Abstract

Nails (fingernails and toenails) are made of keratin. As the nail grows, substances incorporate into the keratin fibers where they can be detected 3–6 months after use. Samples are collected by clipping of 2–3 mm of nail from all fingers (100 mg). We present drug testing results from 10,349 nail samples collected from high-risk cases during a 3-year period of time. Samples were analyzed by validated analytical methods. The initial testing was performed mostly using enzyme-linked immunosorbent assay, but by liquid chromatography–tandem mass spectrometry (LC–MS-MS) as well. Presumptive positive samples were subjected to confirmatory testing with sample preparation procedures including washing, pulverizing, digestion and extraction optimized for each drug class. The total of 7,799 samples was analyzed for amphetamines. The concentrations ranged from 40 to 572,865 pg/mg (median, 100–3,687) for all amphetamine analytes. Amphetamine and methamphetamine were present in 14% of the samples, 22 samples were positive for 3,4-methylenedioxymethamphetamine (0.3%), 7 for methylenedioxyamphetamine (0.09%) and 4 for 3,4-methylenedioxy-N-ethylamphetamine (0.05%). Cocaine and related analytes were found in 5% samples (7,787 total), and the concentration range was 20–265,063 pg/mg (median 84–1,768). Opioids overall ranged from 40 to 118,229 pg/mg (median 123–830). The most prevalent opioid was oxycodone (15.1%) and hydrocodone (11.4%) compared with 1.0–3.6% for the others, including morphine, codeine, hydromorphone, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine and oxymorphone. Carboxy-Δ-9-tetrahydrocannabinol positivity rate was 18.1% (0.04–262 pg/mg, median 6.41). Out of 3,039 samples, 756 were positive (24.9%) for ethyl glucuronide (20–3,754 pg/mg, median 88). Other drugs found in nails included barbiturates, benzodiazepines, ketamine, meperidine, tramadol, zolpidem, propoxyphene, naltrexone and buprenorphine. Nail analyses have become a reliable way of determining the long-term use and abuse of drugs. Extraction techniques are simple and produce accurate and precise results. Sensitive analytical instrumentation, mainly LC–MS-MS, allows for detection of femtogram (10−15 g) quantities of substances in nails. Samples were from a high-risk population, therefore the extraordinary positivity rate was observed.

Introduction

During the last several decades, nails (fingernails and toenails) have become a useful specimen type for detection of drug use and abuse, as well as the exposure to other substances such as heavy metals (1–3). Nails are made of keratin. The average growth rate for fingernails is 3 mm per month (range between 1.9 and 4.4 mm/month) (3). Toenails grow 30–50% slower than fingernails and are much more susceptible to drug contamination from sweat (1, 3). As the nail grows, chemicals (illicit substances, drugs, alcohol biomarkers, etc.) incorporate into the keratin fibers where they can stay for extended periods of time (3–5 months in fingernails, and 8–14 months in toenails) (3). The mechanisms of drug deposition in nails have not been extensively studied. In two recently published reports, the mechanisms of incorporation of zolpidem (ZOL) after a single oral dose were investigated (4, 5). The results revealed potentially three mechanisms of drug incorporation to nails: (i) contamination from sweat detectable 24 h after drug intake, (ii) incorporation from nail bed (vertical growth) detected after 2 weeks and (iii) incorporation from germinal matrix (horizontal growth), where concentration is peaking on average 3 months after drug administration. The authors showed ZOL incorporation from sweat being irremovable by daily hygiene (5). The concentration of ZOL was higher in toenails than in fingernails (4). An extensive review of application of nail testing in drug treatment programs, identification of in utero exposure 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. There are a few differences between nails and hair impacting the usefulness in drug testing. First, both fingernails and toenails grow continuously and do not have the growth cycle characteristic for hair (1, 3). In addition, nails do not contain melanin and for that reason are free of hair-color bias. Physicochemical properties of drugs play an important role in deposition in nails. Specifically, substances not containing a nitrogen atom may accumulate in nails at higher concentrations than in hair (1). For example, the concentrations of ethyl glucuronide (EtG) and 11-nor-Δ9-THC-9-carboxylic acid (THCA) in nails are 3 times and 4.9 times, respectively, higher than in the corresponding hair samples (6, 7).

In this article, we present the survey of drug testing results in 10,349 nail samples collected from high-risk populations for prevalence and concentrations during a 3-year period of time between 2012 and 2014. In addition, we show the differences in concentrations of drugs in fingernails and toenails. All samples were analyzed by validated analytical procedures. The initial testing was performed using either enzyme-linked immunosorbent assay (ELISA), or by liquid chromatography–tandem mass spectrometry (LC–MS-MS). Presumptive positive samples were then subjected to confirmatory testing with sample preparation procedures including washing, pulverizing, digestion and extraction optimized for each drug class.

Experimental

Specimens and analytes

Approximately 3 mm clippings (∼100 mg) from all nails were submitted to the laboratory for analysis for drugs specified by the requestors on the chain of custody forms. The samples came from drug courts, child advocacy centers, drug treatment facilities, drunk driving programs, reference laboratories, physicians/health professionals' programs, lawyers, etc.

