Editorial Special Focus Issue: Forensic and clinical toxicology
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Nails: an adequate alternative matrix in forensic toxicology for drug analysis?
“If no suitable hair sample can be collected … are fingernails and toenails adequate alternative keratinized matrices?”
Keywords: forensic toxicology • incorporation • nail analysis • retrospective monitoring
Long-term monitoring of drug or medicament intake has become an important tool in clinical and forensic toxicology. Repeated sampling and testing of blood or urine samples allows spot checks for abstinence to be conducted over a long period of time. Hair analysis has a variety of advantages over blood or urine testing in terms of ease of use and interpretation. Endogenous substances, xenobiotics and metabolites are continuously incorporated into the solid, keratinized hair matrix, thus allowing their continuous recording over a long time frame. An appropriate extraction procedure allows for quantitative analysis of these substances. The actual time period represented by a hair sample is determined by hair length and the relative share of hair in the various phases of the hair growth cycle with anagen (growing), catagen and telogen (resting) hair. Through segmentation (i.e., the longitudinally sectioned examination of the hair sample), long-term monitoring can be conducted across several shorter time intervals. This leads to an improved time-resolved pattern of the incorporated substances, with increased selectivity and sensitivity and the possibility to record even a single intake. The preferred sample for hair testing is a well-bundled cluster of head hair cut as close to the scalp as possible. Under certain circumstances body hair samples may also be investigated, but the different physiology of non-head hair has to be considered during interpretation.
10.4155/BIO.14.165 © 2014 Future Science Ltd
The examination of hair samples is a routine procedure in a variety of applications [1–3] . Strictly speaking, hair is no longer an alternative matrix, at least in forensic toxicology. However, the examination of a hair sample is also associated with a number of un certainties. With cosmetic treatments, especially bleaching, perming or straightening, the incorporated substances can be degraded or extracted. Furthermore, owing to medical conditions (e.g., alopecia) or beauty treatments, hair may have been removed or is very short. If no suitable hair sample can be collected in such cases, are fingernails and toenails adequate alternative keratinized matrices?
Markus R Baumgartner Center for Forensic Hair Analysis, Zurich Institute of Forensic Medicine, University of Zurich, Kurvenstrasse 17, 8006 Zurich, Switzerland markus.baumgartner@ irm.uzh.ch
“Nail growth and formation is affected by the ingestion of various xenobiotics, such as heavy metals, antibiotics or chemotherapeutic drugs.
”
It has been known for a long time that xenobiotics are stored in nails [4–10] . An important difference between nails and hair is that nails grow continuously and do not have a growth cycle analogous to that of hair. By contrast with hair, nails are normally kept short, and there has never been a case of all of the nails being completely removed to avoid detection of substances. Two different nail sampling techniques are suitable for toxicological analysis: nail clippings, the excess
Bioanalysis (2014) 6(17), 2189–2191
part of
ISSN 1757-6180
2189
Editorial
Baumgartner overhang of the nail, can be collected by cutting with a scissor or a nail clipper; and scrapings obtained by abrasion of the upper layers of the nail using a sharp blade. An entire nail can only be excised and tested in the context of an autopsy. Recently, a range of reports have been published that provide a more refined insight into the mechanisms of the incorporation and storage of substances in the nail matrix [11–13] . Analogous to hair, incorporation occurs during nail growth. Growth occurs in two different areas. Approximately 80% of the nail is continuously being generated in the nail root or germinal matrix [4] . The remaining 20% is formed during the progressive growth of the nail in the nail bed – the flat surface to which the nail plate adheres. This minor formation explains how the growing nail remains connected to the nail bed, and it leads to continuous growth in thickness from the proximal to distal end. Similar to hair, incorporation occurs in nails from external contamination via sweat. External, environmentally dependent contamination can also be observed. These various incorporation pathways were ascertained and confirmed by the examination of nail clippings following a single dose of a drug. However, in cases of continuous intake of a drug or medicament, the concentration determined in clippings reflects a superposition of both internal and external incorporation processes. Consequently, clippings represent a superposed incorporation during the last few months before sample collection. In the case of fingernails, this time frame is approximately 3–5 months, supposing a growth rate between 1.9 and 4.4 mm/month (average 3 mm/month) [14] . Additionally, the growth rate depends on the length of the finger. The growth rate of toenails is approximately 30–50% slower than fingernails. Accordingly, the time frame represented is between 8 and 14 months. By contrast with clippings, scrapings represent a sample material characterized by a very large surface and, consequently, they are highly susceptible to contamination via sweat or environmental influences. Therefore, concentrations determined in scrapings are, to some extent, significantly higher than those in clippings. Some authors prefer toenails over fingernails for nail analysis as these are thought to be less exposed to environmental contamination. Evidence suggests that clippings from fingernails and toenails are suitable for retrospective monitoring for a time frame of up to 5 months (fingernail) or 14 months (toenail) before the collection of the nail sample. Shorter time frames or a shortterm change of consumption cannot be detected by the examination of clippings. Therefore, this form of analysis is most appropriate for the documentation
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Bioanalysis (2014) 6(17)
of average consumption behavior over a longer time period (e.g., for abstinence monitoring). By contrast, the examination of scrapings in antemortem cases is of no or secondary importance. The postmortem evaluation of substance use or abuse by examination of excised whole nails could be an interesting alternative to hair analysis. By an exhaustive, layered segmentation of an excised nail, from the dorsal to ventral side, scraping samples can be collected that represent the nail surface and the bulk matrix, respectively. The analysis of each fraction may be indicative of acute ingestion or contamination shortly before death. Alternatively, it can document the repeated intake of a substance during the weeks or months preceding death [13] .
