Morphological changes in human head hair subjected to various drug testing decontamination strategies.

Morphological changes in hair subjected to decontamination protocols were evaluated by scanning electron microscopy (SEM) as part of the National Laboratory Certification Program's (NLCP) efforts to develop proficiency testing materials in support of Federal Workplace Drug Testing programs. Hair from five different donors was evaluated. Hair samples were subjected to three decontamination protocols: (1) aqueous phosphate buffer, (2) methanol or (3) methylene chloride as models for aqueous, alcohol and polar organic solvent protocols, respectively. Under these protocols, samples of hair were treated for 225 min (aqueous), 15 min (alcohol), or 15 min (polar organic). After decontamination, hair strands were sputter coated with gold/palladium (AuPd) and observed by SEM. Modest lifting of cuticle scales was observed in hair treated with methanol and methylene chloride consistent with some changes to the cell membrane complex (CMC) between cuticle scales. Damage resulting from aqueous buffer treatment ranged from substantial degradation to apparent complete loss of cuticle scales. Fracture structures consistent with cuticle damage were also observed. Each decontamination protocol had a different impact on the cuticle of the hair shaft.

Hair Testing for Drugs - Challenges for Interpretation.

While testing hair for drugs of abuse has become more widely used in the past 30 years, significant challenges still remain to the interpretation of the results from these tests. Of primary concern is the likelihood of unwitting contamination from the environment producing a result indicative of drug use. It is imperative that this possibility be controlled and understood so that results from hair testing can be appropriately interpreted. Presently, truly unique metabolites are needed for many drugs (THC-COOH being a notable exception) since the mechanisms of decontamination processes used for hair are still poorly understood. While there is evidence that many drugs preferentially bind to melanin and that darker hair contains more drug, it is unclear how this translates into the interpretation of results for a population. Cosmetic treatments likely reduce the amount of drug systemically incorporated into hair and thus present a challenge of inappropriately negative interpretation. In addition, hair damaged as a result of cosmetic treatment may be at increased risk for environmental contamination. The asynchronous nature of human hair growth can also complicate results by obscuring the timeline of drug deposition. Lastly, the analytical variation of quantitative values has been demonstrated by several proficiency testing systems throughout the world to be high and method specific. Thus, while hair testing may be able to provide information about exposure to drugs, it is difficult at present to obtain reproducible results that can be unquestionably related to drug ingestion or to infer that the absence of drug in a hair sample is positive proof of no drug usage. Many questions still remain to be addressed by mechanistic understanding of drug deposition and retention in hair.

Occupational exposure to methamphetamine in workers preparing training AIDS for drug detection dogs.

As a part of ongoing testing of personnel preparing training aids for drug detection dogs at the Naval Criminal Investigative Service Regional Forensic Laboratory, personnel handling methamphetamine (MTH) were subject to voluntary urine drug testing. This provided a model of potential unwitting or environmental exposure contribution to MTH concentrations in urine. Urine samples were collected from multiple individuals on the day before, the day of, and the day after the individuals had handled up to 500-g quantities of MTH during the assembly of training aids. Personnel wore gloves, dust masks, and lab coats during the preparation of training aids. A total of 101 urine samples were analyzed for the presence of MTH and amphetamine (AMP) by gas chromatography-mass spectrometry after solid-phase extraction and derivatization. Urine samples collected during and after personnel handled drug yielded a mean MTH concentration of 48 ng/mL with a maximum concentration of 262 ng/mL and a minimum detected concentration of approximately 1.6 ng/mL. Thirty-five of the 52 post drug-handling samples had detectable MTH. Ten of the samples had MTH concentrations above the method limit of quantitation of 15 ng/mL. Only one sample had a concentration greater than 50 ng/mL. None of the samples had detectable AMP. From this limited study, it was evident that handling of MTH under these conditions resulted in minimal exposure and small but detectable concentrations of MTH in urine.

Mechanisms of Drug Deposition in Hair and Issues for Hair Testing.

The purpose of this review is to provide a summary of the knowledge of the mechanisms by which drugs deposit into hair. While much work and many reviews have been produced on methods of analysis of drugs in hair, a smaller body of work and fewer reviews have concentrated on the biochemical mechanisms by which drugs deposit and are retained in hair. Proposed mechanisms are discussed with various studies that have been used to elucidate these mechanisms. While multiple mechanisms are likely involved in the deposition and retention of drugs in hair, melanin and various interactions with melanin including potential covalent linkage of drug with melanin appear to dominate the scheme of interactions. The implications of these interactions are discussed with particular emphasis on the potential difficulties in distinguishing internal deposition from external contamination and the appropriateness of manufacturing control materials. The potential for hair testing to be truly reflective of endogenous deposition may hinge on the ability to isolate and analyze melanin adducts of drugs that would be indicative of endogenous drug deposition during hair growth and active melanogenesis.

