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Articles |
1
National Board of Forensic Medicine, Department of Forensic Chemistry, University Hospital, 581 85 Linköping, Sweden.
2
Department of Clinical Chemistry, Faculty of Health,
University of Linköping, 581 85 Linköping, Sweden.
a Author for correspondence. Fax 46-13-10-48-75; e-mail robert.kronstrand{at}rk.rmv.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Nine human subjects were given codeine as a single oral dose, and plasma codeine concentrations were determined for 24 h, using gas chromatography–mass spectrometry. Hair samples were obtained weekly for a month. Total melanin, eumelanin, and codeine were measured quantitatively in hair samples by spectrophotometry, HPLC, and gas chromatography–mass spectrometry, respectively.
Results: There was an exponential relationship between codeine and melanin concentrations in hair, (r2 = 0.95 with total melanin and r2 = 0.83 with eumelanin). After normalizing the results by the area under the curve for codeine in plasma, we obtained r2 = 0.86 for codeine vs total melanin and r2 = 0.90 vs eumelanin.
Conclusions: Our results stress the importance of melanin determination when measuring drugs in hair. We postulate that analysis of drug concentration in hair may be worthwhile in the monitoring of drug compliance if the results are normalized for melanin content.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hair is a complex epidermal outgrowth synthesized in the hair follicle. It is composed of 65–95% protein, 1–9% lipids, 0.1–5% pigments (melanins), and small amounts of trace elements, polysaccharides, and water. Melanins are polyanion polymers containing negatively charged carboxyl groups and o-semiquinones at physiological pH. The exact structures of the melanin polymers are not known because they can be complex copolymers of both eumelanin and phaeomelanin. The melanin polymers are usually attached to the protein matrix of the hair (1). The subdivision of melanin into two classes (eumelanins and phaeomelanins) is based on their different colors, solubilities, and sulfur content. Eumelanin is a dark brown to black pigment that is insoluble in acid and alkali and contains nitrogen (6–9%) but insignificant amount of sulfur (0–1%). Phaeomelanin is a yellow to reddish-brown pigment that is soluble in alkali and possesses both nitrogen (8–11%) and sulfur (9–12%). The two classes of melanins are also chemically distinct: eumelanin is mainly a polymer of monomer units of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, whereas phaeomelanin contains benzothiazine units derived from cysteinyldopa (2). Hair color is determined by both the quantity and the type of melanin incorporated into the hair shaft. Brown and black hairs have high eumelanin concentrations, whereas blond hair has low concentrations. Red hair usually has a mixture of both phaeomelanin and eumelanin.
Ozeki et al. (3) and Ito and co-workers (4)(5) described methods for quantitative analysis of melanin in hair and other tissues. These methods were applied extensively to pigmentary research. In one of them, the sample was solubilized in an organic base before the melanin concentration is determined by absorbance measurement (3). This method seemed to be a straightforward method to measure total melanin. We wanted not only to estimate the total amount of melanin but to sort out subjects with high eumelanin content in their hair. Several compounds obtained when pigments are degraded by chemical means would be suitable (6), and an appropriate candidate seemed to be pyrrole-2,3,5-tricarboxylic acid (PTCA). A method based on oxidation of the sample followed by determination of PTCA from eumelanin was recently described by Ito and Wakamatsu (4).
The binding of certain drugs and inorganic cations to melanin has been studied extensively both in vitro and in vivo by Larsson and co-workers (7)(8)(9). They concluded that the binding and accumulation of these chemicals in pigmented tissue is a pronounced retention mechanism in the body. Furthermore, the affinities of different chemicals for melanin vary considerably.
Organic amines and metal ions have high melanin affinity (7)(8). These substances are positively charged at physiological pH and interact with the melanin polymer by electrostatic forces between their cationic groups and the negative charges in the melanin polymer. The electrostatic binding of the substances are strengthened by van der Waals forces between aromatic indole rings in the melanin polymers and aromatic rings of the organic amines. Melanin may also be involved in charge transfer when electron-donating complexes interact with the melanin. Hydrophobic interactions with aliphatic molecules are extensive because of the hydrophobic core of the melanin polymer.