“
Our ever-expanding understanding of nail physiology and the incorporation mechanisms of drugs, medicaments or poisons, combined with improvements in technology, provide the opportunity for the development of nail analysis in multiple disciplines of forensic and clinical toxicology.
”
As mentioned previously, the incorporation pathways have been investigated following a single-dose intake of a psychoactive compound. Whether such documentation by weekly collected clippings over months following ingestion of a single dose of a substance can be applied to forensic Drug Facilitated Crime cases [15] has to be demonstrated in future investigations. One single clipping should suffice for the analysis. Such small sample quantities of approximately 2–5 mg require a substance-optimized analysis including an optimized preanalysis protocol (e.g., washing steps and drying), a substance-specific extraction workflow (e.g., pulverizing, and multi-stage or single-stage extraction by different solvents), and highly sensitive and selective detection of the compound typically achieved by GC-MS(/MS) or LC-MS(/MS) methods. Validation experiments have shown that the matrix effects within LC-MS/MS techniques are smaller for nail extracts compared with hair extracts. The question of whether cosmetic treatments influence the concentrations detectable in nail samples remains, to our knowledge, unanswered. Only after clarification of this point will it be possible to conclusively decide whether nail clippings are a real alternative for checking abstinence or for the long-term monitoring of consumption behavior (e.g., regarding alcohol, cannabis, or other drugs or medicaments). However, some laboratories already use nail analysis as a method of abstinence control based on study results [7,16] .
future science group
Nails & drug analysis?
In dermatology, the study of diseases of the nails, known as onychopathology, has a long tradition. Nail growth and formation is affected by the ingestion of various xenobiotics, such as heavy metals, antibiotics or chemotherapeutic drugs. For nearly a century, the presence of Mees’ lines has been used in forensics as an indicator of poisoning with arsenic or thallium weeks to months prior to death. Our everexpanding understanding of nail physiology and the incorporation mechanisms of drugs, medicaments or poisons, combined with improvements in technology, provide the opportunity for the development of
nail analysis in multiple disciplines of forensic and clinical toxicology.
References
9
Ben Khelil M, Tegethoff M, Meinlschmidt G, Jamey C, Ludes B, Raul J-S. Simultaneous measurement of endogenous cortisol, cortisone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in nails by use of UPLC– MS–MS. Anal. Bioanal. Chem. 401(4), 1153–1162 (2011).
10
Krumbiegel F, Hastedt M, Tsokos M. Nails are a potential alternative matrix to hair for drug analysis in general unknown screenings by liquid-chromatography quadrupole time-of-flight mass spectrometry. Forensic Sci. Med. Pathol. doi:10.1007/s12024-014-9588-x (2014) (Epub ahead of print).
11
Hang C, Ping X, Min S. Long-term follow-up analysis of zolpidem in fingernails after a single oral dose. Anal.Bioanal. Chem. 405(23), 7281–7289 (2013).
12
Madry MM, Steuer AE, Binz TM, Baumgartner MR, Kraemer T. Systematic investigation of the incorporation mechanisms of zolpidem in fingernails. Drug Test. Anal.6(6), 533–541 (2014).
13
Madry MM, Steuer AE, Vonmoos M, Quednow BB, Baumgartner MR, Kraemer T. Retrospective monitoring of long-term recreational or dependent cocaine use in toenail clippings/scrapings as an alternative to hair. Bioanalysis (2014) (In Press).