Opioids - Effects on Human Performance and Behavior.

The purpose of the monograph is to provide readers with a summary of the literature relating selected opioids to performance issues, specifically driving. This monograph provides a summary of information to aid expert witnesses in preparing for court testimony. Information for codeine, hydrocodone, hydromorphone, methadone, morphine, and oxycodone is provided. In addition to a review of performance studies, a summary of acute and chronic pharmacology, pharmacokinetics, and metabolism is included. Opioids appear to impair psychomotor functioning likely to be important to the performance of complex, divided attention tasks such as driving. This impairment is notably more prevalent in individuals with no history of opioid use; individuals with long-term opioid use do not demonstrate as extensive of an impairment. Other factors such as personality, environment, and pain control also sharply modulate opioid impairment.

Solid-phase extraction and GC-MS analysis of THC-COOH method optimized for a high-throughput forensic drug-testing laboratory.

In order to facilitate the confirmation analysis of large numbers of urine samples previously screened positive for delta9-tetrahydrocannabinol (THC), an extraction, derivitization, and GC-MS analysis method was developed. This method utilized a positive pressure manifold anion-exchange polymer-based solid-phase extraction followed by elution directly into the automated liquid sampling (ALS) vials. Rapid derivitization was accomplished using pentafluoropropionic anhydride/pentafluoropropanol (PFPA/PFPOH). Recoveries averaged 95% with a limit of detection of 0.875 ng/mL with a 3-mL sample volume. Performance of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH)-d3 and THC-COOH-d9 internal standards were evaluated. The method was linear to 900 ng/mL THC-COOH using THC-COOH-d9 with negligible contribution from the internal standard to very weak samples. Excellent agreement was seen with previous quantitations of human urine samples. More than 1000 human urine samples were analyzed using the method with 300 samples analyzed using an alternate qualifier ion (m/z 622) after some interference was observed with a qualifier ion (m/z 489). The 622 ion did not exhibit any interference even in samples with interfering peaks present in the 489 ion. The method resulted in dramatic reductions in processing time, waste production, and exposure hazards to laboratory personnel.

3H-nicotine, 3H-flunitrazepam, and 3H-cocaine incorporation into melanin: a model for the examination of drug-melanin interactions.

To explore drug-melanin interactions, we examined the in vitro tyrosinase-mediated formation of melanin from tyrosine in the presence of the 3H-cocaine (3H-COC), 3H-flunitrazepam (3H-FLU), and 3H-nicotine (3H-NIC) at 10-100,000 ng/mL. Polymerization in the presence of 10 or 100 ng/mL of each drug resulted in almost complete drug incorporation into the melanin pellet. Only 12% (3H-NIC) to 28% (3H-FLU) of the pellet-associated radioactivity could be released upon treatment with 6 M HCl. At 1000-100,000 ng/mL, between 20 and 50% of label became melanin-associated. In each case a significant percentage of melanin-associated radioactivity was resistant to treatment with 6 M HCl. Nicotine-associated radioactivity in the polymer was subject to much greater quenching than was 3H-COC or 3H-FLU, suggesting a much tighter association with the melanin. The subsequent demonstration of a covalent adduct of a melanin intermediate and nicotine has demonstrated the utility of this polymerization system as a model for further chemical characterization of drug-melanin interactions.

Loss of THCCOOH from urine specimens stored in polypropylene and polyethylene containers at different temperatures.

The loss of delta9-tetrahydrocannabinol (THCCOOH) from urine specimens stored in polypropylene and polyethylene containers at 4 degrees C and 25 degrees C was examined. All specimens were analyzed by GC-MS after sampling at various times over a one-week period. Data were analyzed by one-way analysis of variance and fitted with a first order kinetic equation. Rapid loss of THCCOOH was seen at 4 degrees C for both polypropylene (14% maximal loss, t(1/2) = 0.53 min) and polyethylene (17% maximal loss, t(1/2) = 5.77 min) bottles. At 25 degrees C, a small loss (< 5%) was observed in polypropylene and no significant loss was seen for urine in polyethylene. All losses stabilized within 1 h, and no further losses were seen over one week. The results indicate that THCCOOH binding may be due to decreased solubility of THCCOOH at lower temperatures and subsequent association of THCCOOH with the more lipophilic plastic. The results also indicate that polypropylene and polyethylene do not bind THCCOOH to such an extent as to compromise the integrity of specimens.

