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Hard Evidence

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Fingernail testing is often cited as an alternative specimen to hair. Now, three years and more than 10,000 data points later, fingernails are taking their place as the go-to specimen for long-term alcohol and drug testing.

by Adam Negrusz, Ph.D., F-ABFT

 During the last few decades both fingernails and toenails have become very useful specimens for the detection of drug use and abuse.1,2 Nails are made of a fibrous structural protein known as keratin. The average growth rate for fingernails is 3 mm per month2. Toenails grow 30-50% slower than fingernails and are much more susceptible to drug contamination from sweat. As the nail grows, chemicals (illicit substances, drugs, alcohol biomarkers, etc.) incorporate into the keratin fibers where they are trapped for long periods (3-5 months in fingernails, and 8-14 months in toenails).2

The mechanisms of drug incorporation in nails have not been extensively studied. In two recently published reports, the mechanisms of incorporation of the sleep aid zolpidem after a single oral dose were investigated.3,4 The results indicated three mechanisms of drug incorporation to nails:

1) Contamination from sweat, detectable 24 hours after drug use,

2) Incorporation from the nail bed (vertical growth), detected after 2 weeks, and

3) Incorporation from the germinal matrix (horizontal growth), where concentration is the highest approximately 3 months after drug administration. The studies showed that the concentration of zolpidem was higher in toenails than in fingernails, and the incorporation from sweat was not reversed by daily hygiene.3,4 An extensive review of the application of nail testing in drug treatment programs, identification of in uteroexposure to drugs, therapeutic drug monitoring, forensic toxicology – including postmortem applications and drug facilitated sexual assault – was recently published by Cappelle et al.1

Both hair and nails are keratinized specimens, however, there are a few differences between nails and hair that impact their usefulness in drug testing. Firstly, both fingernails and toenails grow continuously and do not have the growth cycle that is characteristic for hair.1,2 In addition, nails do not contain the color pigment melanin, and so are free of hair-color bias. Physicochemical properties of drugs can play an important role in drug accumulation in nails. Specifically, substances lacking a nitrogen atom may accumulate in nails at higher concentrations than in hair.1 For example, the concentrations of the direct alcohol biomarker ethyl glucuronide (EtG) and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCA, the metabolite of tetrahydrocannabinol [THC], the psychoactive ingredient in marijuana) in nails are 3 times and 4.9 times higher, respectively, than in matching hair samples.5,6

Here at USDTL we have been offering alcohol and drug testing in fingernail and toenail specimens as an alternate to hair testing since 1999. We are excited to be one of the first labs to report the resulting positivity data for more than 10,000 nail specimens tested over a three year span. 

Collecting Fingernail Data

A total of 10,349 nail samples were tested for 52 drugs, drug metabolites, and EtG. The samples were collected from high risk populations between January 1, 2012 and December 31, 2014. Sample sources included drug courts, child advocacy centers, drug treatment facilities, drunk driving programs, reference laboratories, physicians/health professionals’ programs, lawyers, and others. Approximately 3 mm clippings (about 100 mg) from all ten nails were submitted to the laboratory for each nail specimen. Samples were analyzed by validated analytical procedures.

We also examined differences in concentrations of drugs in fingernails and toenails. We started systematically recording the type of nail (fingernail vs. toenail) in May of 2014. Figure 3 (page 15) includes only results where the type of specimen was clearly indicated. As such, sample numbers as well as concentration ranges for the fingernail-toenail comparison may differ from those seen for the total data set.

One of the most fundamental principles of forensic toxicology is that in order to report any given specimen positive for a substance, at least two different analytical techniques have to be used on two separate aliquots from a specimen. In this study, the initial testing of nails was performed using either enzyme linked immunoassay (ELISA), or by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Presumptive positive samples were then subjected to confirmatory testing for targeted analytes. LC-MS-MS was used for confirmatory analysis for the majority of drug classes, except for phencyclidine, barbiturates, tramadol, and normeperidine. For the latter, gas chromatography-mass spectrometry (GC-MS) was employed. The presence of THCA in nails was confirmed by super sensitive GC-GC-MS-MS.