Several authors have made in vivo studies of drug incorporation into hair and differences between pigmented and nonpigmented hair. These studies have been conducted with haloperidol (10)(11), chlorpromazine (12), methadone (13), cocaine (14), and codeine (15)(16)(17). These weak bases are protonated and positively charged at physiological pH. Drugs that do not form cations at physiological pH would be equally distributed in pigmented and nonpigmented hair, and this has been shown with phenobarbital (18).
Rollins et al. (16) investigated the relationship between the ingested dose and the amount of codeine incorporated into human hair. Their subjects were a homogeneous group of dark-haired male volunteers who ingested single or multiple doses of codeine. Within this group, the codeine concentration in hair corresponded well to the ingested dose and to plasma concentration. However, because they did not measure the actual amount and type of melanin in the hair, only arbitrary relationships between incorporation and hair color or ethnic origin were possible.
Slawson et al. (19) measured the amounts of eumelanin and phaeomelanin in hair from rodents given phencyclidine and related the incorporation of phencyclidine to the melanin content. They found 30- to 70-fold more phencyclidine in pigmented hair than in nonpigmented hair from the same animal and concluded that eumelanin rather than phaeomelanin played the major role in the incorporation of phencyclidine into hair.
To evaluate dose-concentration relationships for forensic and clinical applications of hair analyses, controlled dosage studies should be performed in humans and methods for hair melanin and drug analysis should be used. The aim of our study was to investigate the relationship between melanin content of hair and the incorporation of codeine into hair after an oral intake of codeine.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The subjects were allowed to continue their usual hair hygiene
during the study, but no hair treatments such as dyes, permanents, or
bleach were allowed.
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After an overnight fast, the subjects were given 100 mg of codeine orally as a single dose. Blood samples were drawn in 10-mL heparin-containing tubes before and 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, and 24 h after administration of codeine. Plasma was obtained by centrifugation and stored at 4 °C until analyzed. Hair samples were obtained before codeine intake and at days 7, 14, 21, and 28. Samples (triplicates) were obtained from the posterior vertex and cut with scissors as close to the scalp as possible. To ensure parallel alignment, the samples were folded in aluminum foil and stored in the dark at room temperature until analyzed.
The plasma area under the curve (AUC) for codeine was estimated from the plasma determinations during the first 24 h after administration, using the trapezoid rule.
chemicals and reagents
Sepia melanin (99%) and 5-hydroxyindole-2-carboxylic acid were
purchased from Sigma, and Soluene-350 was a product of Packard
(Chemical Instruments AB). The reference materials codeine,
morphine, codeine-d3, and
morphine-d3 were purchased from
Radian. The derivatizing reagent
N-methyl-N-trimethylsilyltrifluoroacetamide
was purchased from Macherey & Nagel. All other solvents and inorganic
chemicals were of analytical grade.
melanin determination
Spectrophotometric assay of total melanin and relative amount of
eumelanin.
The total melanin and relative amount of eumelanin were
determined according to Ozeki et al. (3) with minor changes
in the pretreatment of the hair samples. The hair samples were washed
with isopropanol and isopropanol:water (2:1, by volume) to clean
the hair surface. Approximately 40 mg of hair was then ground to a fine
powder with a Retsch MM 2000 mixer-mill for 20 min with an amplitude of
60. Depending on hair color, 4–8 mg of hair was then weighed directly
into a 10-mL screw-capped glass tube. After addition of 2.0 mL of 100
mL/L water-900 mL/L Soluene-350, the samples were mixed for
10 s and placed in a water bath at 80 °C for 30 min. The
samples were then remixed and placed in the water bath for another 15
min. The samples were cooled to room temperature, and the absorbance
was measured at 500 and 650 nm, using a Perkin-Elmer Lambda 2,
double-beam ultraviolet/visible spectrophotometer. Background
correction was performed with 100 mL/L water-900 mL/L Soluene-350.