Incorporation and retention of radiolabeled S-(+)-and R-(-)-methamphetamine and S(+)- and R(-)-N-(n-butyl)-amphetamine in mouse hair after systemic administration.

We examined the incorporation of unlabeled and tritiated enantiomers of methamphetamine (MA) and a more lipophilic analog N-(n-butyl)-amphetamine (BA) into the hair of pigmented (C57) and nonpigmented (Balb/C) mice after systemic administration. We also compared the ability of extraction methods to remove unlabeled and tritiated MA and BA enantiomers from the hair. R(-)-MA, S(+)-MA, [(3)H]R(-)-MA, [(3)H]S(+)-MA, R(-)-BA, S(+)-BA, [(3)H]R-(-)-BA, and [(3)H]S-(+)-BA were each administered to C57 and Balb/C mice (23 days of age) by i.p. injection at 8.8 mg/kg daily for 3 days. At 44 days of age, hair samples from the animals were treated with a brief methanol wash, a 24-h extraction with pH 6 phosphate buffer, and a final digestion in 1 N NaOH to free residual drugs from the hair. Labeled drugs in the extracts were quantitated by liquid scintillation counting. Unlabeled drugs were quantitated by gas chromatography/mass spectrometry (GC/MS). GC/MS analysis demonstrated MA and BA to be the major (>90%) species present in the blood during the 24 h after administration. Less than 10% of the MA was N-demethylated. No p-hydroxylated metabolites were found. Blood concentrations of tritiated MA and BA enantiomers measured by liquid scintillation counting agreed well with blood concentrations of unlabeled enantiomers measured by GC/MS. Hair concentrations of S(+)-MA were greater than those of R(-)-MA in both mouse strains, paralleling blood concentrations. There were no enantiomeric differences seen with BA hair accumulation in either strain of mouse. Significantly more MA and BA enantiomers were deposited in pigmented than in nonpigmented hair. With labeled and unlabeled compounds, approximately 30% of S(+)-MA and 60% of R(-)-MA in pigmented hair could be removed by a phosphate extraction. A significant amount of drug could not be removed from the hair by extraction. Greater amounts of drug could be extracted from nonpigmented hair than pigmented. Extracted and residual MA and BA concentrations in pigmented hair were significantly greater when labeled compounds were quantitated by liquid scintillation counting than when unlabeled compounds were quantitated by GC/MS. However, radiotracer and unlabeled drug concentrations were the same in nonpigmented hair. The results demonstrate that hair pigmentation is an important determinant in MA and BA deposition, and that MA and BA deposition is not enantioselective. The data demonstrate a significant amount of MA and BA accumulated is not easily amenable to exhaustive aqueous extraction from the hair. The use of tritiated MA and BA enantiomers demonstrates that a significant amount of MA and BA stored in pigmented hair is structurally different from parent MA and BA, perhaps associated with melanin components of hair.

A comparison of sodium hydroxide and sodium sulfide digestion of mouse hair in the recovery of radioactivity following systemic administration of [3H]-nicotine and [3H]-flunitrazepam.

Pigmented (C57BI) and nonpigmented (balb/c) mice, 25 days of age, were treated intraperitoneally with [3H]-nicotine (4 mg/kg, 555 dpm/ng) or [3H]-flunitrazepam (1 mg/kg, 2200 dpm/ng) daily for three days. After 21 days, shaved back hair was digested at 37 degrees C for 24 h with either 1 M sodium hydroxide or 1 M sodium sulfide. With both drugs, sodium sulfide extraction removed the same amount of radioactivity as sodium hydroxide from nonpigmented hair. However, sodium sulfide removed significantly more radioactivity from pigmented hair than did sodium hydroxide. In pigmented hair, sodium sulfide solubilized 35% and 74% of the flunitrazepam- and nicotine-associated radioactivity, respectively. Of this, 12% and 43%, respectively, could be partitioned into ethyl acetate. Microscopic examination of residual pellets after digestion demonstrated a more thorough dissolution of the hair shaft with sodium sulfide with only melanosomes remaining. The results demonstrate the significant interaction of flunitrazepam and nicotine with melanins and the utility of sodium sulfide in increasing drug recovery.

Histologic localization of serum constituents, (45)Ca(2+), (36)Cl(-), [(14)C]urea, and [(35)S]cysteine in forming hair after systemic administration.