All samples were screened for the requested drug classes, and the presumptive positive results were subjected to confirmatory testing for the following targeted analytes: amphetamine (AMP), methamphetamine (MET), 3,4-methylenedioxymethamphetamine (MDMA), methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA), cocaine (COC), benzoylecgonine (BE), norcocaine (NCOC), cocaethylene (CE), 6-monoacetylmorphine (6-MAM), codeine (COD), morphine (MOR), hydromorphone (HYM), hydrocodone (HCOD), oxycodone (OXC), oxymorphone (OXM), methadone (MTD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), fentanyl (FE), norfentanyl (NFE), sufentanil (SUF), norsufentanil (NSUF), propoxyphene (PPX), norpropoxyphene (NPPX), buprenorphine (BUP), norbuprenorphine (NBUP), meperidine (MEP), normeperidine (NMEP), tramadol (TRAM), naltrexone (NALT), naloxone (NAL), 6-beta-naltrexol (6-BNAL), nalbuphine (NALB), butorphanol (BTF), THCA, phencyclidine (PCP), butalbital (BUT), amobarbital (AMO), pentobarbital (PEN), secobarbital (SEC), phenobarbital (PHB), midazolam (MDZ), oxazepam (OX), alprazolam (ALP), temazepam (TEM), diazepam (DIAZ), nordiazepam (NDIAZ), propofol glucuronide (PPFG), ketamine (KET), norketamine (NKET), ZOL and EtG.

Sample preparation

Nail samples were washed with acetone once for the ELISA initial testing and the majority of the confirmatory methods. Some exceptions included THCA, BUP and NBUP, where methylene chloride wash was employed. If EtG or PPFG analysis was requested, the specimens were washed with hexane followed by 10-min sonication in methylene chloride, and finally washed with methanol. All initial washes were discarded.

Immediately after the wash, nail samples were dried at room temperature and pulverized using mini ball beater with 3 mm diameter stainless-steel balls. Calibrations were performed by fortifying pulverized drug-free nail aliquots with standard working solutions of all analytes of interest. In addition, three levels of controls were prepared in naïve pulverized nails fortified with corresponding working control solutions of all analytes. Depending on the analyte, various nail digestion or incubation methods optimized for each drug class were employed. Acid digestion (0.1 N hydrochloric acid at 53°C overnight) was used for amphetamines, COC and metabolites, selected opioids also including NALT, NAL, 6-BNAL, NALB, BTF, and PCP, MTD, EDDP, PPX, NPPX, TRAM, NMEP, NFE, NSUF, KET and NKET. The two-step process was used for 6-MAM to be co-analyzed with the opioids including COD, MOR, HYM, HCOD, OXC and OXM. First, the powdered nail sample was sonicated with methanol for 3 h. After centrifugation, the methanol supernatant was decanted to a separate tube and the remaining nail powder was digested in 0.1 N hydrochloric acid at 53°C overnight. Basic digestion with 1 N sodium hydroxide at 80°C for 1 h was applied for THCA. Sonication for 2 h in deionized water without heat with subsequent overnight incubation at room temperature was utilized for EtG and PPFG analysis. After digestion or incubation, the nail extracts were subjected to the corresponding solid-phase extraction (SPE) methods. The methanol incubation was used for the rest of the drugs: barbiturates (53°C overnight), benzodiazepines (2-h sonication with heat followed by incubation at room temperature overnight) and ZOL (sonication for 4 h with heat). The methanol extracts were then evaporated and reconstituted with the corresponding LC mobile phases for analysis.

Preliminary testing and confirmation

After initial preparation, the samples underwent preliminary testing by ELISA, except for BUP, EtG, KET, NKET, NALT, NAL, 6-BNAL, NALB, BTF and PPFG for which the instrumental screening by LC–MS-MS was employed. LC–MS-MS was also used for confirmatory analysis for the majority of drug classes except for PCP, barbiturates, TRAM and NMEP. For the latter, gas chromatography–mass spectrometry (GC–MS) was employed. The presence of THCA in nails was confirmed by GC–GC–MS-MS. The examples of the entire analytical approach to analysis of nail samples in our laboratory were discussed by Jones et al. (6, 7).

Results and discussion

In this study, we report the results of analysis of 10,349 nail samples acquired between 1 January 2012 and 31 December 2014. All specimens were collected from elevated risk groups such as drug treatment programs, physicians/health professionals' programs, drug courts, and child advocacy centers with the expected high rate of positivity. The results are presented in Table I. The comparison of fingernail and toenail results is presented in Table II. The sample numbers as well as the concentration ranges in Table II may differ from what is presented in Table I. The reason is that our laboratory started systematically recording the type of nail (finger- vs. toenail) in May of 2014. Table II includes only results where the type of specimen was clearly indicated.