Amphetamines

A total of 7,799 samples were analyzed for amphetamines (amphetamine, methamphetamine, MDMA, MDA, and MDEA; Figure 1); 14.4% of the samples were positive for amphetamine, and 13.7% were positive for methamphetamine. The samples we collected did not show obvious concentration differences between fingernails and toenails for amphetamine and methamphetamine (Figure 3), however, previous studies with more limited numbers of samples have shown higher concentrations of the compounds in toenails than in fingernails.1

There were only 22 samples found positive for MDMA (0.3%), 7 for MDA (0.09%), and 4 for MDEA (0.05%). The data set in our study was too limited to compare MDA, MDMA, and MDEA concentrations between fingernails and toenails. A previous study looking at ketamine in fingernail clippings was only able to discover it at the concentration of 0.314 ng/mg (below the limit of quantitation).7 In our study ketamine was found in 2 nail samples at concentrations of 3,772 and 12,632 pg/mg. Ketamine metabolite (norketamine) was found in one sample only at 201 pg/mg. 

Cocaine

7,787 total specimens were tested for cocaine and cocaine metabolites. Cocaine, benzoylecgonine, norcocaine, and cocaethylene were found in 5.3%, 5.2%, 2.6%, and 1.2% of nail samples, respectively. (Figure 2) Cocaine was present in both fingernails and toenails in higher concentrations than benzoylecgonine, and higher concentrations of cocaine and benzoylecgonine were observed in fingernails than in toenails. (Figure 3) Previous studies have found higher concentrations of benzoylecgonine than cocaine in postmortem toenails from subjects of unspecified drug use history.8

Opiates and Opioids

Positivity rates of selected opioids (morphine, codeine, 6-MAM [heroin metabolite], hydrocodone, hydromorphone) in nail samples are presented in Figure 2. The most prevalent of the above five opioids was hydrocodone. It was detected in 11.4% of 7,779 tested nail samples, compared to 1.5-3.6% for the other four tested opioids. 6-MAM was present in 115 samples out of 7,779. (Editor’s note: This paragraph was edited on 8/26/2015 to correct a mistake.)

Other opioids including methadone, EDDP (methadone metabolite), oxycodone, and oxymorphone were found in 1.2%, 1.0%, 15.1%, and 2.6%, respectively, of approximately 3,400 samples analyzed. Methadone and EDDP were found in 44 and 35 samples, respectively, out of 3,567 submitted for analysis for these analytes (Figure 2).

Very limited information is available on the presence of other opioids (and opioid antagonists) such as buprenorphine, norbuprenorphine, naltrexone, naloxone, 6-ß-naltrexol, nalbuphine, or butorphanol in nails. Buprenorphine and norbuprenorphine were found in 15 and 12 nail samples, respectively, out of 40 samples submitted for buprenorphine analysis. We did not receive nail samples with a request for simultaneous testing for both buprenorphine/norbuprenorphine and naloxone. In fact, naloxone was not detected in any nail sample. On the other hand, naltrexone was found in only 2 nail samples (73 and 71 pg/mg), and 6-ß-naltrexol, naltrexone metabolite, was present in 9 out of 18 samples received. To our knowledge, this is the first study reporting naltrexone (used in alcohol dependence treatment) and its metabolite tested in nails.

Marijuana Testing

The detection of THCA is very important to differentiate between marijuana ingestion (intentional and/or passive inhalation) and external contamination.1

In our study the overall positivity rate for THCA was 18.1%. (Figure 1) Mean concentration in toenails were roughly the same as in fingernails (15 pg/mg and 13.2 pg/mg, respectively; Figure 3). The concentration ranges were also very similar suggesting that both specimens are equally useful in revealing cannabis use. Comparing matched hair and nail specimens Jones et al.5 developed a highly sensitive GC-GC-MS-MS method for the quantification of THCA in keratinized specimens with the limit of detection and quantification of 10 and 20 fg/mg, respectively. The mean THCA concentration in nails was 1.8 pg/mg in Jones et al., lower that the mean concentration in this study (13.7 pg/mg). 7,797 nail samples were analyzed for THCA concentrations in our study.

Alcohol Use

Out of 3,039 samples submitted for EtG analysis, 24.9% were positive, with a concentration range from 20-3,754 pg/mg (median 88, mean 177; Figure 2). 