14
Yaemsiri S, Hou N, Slining MM, He K. Growth rate of human fingernails and toenails in healthy American young adults. J. Eur. Acad. Dermatol. Venereol. 24(4), 420–423 (2010).
15
Kintz P.Toxicological Aspects of Drug-Facilitated Crime. Kintz P (Ed.). Elsevier, London, UK (2014).
16
Berger L, Fendrich M, Jones J, Fuhrmann D, Plate C, Lewis D. Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker. Addiction 109(3), 425–431 (2014).
1
Pragst F, Balikova MA. State of the art in hair analysis for detection of drug and alcohol abuse. Clin. Chim. Acta 370(1–2), 17–49 (2006).
2
Kintz P, Villain M, Cirimele V. Hair analysis for drug detection. Ther. Drug Monit. 28(3), 442–446 (2006).
3
Cooper G, Kronstrand R, Kintz P. Society of Hair Testing guidelines for drug testing in hair. Forensic Sci. Int. 218(1–3), 20–24 (2012).
4
Garside D. Drugs-of-abuse in nails. In: Drug Testing in Alternate Biological Specimens. Jenkins AJ (Ed.). Humana Press, Totowa, NJ, USA, 43–65 (2008).
5
6
7
8
Chen H, Xiang P, Shen M. Determination of clozapine in hair and nail: the role of keratinous biological materials in the identification of a bloated cadaver case. J. Forensic Leg. Med. 22, 62–67 (2014). Morini L, Colucci M, Ruberto M, Groppi A. Determination of ethyl glucuronide in nails by liquid chromatography tandem mass spectrometry as a potential new biomarker for chronic alcohol abuse and binge drinking behavior. Anal. Bioanal. Chem. 402(5), 1865–1870 (2012). Jones J, Jones M, Plate C, Lewis D. The detection of THCA using 2-dimensional gas chromatography-tandem mass spectrometry in human fingernail clippings: method validation and comparison with head hair. Am. J. Anal. Chem. 4, 1–8 (2013). Pufal E, Sykutera M, Nowacka T, Stefanowicz A, Sliwka K. Development of a method for estimation of citalopram and desmethylcitalopram in nails and hair and its usefulness in forensic toxicology. Arch. Med. Sadowej Kryminol. 60(4), 216–222 (2009).
future science group
Editorial
Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
www.future-science.com
2191
For reprint orders, please contact [email protected]
Nails: an adequate alternative matrix in forensic toxicology for drug analysis?
“If no suitable hair sample can be collected … are fingernails and toenails adequate alternative keratinized matrices?”
Keywords: forensic toxicology • incorporation • nail analysis • retrospective monitoring
Long-term monitoring of drug or medicament intake has become an important tool in clinical and forensic toxicology. Repeated sampling and testing of blood or urine samples allows spot checks for abstinence to be conducted over a long period of time. Hair analysis has a variety of advantages over blood or urine testing in terms of ease of use and interpretation. Endogenous substances, xenobiotics and metabolites are continuously incorporated into the solid, keratinized hair matrix, thus allowing their continuous recording over a long time frame. An appropriate extraction procedure allows for quantitative analysis of these substances. The actual time period represented by a hair sample is determined by hair length and the relative share of hair in the various phases of the hair growth cycle with anagen (growing), catagen and telogen (resting) hair. Through segmentation (i.e., the longitudinally sectioned examination of the hair sample), long-term monitoring can be conducted across several shorter time intervals. This leads to an improved time-resolved pattern of the incorporated substances, with increased selectivity and sensitivity and the possibility to record even a single intake. The preferred sample for hair testing is a well-bundled cluster of head hair cut as close to the scalp as possible. Under certain circumstances body hair samples may also be investigated, but the different physiology of non-head hair has to be considered during interpretation.
10.4155/BIO.14.165 © 2014 Future Science Ltd
The examination of hair samples is a routine procedure in a variety of applications [1–3] . Strictly speaking, hair is no longer an alternative matrix, at least in forensic toxicology. However, the examination of a hair sample is also associated with a number of un certainties. With cosmetic treatments, especially bleaching, perming or straightening, the incorporated substances can be degraded or extracted. Furthermore, owing to medical conditions (e.g., alopecia) or beauty treatments, hair may have been removed or is very short. If no suitable hair sample can be collected in such cases, are fingernails and toenails adequate alternative keratinized matrices?
Markus R Baumgartner Center for Forensic Hair Analysis, Zurich Institute of Forensic Medicine, University of Zurich, Kurvenstrasse 17, 8006 Zurich, Switzerland markus.baumgartner@ irm.uzh.ch
“Nail growth and formation is affected by the ingestion of various xenobiotics, such as heavy metals, antibiotics or chemotherapeutic drugs.