Table I. Analyte LOQ (pg/mg) Number of samples ≥LOQ/total samples tested % of samples ≥LOQ Minimum–maximum range (pg/mg) Median/mean (pg/mg) AMP 40 1,126/7,799 14.4 40–44,851 831/2,005 MET 40 1,071/7,799 13.7 42–572,865 3,687/13,197 MDMA 40 22/7,799 0.3 43–40,165 298/2,773 MDA 40 7/7,799 0.09 52–706 129/250 MDEA 40 4/7,799 0.05 50–422 100/168 COC 40 415/7,787 5.3 48–265,063 1,768/13,600 BE 20 406/7,787 5.2 23–141,239 971/4,520 NCOC 20 199/7,787 2.6 20–3,875 105/305 CE 20 93/7,787 1.2 20–934 84/145 6-MAM 40 115/7,779 1.5 45–118,229 830/8,774 COD 40 144/7,779 1.9 41–32,358 163/739 MOR 40 283/7,779 3.6 40–105,932 445/3,975 HYM 40 170/7,779 2.2 41–62,857 123/986 HCOD 40 885/7,779 11.4 40–116,341 633/1,351 OXC 40 485/3,202 15.1 41–36,987 614/2,082 OXM 40 83/3,202 2.6 42–47,724 155/1,469 MTD 40 44/3,567 1.2 94–17,859 2,237/3,245 EDDP 40 35/3,567 1.0 49–2,880 527/696 NPPX 40 1/3,567 0.03 45 na BUP 8 15/40 37.5 37–5,491 182/782 NBUP 8 12/40 30.0 9–216 43/65 NMEP 1,000 1/1,344 0.07 1,481 na TRAM 400 53/1,344 3.9 425–158,072 3,553/8,606 NALT 40 2/18 11.1 63–71 na 6-BNAL 40 9/18 50.0 115–2,261 484/749 THCA 0.02 1,412/7,797 18.1 0.04–262 6.41/13.73 PCP 40 3/7,774 0.04 72–13,488 630/4,730 BUT 200 22/4,079 0.5 486–19,101 1,811/3,214 PHB 200 2/4,079 0.05 3,659–4,653 na ALP 40 64/4,083 1.6 45–1,553 152/251 TEM 40 3/4,083 0.07 47–3,769 99/1,305 DIAZ 40 37/4,083 0.9 41–5,338 233/560 NDIAZ 40 35/4,083 0.9 46–1,604 139/260 KET 40 2/122 1.6 3,772–12,632 na NKET 40 1/122 0.8 201 na ZOL 4 1/4 25.0 344 na EtG 8 756/3,039 24.9 20–3,754 88/177 Analyte LOQ (pg/mg) Number of samples ≥LOQ/total samples tested % of samples ≥LOQ Minimum–maximum range (pg/mg) Median/mean (pg/mg) AMP 40 1,126/7,799 14.4 40–44,851 831/2,005 MET 40 1,071/7,799 13.7 42–572,865 3,687/13,197 MDMA 40 22/7,799 0.3 43–40,165 298/2,773 MDA 40 7/7,799 0.09 52–706 129/250 MDEA 40 4/7,799 0.05 50–422 100/168 COC 40 415/7,787 5.3 48–265,063 1,768/13,600 BE 20 406/7,787 5.2 23–141,239 971/4,520 NCOC 20 199/7,787 2.6 20–3,875 105/305 CE 20 93/7,787 1.2 20–934 84/145 6-MAM 40 115/7,779 1.5 45–118,229 830/8,774 COD 40 144/7,779 1.9 41–32,358 163/739 MOR 40 283/7,779 3.6 40–105,932 445/3,975 HYM 40 170/7,779 2.2 41–62,857 123/986 HCOD 40 885/7,779 11.4 40–116,341 633/1,351 OXC 40 485/3,202 15.1 41–36,987 614/2,082 OXM 40 83/3,202 2.6 42–47,724 155/1,469 MTD 40 44/3,567 1.2 94–17,859 2,237/3,245 EDDP 40 35/3,567 1.0 49–2,880 527/696 NPPX 40 1/3,567 0.03 45 na BUP 8 15/40 37.5 37–5,491 182/782 NBUP 8 12/40 30.0 9–216 43/65 NMEP 1,000 1/1,344 0.07 1,481 na TRAM 400 53/1,344 3.9 425–158,072 3,553/8,606 NALT 40 2/18 11.1 63–71 na 6-BNAL 40 9/18 50.0 115–2,261 484/749 THCA 0.02 1,412/7,797 18.1 0.04–262 6.41/13.73 PCP 40 3/7,774 0.04 72–13,488 630/4,730 BUT 200 22/4,079 0.5 486–19,101 1,811/3,214 PHB 200 2/4,079 0.05 3,659–4,653 na ALP 40 64/4,083 1.6 45–1,553 152/251 TEM 40 3/4,083 0.07 47–3,769 99/1,305 DIAZ 40 37/4,083 0.9 41–5,338 233/560 NDIAZ 40 35/4,083 0.9 46–1,604 139/260 KET 40 2/122 1.6 3,772–12,632 na NKET 40 1/122 0.8 201 na ZOL 4 1/4 25.0 344 na EtG 8 756/3,039 24.9 20–3,754 88/177 View Large

