, diffusion of zolpidem through the nail
bed to the free edge could also be considered.
79
After 2 weeks, a sec-
ond peak is detected. At this point in time, the part of the nail that
was in contact with the nail bed during zolpidem intake should be at
the free edge, and the wash out effect should have eliminated the zol-
pidem incorporated through sweat. After peak two, there is a
decrease in concentrations attributed to wash-out by daily hygiene.
Finally, 10 to 18 weeks after the intake, a third peak of concentrations
can be detected, corresponding to the drug incorporated from the nail
matrix into the forming nails, in line with the average nail growth. In
single nails, zolpidem was detected until the 20th week. This differ-
ence in incorporation pathways can also obscure the detection win-
dow of the nails. In fact, in most studies, paired hair and nail samples
were collected at the same time, with no detailed information about
patterns of consumption, which hinders direct comparison of concen-
trations. A possible solution is performing controlled studies, where
one or more doses are administered, and samples are collected over a
period of time. Hang et al.
28
compared zolpidem concentrations at the
proximal 2 cm of hair with the peak concentrations observed in nails
(10–15 weeks) and found higher concentrations in hair. Madry et al.
29
also compared zolpidem peak concentrations found in hair and nails
and reported a big interindividual variability also with higher concen-
trations in hair, although when comparing total concentrations (sum
of concentrations of each hair sample and nail sample), they did not
find a difference between hair and nails. Ropero-Miller et al.
49
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administered multiple doses of cocaine and codeine and collected
multiple hair and nail samples, making it possible to determine a time
when the maximum concentration was observed (3–4 weeks for hair
and 1–3 weeks for nails) and to compare maximum concentrations. In
another study, Madry et al.
40
administered two doses of MDMA to
15 subjects and collected samples a median of 20 days after the last
administration. The concentrations compared corresponded to the
last 5 months in hair and 3.5 months in nails. This included the time of
administration, but some amount of drug from before the start of the
study could be influencing the results. Furthermore, these stud-
ies
28,29,40
show that incorporation through the nail bed makes some
amount of the drug appear in nail clippings before the peak of maxi-
mum concentration, making temporal interpretation of concentrations
in nails, and the comparison with hair, very difficult.
Finally, the absence of cuticle in nails can also contribute to a dif-
ference in concentrations. Incorporation through sweat can be more
relevant in toenail samples, and contamination through contact during
drug manipulation can affect fingernail concentrations.
48
On the other
hand, the lack of a protective layer can also allow for drug extraction
from the inside of the nails during normal hygiene
40
or in the decon-
tamination step during the analysis.
49
The choice of fingernails or toenails for the analysis must also be
considered when interpreting the results. In general, concentrations in
fingernails and toenails tend to be similar, although in most cases
(amphetamines, antidepressants, benzodiazepines, antipsychotics),
concentrations in toenails are slightly higher. This can be explained by
the differences detailed for the comparison of hair and nails: an accu-
mulation due to a slower growth rate, a higher incorporation through
sweat, and a lower extraction compared with the frequent
handwashing.
However, other drugs such as cannabinoids, PCP, cocaine, and
opioids were detected in higher concentrations in fingernails in almost
all studies. For THC, CBD, and CBN, the proposed explanation was
that fingernails could have been contaminated during drug manipula-
tion, and the washing procedure was not enough to mitigate this
contamination.
48
In the end, it is difficult to compare the different studies because
of the multitude of variables that can affect concentrations in hair and
nails. One of them is the dosage, as some authors study single dose
consumption,
28,29
others study concentrations in chronic users (with
different patterns of consumption, that are usually unknown
30,36–
39,47,51,53
or estimated retrospectively
33,41,48,54–56
) and, in the case of
pharmaceutical drugs, the prescribed doses also varied in time and
between participants.
34,46
The election of fingernails or toenails as the
nail matrix for comparison is also an important variable that is some-
times not correctly specified or considered when interpreting the
results, and as described previously, concentrations can be different in
these two types of nail samples, thus leading to an incorrect compari-
son between nails and hair. Finally, the most important condition for
comparing nail and hair results is the selection of paired nails and hair
samples from the same individuals, with a window of detection corre-
sponding to the same time period. While it is difficult to compare sam-
ples corresponding to the same period of consumption, the
comparison of nails and hair can be made for chronic consumers when
the pattern of consumption has not changed in the last months before
sample collection. In any case, considering the high interindividual var-
iability in concentrations, the comparison of concentrations found in
nail and hair samples taken from different individuals does not offer a
reliable result.
5|CONCLUSION
Although for some compounds a clear pattern was observed in terms
of the matrix (hair, fingernails, or toenails) in which higher concentra-
tions are detected, for the majority of the drugs, no conclusive results
could be obtained. In any case, the number of publications comparing
nails to hair is still limited, and in most cases, comparisons were made
with a low number of paired samples. In addition, the lack of standard-
ized sampling techniques, preanalytical procedures, and analytical
methods for hair and nail analysis makes it difficult to compare the
results of the available studies. Moreover, in most cases, the dosages
taken by the sample donors were unknown, so, to date, a direct
extrapolation of the amount of drug used cannot be performed based
on concentrations found in nails or hair samples. Furthermore, choos-
ing fingernails or toenails as the nail sample is important to highlight,
because the contamination pathways and windows of detection
reflected by these samples are different. Some of the aforementioned
publications analyzed only one of these nail matrices or did not spec-
ify if fingernails or toenails were used in the study. This adds another
variable to the interpretation of the results that should be considered
in future studies. Nevertheless, nails are a promising matrix for the
determination of long-term drug use, and of special interest as a sub-
stitute of hair analysis when this is not available, hence the need for
further, well planned studies to characterize this matrix.
AUTHOR CONTRIBUTIONS
All authors contributed to the article conceptualization. Literature
search and data analysis was performed by María Cobo Golpe under
the supervision of Elena Lendoiro. Original draft was written by María
Cobo Golpe and critically revised by Ana de-Castro-Ríos and Elena
Lendoiro. All authors read and approved the final manuscript.
ORCID
M. Cobo-Golpe https://orcid.org/0000-0002-3158-6118
A. de-Castro-Ríos https://orcid.org/0000-0002-9832-012X
E. Lendoiro https://orcid.org/0000-0002-3414-0509
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