Human albino hair was analyzed in each series, and its absorbance was
subtracted from the hair samples.
The total concentration of melanin in the hair samples was obtained from a sepia melanin calibration curve that we constructed by solubilizing 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 200 µg of sepia melanin together with rabbit albino hair. After subtraction of the appropriate blank, the same response was obtained as with pure melanin but with better precision. Rabbit albino hair was used in the calibrators because of limited availability of human albino hair. Although the reason that albino hair was included in the calibrators was that it gave better reproducibility of the calibration curve, it also gave a matrix similar to that of the human samples. Values for unknown samples were converted to µg melanin/mg hair by dividing by the actual amount (4–8 mg) of hair used.
The spectral curves of phaeomelanin and eumelanin differ in such a way
that the absorbance of phaeomelanin is lower than the absorbance of
eumelanin at 650 nm (Fig. 1
.) Taking this into account, we obtained the relative amount of
eumelanin in the hair samples by dividing the absorbance at 650 nm
(A650) with the absorbance at 500 nm
(A500). A low
A650/A500
ratio indicates a phaeomelaninic hair sample, and a high ratio
indicates a eumelaninic hair sample.
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The sample amount was adjusted so that the absorbance of the hair samples was 0.2–0.8 absorbance units at 500 nm.
HPLC analysis of PTCA.
The hair samples were prepared
according to Ozeki et al. (3). A 30-mg sample of hair was
homogenized in 3 mL of water with a Potter Elvehjem glass homogenizer
(Kebo Laboratory). Aliquots of the hair suspensions were oxidized
according to Ito and Fujita (20).
A 200-µL suspension containing 2 mg of hair was added to 1.0 mL of 1 mol/L H2SO4. The samples were mixed, and then a 15-µL aliquot of a 30 g/L potassium permanganate solution was added. The samples were repeatedly mixed, and a new aliquot of permanganate was added as soon as the purple color disappeared.
Ten minutes after the first addition of potassium permanganate, the oxidation was stopped by addition of 100 µL of 100 g/L Na2SO3. PTCA was then extracted with 5 mL of diethyl ether. The samples were centrifuged at 4000g for 5 min, and the organic phase was removed and evaporated under a stream of nitrogen. The residue was reconstituted in 200 or 400 µL of deionized water, depending on the subject hair color.
Together with each series of hair samples, a sample containing 5-hydroxyindole-2-carboxylic acid and 2 mg of albino hair was oxidized to establish the retention time of the PTCA peak. A blank sample with albino hair was oxidized and analyzed in each series.
The calibration samples were prepared and analyzed according to the same procedure. A calibration curve for eumelanin was constructed by oxidation of 0.5, 1.0, 1.5, 2.0, 2.5, 5.0, 7.5, 10.0, 12.5, and 15 µg of sepia melanin per milligram of albino hair. Because we obtained an exponential calibration curve for melanin in our initial analytical experiments, we added serum albumin to the calibrators according to the procedure described by Ito et al. (4) and obtained a linear response (r2 = 0.990). Substitution of albino hair for albumin produced higher recoveries and kept the curve linear (r2 = 0.996). Rabbit albino hair was used in the calibrators because of limited availability of human albino hair. The sample amount (2 mg of hair) was kept constant because the recoveries of PTCA from permanganate oxidation varied with sample amount.
PTCA was determined with HPLC and ultraviolet detection. The pump was a Waters 501 pump, and the detector was a Waters 480 lambda-max detector set at 270 nm. We used a 150 x 4.6 mm analytical column containing Phenomenex Ultracarb 20% ODS (Scandinaviska GeneTec AB) with a 5-µm particle size. The mobile phase was a mixture of 900 mL of 5 mmol/L ammonium formate solution, pH 2.7, and 100 mL of methanol per liter, delivered at a flow rate of 0.8 mL/min. A 50-µL aliquot of the sample was injected with a Perkin-Elmer ISS-100 autoinjector. Chromatograms were evaluated with a Shimadzu C-R3A Chromatopac integrator.
codeine determination
Determination of codeine in hair.