Table I. Analyte LOQ (pg/mg) Number of samples ≥LOQ/total samples tested % of samples ≥LOQ Minimum–maximum range (pg/mg) Median/mean (pg/mg) AMP 40 1,126/7,799 14.4 40–44,851 831/2,005 MET 40 1,071/7,799 13.7 42–572,865 3,687/13,197 MDMA 40 22/7,799 0.3 43–40,165 298/2,773 MDA 40 7/7,799 0.09 52–706 129/250 MDEA 40 4/7,799 0.05 50–422 100/168 COC 40 415/7,787 5.3 48–265,063 1,768/13,600 BE 20 406/7,787 5.2 23–141,239 971/4,520 NCOC 20 199/7,787 2.6 20–3,875 105/305 CE 20 93/7,787 1.2 20–934 84/145 6-MAM 40 115/7,779 1.5 45–118,229 830/8,774 COD 40 144/7,779 1.9 41–32,358 163/739 MOR 40 283/7,779 3.6 40–105,932 445/3,975 HYM 40 170/7,779 2.2 41–62,857 123/986 HCOD 40 885/7,779 11.4 40–116,341 633/1,351 OXC 40 485/3,202 15.1 41–36,987 614/2,082 OXM 40 83/3,202 2.6 42–47,724 155/1,469 MTD 40 44/3,567 1.2 94–17,859 2,237/3,245 EDDP 40 35/3,567 1.0 49–2,880 527/696 NPPX 40 1/3,567 0.03 45 na BUP 8 15/40 37.5 37–5,491 182/782 NBUP 8 12/40 30.0 9–216 43/65 NMEP 1,000 1/1,344 0.07 1,481 na TRAM 400 53/1,344 3.9 425–158,072 3,553/8,606 NALT 40 2/18 11.1 63–71 na 6-BNAL 40 9/18 50.0 115–2,261 484/749 THCA 0.02 1,412/7,797 18.1 0.04–262 6.41/13.73 PCP 40 3/7,774 0.04 72–13,488 630/4,730 BUT 200 22/4,079 0.5 486–19,101 1,811/3,214 PHB 200 2/4,079 0.05 3,659–4,653 na ALP 40 64/4,083 1.6 45–1,553 152/251 TEM 40 3/4,083 0.07 47–3,769 99/1,305 DIAZ 40 37/4,083 0.9 41–5,338 233/560 NDIAZ 40 35/4,083 0.9 46–1,604 139/260 KET 40 2/122 1.6 3,772–12,632 na NKET 40 1/122 0.8 201 na ZOL 4 1/4 25.0 344 na EtG 8 756/3,039 24.9 20–3,754 88/177 Analyte LOQ (pg/mg) Number of samples ≥LOQ/total samples tested % of samples ≥LOQ Minimum–maximum range (pg/mg) Median/mean (pg/mg) AMP 40 1,126/7,799 14.4 40–44,851 831/2,005 MET 40 1,071/7,799 13.7 42–572,865 3,687/13,197 MDMA 40 22/7,799 0.3 43–40,165 298/2,773 MDA 40 7/7,799 0.09 52–706 129/250 MDEA 40 4/7,799 0.05 50–422 100/168 COC 40 415/7,787 5.3 48–265,063 1,768/13,600 BE 20 406/7,787 5.2 23–141,239 971/4,520 NCOC 20 199/7,787 2.6 20–3,875 105/305 CE 20 93/7,787 1.2 20–934 84/145 6-MAM 40 115/7,779 1.5 45–118,229 830/8,774 COD 40 144/7,779 1.9 41–32,358 163/739 MOR 40 283/7,779 3.6 40–105,932 445/3,975 HYM 40 170/7,779 2.2 41–62,857 123/986 HCOD 40 885/7,779 11.4 40–116,341 633/1,351 OXC 40 485/3,202 15.1 41–36,987 614/2,082 OXM 40 83/3,202 2.6 42–47,724 155/1,469 MTD 40 44/3,567 1.2 94–17,859 2,237/3,245 EDDP 40 35/3,567 1.0 49–2,880 527/696 NPPX 40 1/3,567 0.03 45 na BUP 8 15/40 37.5 37–5,491 182/782 NBUP 8 12/40 30.0 9–216 43/65 NMEP 1,000 1/1,344 0.07 1,481 na TRAM 400 53/1,344 3.9 425–158,072 3,553/8,606 NALT 40 2/18 11.1 63–71 na 6-BNAL 40 9/18 50.0 115–2,261 484/749 THCA 0.02 1,412/7,797 18.1 0.04–262 6.41/13.73 PCP 40 3/7,774 0.04 72–13,488 630/4,730 BUT 200 22/4,079 0.5 486–19,101 1,811/3,214 PHB 200 2/4,079 0.05 3,659–4,653 na ALP 40 64/4,083 1.6 45–1,553 152/251 TEM 40 3/4,083 0.07 47–3,769 99/1,305 DIAZ 40 37/4,083 0.9 41–5,338 233/560 NDIAZ 40 35/4,083 0.9 46–1,604 139/260 KET 40 2/122 1.6 3,772–12,632 na NKET 40 1/122 0.8 201 na ZOL 4 1/4 25.0 344 na EtG 8 756/3,039 24.9 20–3,754 88/177 View Large