To further investigate the chemical mechanisms involved in the accumulation of drugs or other compounds in hair, we characterized histologically the deposition of radiolabeled serum constituents in the hair of BALB/c (albino) and C57 (pigmented) mice. The extent and location of the incorporation of a normal serum cation ((45)Ca(2+)), a serum anion ((36)Cl(-)), a neutral constituent ([(14)C]urea), and a structural component of hair ([(35)S]cysteine) were studied to provide a comparative framework for the examination of drugs deposited in hair from serum. Two mouse strains were used to evaluate the effect of hair pigmentation on deposition. Localization of deposition was observed using microautoradiography of skin sections from animals given a systemic dose of each tracer. The cation, (45)Ca(2+), associated with melanocytes and melanosomes of forming C57 hair within 5 min of dosing, but did not associate with the cells of forming BALB/c hair. This was consistent with previous results that indicated greater concentrations of Ca(2+) in mature C57 mouse hair when compared with mature BALB/c hair. Both [(14)C]urea and [(35)S]cysteine associated with all cells in the papilla of the forming hair of both C57 and BALB/c mice. This again was consistent with previous results that indicated that similar concentrations of cysteine and urea were incorporated into mature C57 and BALB/c hair. The anion, (36)Cl(-), did not associate with either C57 or BALB/c hair. The lack of deposition of (36)Cl(-) may be due to the loss of the tracer during sample processing and suggests that Cl(-) could be removed from mature hair. These data confirm previous results that suggested that the melanin component of hair was capable of ionic interactions and that the protein component was capable of neutral, lipophilic interactions. Our findings suggest a multicompartmental model of drug deposition in hair.

Deposition of [3H]cocaine, [3H]nicotine, and [3H]flunitrazepam in mouse hair melanosomes after systemic administration.

Microautoradiography was employed to show that association of drugs from the serum directly with forming hair pigment is a primary pathway of deposition into the hair. After systemic administration of [3H]flunitrazepam, [3H]nicotine, and [3H]cocaine, association of all three drugs with melanin in the forming hair was observed within minutes of dosage. Sebum was determined to be an insignificant deposition route for all three drugs. Pigmented mice had significantly higher concentrations of all three drugs than did nonpigmented mice. The results provide a better basis for ultimately using hair for reliable analysis of drug and environmental toxin exposure.

Comparison of in vivo and in vitro deposition of rhodamine and fluorescein in hair.

A direct differentiation of the internal and external drug-deposition pattern into hair was made using two fluorescent dyes and fluorescence microscopy after systemic administration to mice or external exposure of untreated hair. Mice (23 days old, C57 and Balb/C) were administered either rhodamine or fluorescein intraperitoneally at varied doses on 3 consecutive days of 3 weeks, and hair was sampled 1 week later. Another group was given 10 mg/kg rhodamine or 100 mg/kg fluorescein and sampled at time points from 5 min to 168 hr. The time courses of external deposition of rhodamine and fluorescein into untreated hair were examined after hair was soaked in 0.1 mg/ml solutions at pH 3, pH 6, and pH 9 aqueous buffer or methanol. The hair was then extracted in pH 6 phosphate buffer or methanol for 24 hr. In vivo accumulation was distinguishable as fluorescent bands along the length of the hair for rhodamine and fluorescein. The pattern of in vivo deposition appears to arise from the rapid accumulation within the cortex and medulla, with little deposition evident in the cuticle. Neither phosphate buffer nor methanol washes affected the intensity of fluorescence in the hair. External loading of rhodamine into the hair resulted in staining of the junctions of cuticle scales. This pattern persisted even after 12 hr of solution exposure. Extraction with pH 6 phosphate buffer or methanol did not remove rhodamine. Fluoroscein followed a similar pattern, with maximum fluorescence when hair was loaded in pH 6 100mM phosphate buffer and nominal staining when loaded in pH 9 100 mM Tris buffer or methanol. Soaking the hair in pH 6 buffer, but not methanol, removed some fluorescein. These results demonstrate that compounds in the circulation can rapidly diffuse into the forming cortex and medulla, where rapid associations occur with elongating intermediate filaments specific to the medulla and cortex. These compounds can become significantly occluded within the mature matrix and are resistant to removal in aqueous or methanolic solutions.

Deposition and retention of radiolabeled serum constituents in hair after systemic administration.