”
It has been known for a long time that xenobiotics are stored in nails [4–10] . An important difference between nails and hair is that nails grow continuously and do not have a growth cycle analogous to that of hair. By contrast with hair, nails are normally kept short, and there has never been a case of all of the nails being completely removed to avoid detection of substances. Two different nail sampling techniques are suitable for toxicological analysis: nail clippings, the excess
Bioanalysis (2014) 6(17), 2189–2191
part of
ISSN 1757-6180
2189
Editorial
Baumgartner overhang of the nail, can be collected by cutting with a scissor or a nail clipper; and scrapings obtained by abrasion of the upper layers of the nail using a sharp blade. An entire nail can only be excised and tested in the context of an autopsy. Recently, a range of reports have been published that provide a more refined insight into the mechanisms of the incorporation and storage of substances in the nail matrix [11–13] . Analogous to hair, incorporation occurs during nail growth. Growth occurs in two different areas. Approximately 80% of the nail is continuously being generated in the nail root or germinal matrix [4] . The remaining 20% is formed during the progressive growth of the nail in the nail bed – the flat surface to which the nail plate adheres. This minor formation explains how the growing nail remains connected to the nail bed, and it leads to continuous growth in thickness from the proximal to distal end. Similar to hair, incorporation occurs in nails from external contamination via sweat. External, environmentally dependent contamination can also be observed. These various incorporation pathways were ascertained and confirmed by the examination of nail clippings following a single dose of a drug. However, in cases of continuous intake of a drug or medicament, the concentration determined in clippings reflects a superposition of both internal and external incorporation processes. Consequently, clippings represent a superposed incorporation during the last few months before sample collection. In the case of fingernails, this time frame is approximately 3–5 months, supposing a growth rate between 1.9 and 4.4 mm/month (average 3 mm/month) [14] . Additionally, the growth rate depends on the length of the finger. The growth rate of toenails is approximately 30–50% slower than fingernails. Accordingly, the time frame represented is between 8 and 14 months. By contrast with clippings, scrapings represent a sample material characterized by a very large surface and, consequently, they are highly susceptible to contamination via sweat or environmental influences. Therefore, concentrations determined in scrapings are, to some extent, significantly higher than those in clippings. Some authors prefer toenails over fingernails for nail analysis as these are thought to be less exposed to environmental contamination. Evidence suggests that clippings from fingernails and toenails are suitable for retrospective monitoring for a time frame of up to 5 months (fingernail) or 14 months (toenail) before the collection of the nail sample. Shorter time frames or a shortterm change of consumption cannot be detected by the examination of clippings. Therefore, this form of analysis is most appropriate for the documentation
2190
Bioanalysis (2014) 6(17)
of average consumption behavior over a longer time period (e.g., for abstinence monitoring). By contrast, the examination of scrapings in antemortem cases is of no or secondary importance. The postmortem evaluation of substance use or abuse by examination of excised whole nails could be an interesting alternative to hair analysis. By an exhaustive, layered segmentation of an excised nail, from the dorsal to ventral side, scraping samples can be collected that represent the nail surface and the bulk matrix, respectively. The analysis of each fraction may be indicative of acute ingestion or contamination shortly before death. Alternatively, it can document the repeated intake of a substance during the weeks or months preceding death [13] .
“
Our ever-expanding understanding of nail physiology and the incorporation mechanisms of drugs, medicaments or poisons, combined with improvements in technology, provide the opportunity for the development of nail analysis in multiple disciplines of forensic and clinical toxicology.
”
As mentioned previously, the incorporation pathways have been investigated following a single-dose intake of a psychoactive compound. Whether such documentation by weekly collected clippings over months following ingestion of a single dose of a substance can be applied to forensic Drug Facilitated Crime cases [15] has to be demonstrated in future investigations. One single clipping should suffice for the analysis. Such small sample quantities of approximately 2–5 mg require a substance-optimized analysis including an optimized preanalysis protocol (e.g., washing steps and drying), a substance-specific extraction workflow (e.g., pulverizing, and multi-stage or single-stage extraction by different solvents), and highly sensitive and selective detection of the compound typically achieved by GC-MS(/MS) or LC-MS(/MS) methods. Validation experiments have shown that the matrix effects within LC-MS/MS techniques are smaller for nail extracts compared with hair extracts. The question of whether cosmetic treatments influence the concentrations detectable in nail samples remains, to our knowledge, unanswered. Only after clarification of this point will it be possible to conclusively decide whether nail clippings are a real alternative for checking abstinence or for the long-term monitoring of consumption behavior (e.g., regarding alcohol, cannabis, or other drugs or medicaments). However, some laboratories already use nail analysis as a method of abstinence control based on study results [7,16] .
future science group
Nails & drug analysis?