Untreated hair samples
(10–40 mg) were weighed into 10-mL screw-capped glass tubes. One
milliliter of 2 mol/L sodium hydroxide and 25 µL of internal standard
(1.0 mg/L of codeine-d3 and
morphine-d3 in methanol) were added to
the tube, which was then heated at 80 °C for 20 min in a water bath.
After a sample was cooled to room temperature, 0.2–0.3 mL of 5 mol/L
hydrochloric acid was added to neutralize the sample. The sample was
then buffered with 5 mL of 0.2 mol/L phosphate buffer, pH 6.1. A
solid-phase extraction cartridge (Bond Elute Certify) was activated and
conditioned with 2 mL of methanol followed by 2 mL of deionized water.
The sample was then allowed to pass through the cartridge at a maximum
flow rate of 2 mL/min. The cartridge was washed with 2 mL of deionized
water, 1 mL of 0.1 mol/L citrate buffer, pH 4.7, and finally with 2 mL
of methanol before drying under reduced pressure (<10 mmHg) for
5 min. Analytes were eluted with a mixture of 800 mL/L methylene
chloride and 200 mL/L 2-propanol with 20 mL/L ammonia added. The
eluate was evaporated under a gentle stream of nitrogen, and the
residue was reconstituted in 30 µL of acetonitrile.
N-methyl-N-trimethylsilyltrifluoroacetamide (10
µL) was added, and the sample was mixed before transfer to a gas
chromatography micro vial.
The determination of codeine in hair was performed on a Hewlett-Packard gas chromatography–mass spectrometry system with an HP5890 series II gas chromatograph equipped with an HP7673A autosampler and an HP5972 mass selective detector. The column was a 30-m HP-5 MS column (0.25 mm i.d. and 0.25 µm film thickness). Helium was used as the carrier gas at a constant flow of 1.0 mL/min. Splitless injection was used with a splitless time of 60 s. The injector and interface temperatures were 210 and 280 °C, respectively. The oven temperature was held at 150 °C during the splitless time and then increased at 30 °C/min to 260 °C and 5 °C/min to 285 °C. Ions monitored for the analytes were m/z 178.2, 234.2, and 371.2 for codeine; m/z 181.2 and 374.2 for codeine-d3; m/z 236.2, 414.2, and 429.2 for morphine; and m/z 239.2 and 432.2 for morphine-d3.
Calibration samples were prepared by the addition of codeine and morphine calibration solutions to drug-free hair before extraction. Concentrations were set at 0.025, 0.050, 0.075, 0.100, 0.200, 0.250, 1.000, and 2.000 ng/mg hair. The detection limit was considered the concentration of analyte that gave a signal-to-noise ratio of 3 for the quantification ion. The within-day and total imprecision (CV) and the recoveries were determined with drug-free hair samples supplemented with both codeine and morphine at 0.050 and 0.250 ng/mg hair.
The within-day imprecision and recovery were also determined with the reference material RM8448 obtained from NIST. The initial values for codeine and morphine in this material was determined by NIST in 1991 and re-established in 1994 (21).
Measurement of codeine in plasma.
The measurement of codeine
and morphine in plasma was performed with the routine method in the
laboratory using gas chromatography–mass spectrometry. Briefly, the
plasma samples were extracted with Bond Elute Certify cartridges
according to the procedure for the hair samples, but the compounds were
detected as their perfluoroacyl derivatives. The limit of detection for
both codeine and morphine was 1.0 ng/g. The total CVs (n = 53) for
codeine and morphine were 5.9% and 6.3% at 20.0 ng/g.
analysis of cyp2d6
DNA was prepared from blood using the QIAamp Blood Kit (Qiagen) as
recommended by the manufacturer. The Taq DNA polymerase amplification
of DNA for the identification of the CYP2D6 wild-type
gene and two mutant alleles, CYP2D6A and CYP2D6B,
was carried out as described by Heim and Meyer (22) with
minor modifications.
statistics and calculations
The imprecision for the melanin analyses was determined from
triplicate analysis of hair from one individual in five separate
series. For each series, mean and variance were calculated and used for
the within-series calculation. Total imprecision was determined by
calculating the variance for all parallel samples from the individual
from all five series.