To investigate the chemical mechanisms involved in the accumulation of drugs or other compounds in hair, we examined the deposition of radiolabeled serum constituents in the hair of BALB/c (albino) and C57 (pigmented) mice. The extents of in vivo incorporation of a normal serum cation (45Ca2+), a serum anion (36C1-), a neutral constituent ([14C]urea), and a structural component of hair ([35S]cysteine) were studied to provide a reference framework for the examination of foreign substances deposited in hair from serum. The use of two mouse strains allowed evaluation of the effect of hair pigmentation on levels of accumulation. Additionally, the endogenous contents of Mg2+, Na+, and K+ (measured by inductively coupled plasma-atomic emission spectroscopy) were determined, as was their stability to removal. Hair concentrations of isotopes were calculated from mean specific activities determined over the treatment period and corrected for quenching and decay. 45Ca2+ accumulation (500 ng/mg of hair in C57 mice and 25 ng/mg of hair in BALB/c mice) was unaffected by 24-hr phosphate buffer extraction. Of the [14C]urea accumulated (3500 ng/mg in C57 and BALB/c mice), 50% was removed by 24-hr extraction in phosphate buffer. Of the 36C1- accumulated (65 ng/mg in C57 mice and 30 ng/mg in BALB/c mice), one half was removed by 24-hr extraction in phosphate buffer. The accumulated [35S]cysteine (210 ng/mg in C57 mice and 110 ng/mg in BALB/c mice) could not be removed. Endogenous Mg2+ (350 ng/mg in C57 mice and 75 ng/mg in BALB/c mice) was stable to 24-hr extraction with phosphate buffer. K+ (2500 ng/mg) and Na+ (400 ng/mg) concentrations were approximately equal in the two strains and were largely extractable. Based on the accumulation of a neutral serum constituent (urea), the data suggest that factors other than ionic binding are important in the deposition of circulating molecules into hair. The extent and reversibility of ionic binding are dependent on the chemical nature of the binding substance. The presence of hair pigmentation greatly increased the accumulation of 45Ca2+, 36C1-, and [35S]cysteine. These data suggest a multicompartmental nature of drug storage in hair.

Fentanyl in hair. Chemical factors involved in accumulation and retention of fentanyl in hair after external exposure or in vivo deposition.

The levels of fentanyl extractable from mouse hair after chronic systemic administration and the suitability of externally loaded hair samples for establishing control and comparison samples were determined. Additionally, the effects of chemical modification of specific polar functionalities within the hair protein matrix on the deposition and recovery of fentanyl in hair subjected to external loading were determined. BALB/c mice entering a second phase of synchronized hair growth were treated ip with fentanyl (0.02, 0.05, or 0.10 mg/kg) on Monday, Wednesday, and Friday for 3 weeks. At that time, fentanyl concentrations in hair, as determined by GC/MS, were 0.025-0.050 ng/mg of hair. Hair samples exposed to fentanyl in phosphate buffer (ionized drug) showed no significant accumulation of drug into the hair, as determined by loss of fentanyl from the loading solution or by extraction of the hair. Hair samples exposed to nonionized fentanyl in methanolic solution (10, 50, and 100 ng/ml) showed significant accumulation of drug in the hair and significant removal of drug from the incubation solution. Fentanyl removal from solution plateaued after 24 hr, suggesting equilibration between fentanyl in solution and fentanyl in the hair. A mass balance between drug lost from the incubation solution and drug recovered from hair samples suggests that 94% of accumulated fentanyl is tightly bound to the hair matrix or resides in water-inaccessible compartments within the hair. These results suggest that fentanyl accumulation after in vivo administration differs, in the nature of storage, from fentanyl accumulation from external solutions and that external spiking of hair may not provide suitable control samples. Chemical modification of hair protein functionalities (reaction with diazomethane to esterify carboxylic acid groups or with acetic anhydride and pyridine to acetylate amine and hydroxyl functionalities) led to reproducible protein structure modification, as demonstrated by Fourier transform-IR and by pH titration. Hair from BALB/c mice was used. The accumulation of fentanyl was examined in hair samples exposed to fentanyl in methanol or methylene chloride solutions (10 ng/ml, 24 hr). Fentanyl was recovered from hair by 24-hr extraction in phosphate buffer, pH 6. Esterification of hair resulted in significantly less uptake of nonionized fentanyl from a methanolic solution and significantly lower recovery of drug from hair, relative to untreated hair, suggesting that carboxylic acid functionalities are necessary for the incorporation of drug. Acetylation of hair resulted in increased removal of fentanyl from methylene chloride solutions and increased recovery of fentanyl. This is consistent with the creation or expansion of a less polar compartment. Fentanyl uptake from a methanolic solution was also greater in acetylated hair. These results demonstrate that solution-accessible ionizable functionalities of hair play a significant role in the accumulation and retention of nonionized fentanyl from organic solutions.