Table II. Analyte Number of samples ≥LOQ/total samples tested (% of samples ≥LOQ) Minimum–maximum range (pg/mg) Median/mean (pg/mg) Fingernails Toenails Fingernails Toenails Fingernails Toenails AMP 212/1,542 (13.7%) 99/550 (18.0%) 42–29,105 40–44,851 980/1,879 843/2,906 MET 190/1,542 (12.3%) 73/550 (13.3%) 80–249,706 47–344,619 3,784/16,148 3,021/15,717 MDMA 4/1,542 (0.3%) 1/550 (0.2%) 70–3,834 99 2,564/2,258 na/na COC 84/1,540 (5.5%) 16/550 (2.9%) 159–265,063 306–6,067 2,101/18,510 1,281/2,143 BE 82/1,540 (5.3%) 16/550 (2.9%) 41–45,939 166–6,887 1,285/4,803 594/1,293 NCOC 38/1,540 (2.5%) 3/550 (0.5%) 20–2,357 68–144 120/289 71/94 CE 21/1,540 (1.4%) 2/550 (0.4%) 22–463 221–252 61/90 na/na 6-MAM 30/1,540 (1.9%) 6/550 (1.1%) 51–118,229 122–22,935 2,959/18,645 1,051/4,681 COD 31/1,540 (2.0%) 11/550 (2.0%) 41–6,882 45–805 192/816 130/201 MOR 57/1,540 (3.7%) 15/550 (2.7%) 47–66,666 40–11,857 780/7,209 285/1,466 HYM 41/1,540 (2.7%) 12/550 (2.2%) 41–62,857 53–1,188 127/1,813 115/283 HCOD 134/1,540 (8.7%) 44/550 (8.0%) 59–13,473 113–8,611 913/1,663 753/1,363 OXC 107/606 (17.7%) 29/214 (13.6%) 43–18,482 50–3,827 737/2,148 385/879 OXM 25/606 (4.1%) 5/214 (2.3%) 42–24,902 42–280 124/1,278 108/119 MTD 7/703 (1.0%) 1/251 (0.4%) 1,534–4,640 11,896 3,401/3,141 na/na EDDP 7/703 (1.0%) 1/251 (0.4%) 139–1,277 1,721 370/582 na/na BUP 5/14 (35.7%) 2/6 (33.3%) 52–5,491 37–175 299/1,271 na/na NBUP 4/14 (28.6%) 2/6 (33.3%) 9–54 35–62 23/28 na/na THCA 255/1,539 (16.6%) 110/551 (20.0%) 0.12–146 0.15–106 6.4/13.2 8.4/15 ALP 22/835 (2.6%) 2/304 (0.7%) 57–1,117 61–148 207/315 na/na DIAZ 8/835 (1.0%) 2/304 (0.7%) 43–461 43–247 190/216 na/na NDIAZ 9/835 (1.1%) 2/304 (0.7%) 55–638 389–438 93/179 na/na EtG 157/650 (24.2%) 16/66 (24.2%) 20–3,121 23–254 82/186 59/75 Analyte Number of samples ≥LOQ/total samples tested (% of samples ≥LOQ) Minimum–maximum range (pg/mg) Median/mean (pg/mg) Fingernails Toenails Fingernails Toenails Fingernails Toenails AMP 212/1,542 (13.7%) 99/550 (18.0%) 42–29,105 40–44,851 980/1,879 843/2,906 MET 190/1,542 (12.3%) 73/550 (13.3%) 80–249,706 47–344,619 3,784/16,148 3,021/15,717 MDMA 4/1,542 (0.3%) 1/550 (0.2%) 70–3,834 99 2,564/2,258 na/na COC 84/1,540 (5.5%) 16/550 (2.9%) 159–265,063 306–6,067 2,101/18,510 1,281/2,143 BE 82/1,540 (5.3%) 16/550 (2.9%) 41–45,939 166–6,887 1,285/4,803 594/1,293 NCOC 38/1,540 (2.5%) 3/550 (0.5%) 20–2,357 68–144 120/289 71/94 CE 21/1,540 (1.4%) 2/550 (0.4%) 22–463 221–252 61/90 na/na 6-MAM 30/1,540 (1.9%) 6/550 (1.1%) 51–118,229 122–22,935 2,959/18,645 1,051/4,681 COD 31/1,540 (2.0%) 11/550 (2.0%) 41–6,882 45–805 192/816 130/201 MOR 57/1,540 (3.7%) 15/550 (2.7%) 47–66,666 40–11,857 780/7,209 285/1,466 HYM 41/1,540 (2.7%) 12/550 (2.2%) 41–62,857 53–1,188 127/1,813 115/283 HCOD 134/1,540 (8.7%) 44/550 (8.0%) 59–13,473 113–8,611 913/1,663 753/1,363 OXC 107/606 (17.7%) 29/214 (13.6%) 43–18,482 50–3,827 737/2,148 385/879 OXM 25/606 (4.1%) 5/214 (2.3%) 42–24,902 42–280 124/1,278 108/119 MTD 7/703 (1.0%) 1/251 (0.4%) 1,534–4,640 11,896 3,401/3,141 na/na EDDP 7/703 (1.0%) 1/251 (0.4%) 139–1,277 1,721 370/582 na/na BUP 5/14 (35.7%) 2/6 (33.3%) 52–5,491 37–175 299/1,271 na/na NBUP 4/14 (28.6%) 2/6 (33.3%) 9–54 35–62 23/28 na/na THCA 255/1,539 (16.6%) 110/551 (20.0%) 0.12–146 0.15–106 6.4/13.2 8.4/15 ALP 22/835 (2.6%) 2/304 (0.7%) 57–1,117 61–148 207/315 na/na DIAZ 8/835 (1.0%) 2/304 (0.7%) 43–461 43–247 190/216 na/na NDIAZ 9/835 (1.1%) 2/304 (0.7%) 55–638 389–438 93/179 na/na EtG 157/650 (24.2%) 16/66 (24.2%) 20–3,121 23–254 82/186 59/75 View Large