In dermatology, the study of diseases of the nails, known as onychopathology, has a long tradition. Nail growth and formation is affected by the ingestion of various xenobiotics, such as heavy metals, antibiotics or chemotherapeutic drugs. For nearly a century, the presence of Mees’ lines has been used in forensics as an indicator of poisoning with arsenic or thallium weeks to months prior to death. Our everexpanding understanding of nail physiology and the incorporation mechanisms of drugs, medicaments or poisons, combined with improvements in technology, provide the opportunity for the development of
nail analysis in multiple disciplines of forensic and clinical toxicology.
References
9
Ben Khelil M, Tegethoff M, Meinlschmidt G, Jamey C, Ludes B, Raul J-S. Simultaneous measurement of endogenous cortisol, cortisone, dehydroepiandrosterone, and dehydroepiandrosterone sulfate in nails by use of UPLC– MS–MS. Anal. Bioanal. Chem. 401(4), 1153–1162 (2011).
10
Krumbiegel F, Hastedt M, Tsokos M. Nails are a potential alternative matrix to hair for drug analysis in general unknown screenings by liquid-chromatography quadrupole time-of-flight mass spectrometry. Forensic Sci. Med. Pathol. doi:10.1007/s12024-014-9588-x (2014) (Epub ahead of print).
11
Hang C, Ping X, Min S. Long-term follow-up analysis of zolpidem in fingernails after a single oral dose. Anal.Bioanal. Chem. 405(23), 7281–7289 (2013).
12
Madry MM, Steuer AE, Binz TM, Baumgartner MR, Kraemer T. Systematic investigation of the incorporation mechanisms of zolpidem in fingernails. Drug Test. Anal.6(6), 533–541 (2014).
13
Madry MM, Steuer AE, Vonmoos M, Quednow BB, Baumgartner MR, Kraemer T. Retrospective monitoring of long-term recreational or dependent cocaine use in toenail clippings/scrapings as an alternative to hair. Bioanalysis (2014) (In Press).
14
Yaemsiri S, Hou N, Slining MM, He K. Growth rate of human fingernails and toenails in healthy American young adults. J. Eur. Acad. Dermatol. Venereol. 24(4), 420–423 (2010).
15
Kintz P.Toxicological Aspects of Drug-Facilitated Crime. Kintz P (Ed.). Elsevier, London, UK (2014).
16
Berger L, Fendrich M, Jones J, Fuhrmann D, Plate C, Lewis D. Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker. Addiction 109(3), 425–431 (2014).
1
Pragst F, Balikova MA. State of the art in hair analysis for detection of drug and alcohol abuse. Clin. Chim. Acta 370(1–2), 17–49 (2006).
2
Kintz P, Villain M, Cirimele V. Hair analysis for drug detection. Ther. Drug Monit. 28(3), 442–446 (2006).
3
Cooper G, Kronstrand R, Kintz P. Society of Hair Testing guidelines for drug testing in hair. Forensic Sci. Int. 218(1–3), 20–24 (2012).
4
Garside D. Drugs-of-abuse in nails. In: Drug Testing in Alternate Biological Specimens. Jenkins AJ (Ed.). Humana Press, Totowa, NJ, USA, 43–65 (2008).
5
6
7
8
Chen H, Xiang P, Shen M. Determination of clozapine in hair and nail: the role of keratinous biological materials in the identification of a bloated cadaver case. J. Forensic Leg. Med. 22, 62–67 (2014). Morini L, Colucci M, Ruberto M, Groppi A. Determination of ethyl glucuronide in nails by liquid chromatography tandem mass spectrometry as a potential new biomarker for chronic alcohol abuse and binge drinking behavior. Anal. Bioanal. Chem. 402(5), 1865–1870 (2012). Jones J, Jones M, Plate C, Lewis D. The detection of THCA using 2-dimensional gas chromatography-tandem mass spectrometry in human fingernail clippings: method validation and comparison with head hair. Am. J. Anal. Chem. 4, 1–8 (2013). Pufal E, Sykutera M, Nowacka T, Stefanowicz A, Sliwka K. Development of a method for estimation of citalopram and desmethylcitalopram in nails and hair and its usefulness in forensic toxicology. Arch. Med. Sadowej Kryminol. 60(4), 216–222 (2009).
future science group
Editorial
Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.
www.future-science.com
2191