For codeine and morphine in hair, the within-series imprecision was determined by analysis of five control samples at low and a high concentrations in one series and calculation of the mean and variance. The total imprecision was determined by calculation of the mean and variance for control samples analyzed during a 2-month period.
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Results |
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Chromatograms from human albino hair (A), oxidized sepia melanin (B),
and subject 8 (C) are shown in Fig. 2
. Chromatogram A shows no interferences at the retention time of
PTCA. The
A650/A500
ratios for hair samples were 0.21–0.30 for blond hair, 0.12–0.23 for
red hair, and 0.28–0.40 for dark brown/black hair.
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codeine and morphine
Linear calibration curves were established for both codeine and
morphine from 0.025 to 2.0 ng/mg hair. The equation for codeine was
y = 1.15x + 0.0013
(r2 = 0.999); the equation for
morphine was y = 1.10x + 0.0340
(r2 = 0.999).
The within-day and total CVs and recoveries of codeine and morphine
from hair are presented in Table 2
. We also compared our method using a reference material from
NIST. The values for RM 8448 were established by NIST in 1991 to 6.7
ng/mg hair for codeine and 11.9 ng/mg hair for morphine, and
reestablished to 5.6 ng/mg hair for codeine and 9.4 ng/mg hair for
morphine in 1994 (19). Our results for codeine were
4.87 ± 0.13 ng/mg hair (87.0%; n = 9) and for morphine were
8.68 ± 0.24 ng/mg hair (92.4%; n = 9). Considering the
decrease in concentration found by NIST between 1991 and 1994, our
results from 1998 agreed well.
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The detection limit for codeine and morphine in hair was 0.025 ng/mg
when a sample amount of 20 mg of hair was used. The use of more hair
for the analysis could lower the detection limit. Selected-ion
monitoring chromatograms for a negative control, a positive
control, and an authentic sample (subject 8) are shown in Fig. 3
. There was no interference at the retention time for codeine
for the quantification of ion m/z 371.2, as shown in Fig. 3A
.
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Spectra from codeine and morphine reference substances are shown in
Fig. 4
as well as a spectrum obtained from the second sample (week 2)
from subject 8 at the retention time of codeine. The characteristic
ions of codeine (m/z 371, 234, 196, 178, and 146) were
detected, although the spectra also contained at least one prominent
ion not derived from codeine (m/z 204) because of the low
codeine concentration and the background noise.
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in vivo study of codeine incorporation
Individual plasma codeine concentration curves are presented in
Fig. 5
. Some plasma curves did not show a distinct peak, which could
lead to underestimation of the AUC for these subjects. Both blood and
hair samples obtained before drug administration were negative for
codeine and morphine.
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Hair concentrations of melanin and codeine are presented in Table 3
. The melanin analyses were performed on the predose sample, and
codeine analyses were performed on 2-cm segments of the hair samples
taken 1, 2, 3, and 4 weeks after intake.
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The relationships between the incorporated codeine (in samples from
weeks 2–4) and the total melanin and eumelanin (based on PTCA)
concentration of hair are shown in Figs. 6
and 7
. The y-axes are logarithmic. There were
strong exponential correlations, with
r2 = 0.95 for total melanin and
r2 = 0.83 for eumelanin. When
normalizing with the AUC for codeine in plasma,
r2 = 0.86 for total melanin and
r2 = 0.90 for eumelanin.
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There was a much higher codeine concentration in the hair of the only black-haired subject in the study (subject 9) than in the hair of the other subjects. We think this is a cause-effect relationship, and this is supported by studies performed earlier. However, subject 9 could be an outlier; to address this, we omitted data from subject 9 and replotted the data. The correlation between codeine and total melanin was excellent, with r2 = 0.93 for an exponential function, but we also obtained a very good fit for a linear function (r2 = 0.90). After omission of subject 9, the correlation between codeine and eumelanin was lower, with r2 = 0.70 for both exponential and linear functions.