Table II. Analyte Number of samples ≥LOQ/total samples tested (% of samples ≥LOQ) Minimum–maximum range (pg/mg) Median/mean (pg/mg) Fingernails Toenails Fingernails Toenails Fingernails Toenails AMP 212/1,542 (13.7%) 99/550 (18.0%) 42–29,105 40–44,851 980/1,879 843/2,906 MET 190/1,542 (12.3%) 73/550 (13.3%) 80–249,706 47–344,619 3,784/16,148 3,021/15,717 MDMA 4/1,542 (0.3%) 1/550 (0.2%) 70–3,834 99 2,564/2,258 na/na COC 84/1,540 (5.5%) 16/550 (2.9%) 159–265,063 306–6,067 2,101/18,510 1,281/2,143 BE 82/1,540 (5.3%) 16/550 (2.9%) 41–45,939 166–6,887 1,285/4,803 594/1,293 NCOC 38/1,540 (2.5%) 3/550 (0.5%) 20–2,357 68–144 120/289 71/94 CE 21/1,540 (1.4%) 2/550 (0.4%) 22–463 221–252 61/90 na/na 6-MAM 30/1,540 (1.9%) 6/550 (1.1%) 51–118,229 122–22,935 2,959/18,645 1,051/4,681 COD 31/1,540 (2.0%) 11/550 (2.0%) 41–6,882 45–805 192/816 130/201 MOR 57/1,540 (3.7%) 15/550 (2.7%) 47–66,666 40–11,857 780/7,209 285/1,466 HYM 41/1,540 (2.7%) 12/550 (2.2%) 41–62,857 53–1,188 127/1,813 115/283 HCOD 134/1,540 (8.7%) 44/550 (8.0%) 59–13,473 113–8,611 913/1,663 753/1,363 OXC 107/606 (17.7%) 29/214 (13.6%) 43–18,482 50–3,827 737/2,148 385/879 OXM 25/606 (4.1%) 5/214 (2.3%) 42–24,902 42–280 124/1,278 108/119 MTD 7/703 (1.0%) 1/251 (0.4%) 1,534–4,640 11,896 3,401/3,141 na/na EDDP 7/703 (1.0%) 1/251 (0.4%) 139–1,277 1,721 370/582 na/na BUP 5/14 (35.7%) 2/6 (33.3%) 52–5,491 37–175 299/1,271 na/na NBUP 4/14 (28.6%) 2/6 (33.3%) 9–54 35–62 23/28 na/na THCA 255/1,539 (16.6%) 110/551 (20.0%) 0.12–146 0.15–106 6.4/13.2 8.4/15 ALP 22/835 (2.6%) 2/304 (0.7%) 57–1,117 61–148 207/315 na/na DIAZ 8/835 (1.0%) 2/304 (0.7%) 43–461 43–247 190/216 na/na NDIAZ 9/835 (1.1%) 2/304 (0.7%) 55–638 389–438 93/179 na/na EtG 157/650 (24.2%) 16/66 (24.2%) 20–3,121 23–254 82/186 59/75 Analyte Number of samples ≥LOQ/total samples tested (% of samples ≥LOQ) Minimum–maximum range (pg/mg) Median/mean (pg/mg) Fingernails Toenails Fingernails Toenails Fingernails Toenails AMP 212/1,542 (13.7%) 99/550 (18.0%) 42–29,105 40–44,851 980/1,879 843/2,906 MET 190/1,542 (12.3%) 73/550 (13.3%) 80–249,706 47–344,619 3,784/16,148 3,021/15,717 MDMA 4/1,542 (0.3%) 1/550 (0.2%) 70–3,834 99 2,564/2,258 na/na COC 84/1,540 (5.5%) 16/550 (2.9%) 159–265,063 306–6,067 2,101/18,510 1,281/2,143 BE 82/1,540 (5.3%) 16/550 (2.9%) 41–45,939 166–6,887 1,285/4,803 594/1,293 NCOC 38/1,540 (2.5%) 3/550 (0.5%) 20–2,357 68–144 120/289 71/94 CE 21/1,540 (1.4%) 2/550 (0.4%) 22–463 221–252 61/90 na/na 6-MAM 30/1,540 (1.9%) 6/550 (1.1%) 51–118,229 122–22,935 2,959/18,645 1,051/4,681 COD 31/1,540 (2.0%) 11/550 (2.0%) 41–6,882 45–805 192/816 130/201 MOR 57/1,540 (3.7%) 15/550 (2.7%) 47–66,666 40–11,857 780/7,209 285/1,466 HYM 41/1,540 (2.7%) 12/550 (2.2%) 41–62,857 53–1,188 127/1,813 115/283 HCOD 134/1,540 (8.7%) 44/550 (8.0%) 59–13,473 113–8,611 913/1,663 753/1,363 OXC 107/606 (17.7%) 29/214 (13.6%) 43–18,482 50–3,827 737/2,148 385/879 OXM 25/606 (4.1%) 5/214 (2.3%) 42–24,902 42–280 124/1,278 108/119 MTD 7/703 (1.0%) 1/251 (0.4%) 1,534–4,640 11,896 3,401/3,141 na/na EDDP 7/703 (1.0%) 1/251 (0.4%) 139–1,277 1,721 370/582 na/na BUP 5/14 (35.7%) 2/6 (33.3%) 52–5,491 37–175 299/1,271 na/na NBUP 4/14 (28.6%) 2/6 (33.3%) 9–54 35–62 23/28 na/na THCA 255/1,539 (16.6%) 110/551 (20.0%) 0.12–146 0.15–106 6.4/13.2 8.4/15 ALP 22/835 (2.6%) 2/304 (0.7%) 57–1,117 61–148 207/315 na/na DIAZ 8/835 (1.0%) 2/304 (0.7%) 43–461 43–247 190/216 na/na NDIAZ 9/835 (1.1%) 2/304 (0.7%) 55–638 389–438 93/179 na/na EtG 157/650 (24.2%) 16/66 (24.2%) 20–3,121 23–254 82/186 59/75 View Large