In another study by Jones et al.,6 the reported EtG mean concentration was 29.1 pg/mg, and it was 3 times higher than in corresponding hair samples of college students (age 18-26 years). The mean concentration was significantly lower than in this report where subjects expressed risky alcohol drinking behavior. Morini et al.9 found a correlation between EtG concentration in fingernails and self-reported alcohol consumption. The highest EtG concentration was 92.6 pg/mg for a person consuming >60 g of alcohol per day. In a separate study, Berger et al.10 also found that EtG in fingernails at 30 pg/mg has 100% sensitivity to identify high-risk drinking behavior (≥30 standard drinks per week), and 100% specificity to rule out abstinence, compared to hair EtG at 30 pg/mg. In both studies, the highest EtG concentration in fingernails was 397.08 pg/mg. Both studies concluded that the detection of EtG in nails is a better alcohol use biomarker than in hair. 

Other Drugs

Examples of other drugs and metabolites found in nails include barbiturates, benzodiazepines, normeperidine, phencyclidine, tramadol, and norpropoxyphene. The positivity rate for benzodiazepines in nails was below 1%, except for alprazolam which showed a positivity rate of 1.6%.

Summary

In summary, this data demonstrates nails are a very useful matrix for the detection of drugs and illicit substances, and may be a superior alternative specimen type to hair samples. Nail sample preparation is relatively simple and the advancement in analytical instrument technology allows for accurate measurements of extremely low quantities (femtograms) of parent compounds as well as their metabolites. Most parent drugs are present at greater concentrations than their metabolites. Naltrexone is an exceptional case where its metabolite, 6-ß-naltrexone, is the predominant form present in nails. It is worth pointing out that the concentrations of substances found antemortem in our study are largely comparable with concentrations reported in postmortem cases, whether or not the causes or manners of death were drug-related. More research is necessary to better understand and interpret these analytical findings. 

References

1. Cappelle, D., Yegles, M., Neels, H., van Nuijs, A.L.N., De Doncker, M., Maudens, K., Covaci, A., Crunelle, C.L. (2015) Nail analysis for the detection of drugs of abuse and pharmaceuticals: a review. Forensic Toxicology, 33, 12-36.

2. Baumgartner, M.R. (2014) Nails: an adequate alternative matrix in forensic toxicology for drug analysis? Bioanalysis, 6, 2189-2191.

3. Hang, C., Ping, X., Min, S. (2013) Long-term follow-up analysis of zolpidem in fingernails after a single oral dose. Analytical and Bioanalytical Chemistry, 405, 7281-7289.

4. Madry, M.M., Steuer, A.E., Binz, T.M., Baumgartner, M.R., Kreamer, T. (2014) Systemic investigation of the incorporation mechanism of zolpidem in fingernails. Drug Testing and Analysis, 6, 533-541.

5. Jones, J., Jones, M., Plate, C., Lewis, D. (2013) The detection of THCA using 2-dimensional gas chromatography-tandem mass spectrometry in human fingernail clippings: method validation and comparison with head hair. American Journal of Analytical Chemistry, 4, 1-8.

6. Jones, J., Jones, M., Plate, C., Lewis, D., Fendrich, M., Berger, L., Fuhrmann, D. (2012) Liquid chromatography-tandem mass spectrometry assay to detect ethyl glucuronide in human fingernail: comparison to hair and gender differences. American Journal of Analytical Chemistry, 3, 83-91.

7. Cirimele, V., Kintz, P., Mangin, P. (1995) Detection of amphetamines in fingernails: an alternative to hair analysis. Archives of Toxicology, 70, 68-69.

8. Engelhart, D.A., Lavins, E.S., Sutheimer, C.A. (1998) Detection of drugs of abuse in nails. Journal of Analytical Toxicology, 22, 314-318.

9. Morini, L., Calucci, M., Ruberto M.G., Groppi, A. (2012) 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. Analytical and Bioanalytical Chemistry, 402, 1865-1870.

10. Berger, L., Fendrich, M., Jones, J., Fuhrmann, D., Plate, C., Lewis, D. (2014) Ethyl glucuronide in hair and fingernails as a long-term alcohol biomarker. Addiction, 109, 425-431.


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