The total of 7,799 samples was analyzed for amphetamines (AMP, MET, MDMA, MDA and MDEA). AMP and MET were present in 14.4 and 13.7%, respectively, of the nail samples, and the concentration ranges were 40–44,851 pg/mg (median 831) and 42–572,865 pg/mg (median 3,687), respectively. As in our study, higher concentrations of MET than AMP in nails were observed by Suzuki et al. (8) with the highest MET concentration of 642 ng/mg. The authors employed incubation of specimens in 0.6 M hydrochloric acid followed by liquid–liquid extraction (chloroform–isopropanol, 3:1, v/v). The samples we collected did not show obvious concentration difference between fingernails and toenails for AMP and MET (Table II). However, previous studies showed higher concentrations of the compounds in toenails than in fingernails from limited number of tested subjects (1). There were only 22 samples found positive for MDMA (0.3%), 7 for MDA (0.09%) and 4 for MDEA (0.05%), and the concentration ranges were 43–40,165 pg/mg (median 298), 52–706 pg/mg (median 129) and 50–422 pg/mg (median 100), respectively. There was limited information in our study to compare MDA, MDMA and MDEA concentrations between fingernails and toenails. Cirimele et al. (9) reported MDA and MDMA concentrations in fingernails of 9.7 and 60.2 ng/mg, respectively, which for MDA is significantly higher than that in our study. MDMA results corroborate with our findings. The authors also noticed that the concentrations of AMP, MDA and MDMA in fingernails were slightly higher than in head hair (9). In the study by Kim et al. (10), MDA concentration in one positive sample was <143 pg/mg. The study also found KET in fingernail clippings at the concentration of 0.314 ng/mg (below the limit of quantitation). In our study, KET was found in two nail samples at concentrations of 3,772 and 12,632 pg/mg. NKET was found in one sample only and the concentration was 201 pg/mg.

COC, BE, NCOC and CE were found in 5.3, 5.2, 2.6, and 1.2% of nail samples, respectively, out of 7,787 total specimens where these analytes were requested. COC concentrations ranged from 48 to 265,063 pg/mg (median 1,768), BE from 23 to141,239 pg/mg (median 971), NCOC between 20 and 3,875 pg/mg (median 105) and the range for CE was 20–934 pg/mg (median 84). The data show that COC was present in both fingernails and toenails in higher concentrations than BE (Table I). As presented in Table II, higher concentrations of COC and BE were observed in fingernails than in toenails. Engelhart et al. (11) found higher concentrations of BE than COC in postmortem toenails from subjects of unspecified drug use history. The highest concentrations of COC and BE were 140.17 ng/g and 315.44 ng/mg, respectively. The highest concentration of NCOC was 6.78 ng/mg, and CE 2.6 ng/mg (11). Garside et al. (12) reported COC and BE to be predominant analytes in all postmortem positive nail specimens from suspected cocaine users. The application of SPE and solvent extraction followed by GC–MS quantification revealed the ratio of COC to BE to be 2–10:1. The analysis of nails in postmortem cases by Engelhart and Jenkins (13) showed maximum COC concentrations to be much higher than BE (414.1 and 170.3 ng/mg, respectively). In addition, concentrations of all COC analytes were higher in fingernails than in toenails. The NCOC concentration range was 0.11–32.7 ng/mg, much higher than in Garside's study (12) and in this report (20–3,875 pg/mg). CE levels were similar to reported by Garside et al. (12) and here.

Concentrations of selected opioids (MOR, COD, 6-MAM, HCOD and HYM) in nail samples are presented in Table I. The levels overall ranged from 40 to 118,229 pg/mg (median 123–830). The most prevalent of the above five opioids was HCOD. It was detected in 11.4% of 7,779 tested nail samples, compared with 1.5–3.6% for the other four. 6-MAM, heroin metabolite, was present in 115 samples out of 7,779 and the concentration range was 45–118,229 pg/mg. Concentrations of all opioids were much higher in fingernails than in toenails (Table II). Engelhart et al. (11) found MOR, 6-MAM and HCOD in postmortem toenails from subjects of unspecified drug use history at average concentrations 0.37, 0.89 and 0.62 ng/mg, respectively, and they were significantly lower than in our report (Table II). In another study, the same author (13) reported the presence of MOR, 6-MAM, COD, HYM, OXC and HCOD in postmortem nail clippings from subjects with documented drug abuse history and/or drug-related homicide. Concentrations of MOR and 6-MAM were this time significantly higher than in our report (0.05–407.9 and 0.04–504 ng/mg, respectively). COD concentrations in postmortem nails corresponded well to concentrations observed by us. Other opioids including MTD, EDDP, OXC and OXM were found in 1.2, 1.0, 15.1, and 2.6%, respectively, of approximately 3,400 samples analyzed. MTD and its metabolite EDDP were found in 44 and 35 samples, respectively, out of 3,567 submitted for analysis for these analytes. The concentrations ranged from 94 to 17,859 pg/mg for MTD, and from 49 to 2,880 pg/mg for EDDP. Lemos et al. (14) found MTD in fingernail clippings collected from patients in a methadone-maintenance program. The mean MTD concentration was 26.9 ng/mg and was significantly higher than the mean concentration in this report (3,245 pg/mg).

Very limited information is available on the presence of other opioids (and opioid antagonists) such as BUP, NBUP, NALT, NAL, 6-BNAL, NALB or BTF in nails. BUP and NBUP were found in 15 and 12 nail samples, respectively, out of 40 submitted for analysis. The concentration range for BUP was 37–5,491 pg/mg (mean 782 pg/mg) and 9–216 pg/mg (mean 65 pg/mg) for NBUP. Very recently, Tzatzarakis et al. (15) reported the presence of BUP, NBUP and NAL in fingernails and urine collected from heroin users undergoing suboxone substitution therapy. The mean nail concentrations were as follows: BUP 163 pg/mg, NBUP 129 pg/mg and NAL 47.4 pg/mg. We did not receive nail samples with concomitant request for testing for both BUP/NBUP and NAL. Also, NAL was not detected in any nail sample. On the other hand, NALT was found in only two nail samples (63 and 71 pg/mg), and 6-BNAL, NALT metabolite, was present in 9 out of 18 samples received, in concentrations ranging from 115 to 2,261 pg/mg. To date, there were no prior studies reporting NALT (used in alcohol dependence treatment) and its metabolite tested in nails.

The detection of THCA is very important to differentiate between marijuana ingestion (intentional and/or passive inhalation) and external contamination (1). In our study, concentrations of THCA in nails ranged from 0.04 to 262 pg/mg (median 6.41), and the overall positivity rate was 18.1%. The mean concentration in fingernails was 13.2 pg/mg, and in toenails 15 pg/mg. The concentration ranges were also very similar suggesting that both specimens are equally useful in revealing cannabis use. Jones et al. (6) developed a highly sensitive GC–GC–MS-MS method with the limit of detection and quantification of 10 and 20 fg/mg, respectively, for quantification of THCA in 60 matched hair and nail specimens. The mean THCA concentration in nails was 1,813 fg/mg, lower than the mean concentration in this study (13.7 pg/mg).

Out of 3,039 samples submitted for analysis for EtG, 756 were positive (24.9%), and a concentration range was 20–3,754 pg/mg (median 88, mean 177). In another study by Jones et al. (7), the reported EtG mean concentration was 29.1 pg/mg, and it was three times higher than in the 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. (16) 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. Berger et al. (17) 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 with hair EtG at 30 pg/mg. 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.

Examples of other drugs found in nails include barbiturates, benzodiazepines, NMEP, PCP, TRAM and NPPX. The positivity rate for benzodiazepines in nails was below 1% except for ALP (1.6%) (Tables I and II).

In summary, as this report indicates, nails are a very useful matrix for detection of drugs and illicit substances, and for analytes such as THCA or EtG, may be a superior alternative specimen type to hair samples. Nail sample preparation is relatively simple and the advancement in analytical equipment technology allows for accurate measurements of extremely low quantities of parent compounds as well as the metabolites. Most drugs are present at greater concentration than their metabolites. NALT is an exceptional case where its metabolite 6-BNAL, not a parent drug, 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 the analytical findings.

Conclusion

Analysis of nails is a reliable way of determining the long-term use and abuse of drugs. Extraction techniques are simple and produce accurate and precise results. Sensitive analytical instrumentation allows for detection of femtogram quantities of substances in nails. Prescription drugs, drugs of abuse and illicit substances can be detected and quantified in both fingernails and toenails in a wide range of concentrations. Except for AMP, the mean concentrations of drugs in fingernails are higher than in toenails. With very few exceptions, parent compounds typically reach higher concentrations in nails than their metabolites. More research studies are necessary to better understand the significance and the meaning of nail results.

References

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