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Discussion |
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Tracqui et al. (23) expressed concern regarding the lack of correlation between administered drug and hair concentration in the studies performed to date and consequently about the usefulness of hair analysis in compliance measurements. However, they state that the main reasons for the lack of correlation were difficulty in obtaining accurate retrospective data on drug consumption, the unknown hair pigmentation of the subjects, and the influence of genetic polymorphism on metabolism. However, studies addressing these questions to some extent have shown a correlation between dose and drug concentration in hair (10)(11)(12).
In addition to controlled dosage and quantitative determination of melanin in hair, we included genotyping of CYP2D6 to relate possible outlying codeine hair concentrations to poor metabolizers. One could speculate that poor metabolizers should have disproportionately higher incorporation of codeine in hair even after normalization for melanin content. Normalization for AUC should also eliminate such an effect. Only a minority of the population lacks the metabolizing enzyme coded for by the CYP2D6 gene, and our group was homogeneous for extensive metabolizers. Although we had only a limited number of subjects, we could eliminate the possibility that the individual with highest incorporation of codeine was a poor metabolizer, strengthening the view that this incorporation was indeed dependent on high amounts of melanin pigments in the hair.
The dose or plasma concentration of the drug will affect the amount of drug incorporated into hair, as shown for codeine by Rollins et al. (16) and for carbamazepine by Williams et al. (24). The study by Rollins et al. showed a correlation between AUC and drug concentration in hair of a homogeneous group of dark-haired male volunteers. In our study, subjects with different hair color participated and no correlation was found between AUC and hair concentration of codeine.
Several animal studies on drug incorporation in hair have shown that the hair color influences the incorporation of drugs (13)(17)(18). Pötsch et al. (17) studied the incorporation of codeine in the tortoise shell guinea pig and compared results from black, red, and white hair from the same animal. They found that eumelanin rather than phaeomelanin was the major factor influencing incorporation. In addition, studies of gray-haired human subjects have shown a similar effect with haloperidol and chlorpromazine (12) as well as with cocaine and its metabolites (25). The authors of these animal and human studies concluded that brown and black hair accumulate higher drug concentrations than white (nonpigmented) or red hair.
Slawson et al. (19) measured both eumelanin and phaeomelanin in addition to phencyclidine and showed that the incorporation of phencyclidine into rodent hair was related to eumelanin rather than phaeomelanin, and Mårs (26) showed that the in vitro binding capacity of eumelanin is somewhat higher than that of phaeomelanin.
In humans, the shades of hair color are the result of a mixed melanogenesis, and the resulting pigment is a combination of eumelanin and phaeomelanin. Considering this wide range of hair color shades in humans, it is necessary not only to accurately determine the color shade of the hair but also to quantitatively measure the pigmentation and at least qualitatively determine the type of pigmentation.
In our study, a hair color chart from Schwartzkopf including numerous shades was used to estimate the hair color of the subjects. However, we could not differentiate the hair colors between four of the blond subjects. On the other hand, the hair melanin content was different in these subjects over a close range (3.1–5.2 µg/mg hair). This stresses the importance of measuring the melanin content of hair and not only determining the hair color.
We quantified the eumelanin contents of the hair samples by
permanganate oxidation and HPLC analysis of PTCA. According to
Pötsch et al. (17), Mårs (26), and Slawson
et al. (19), eumelanin has a higher binding capacity than
phaeomelanin, and measurement of eumelanin could be a better way to
normalize for pigmentation than measuring total melanin. In our
experiments, the correlation between codeine and eumelanin in hair was
stronger than that between codeine and total melanin when hair codeine
was normalized for the AUC for codeine, but it was the other way around
when the AUC normalization was excluded. Figs. 6
and 7
show that the
r2 was high, and the variation in
melanin content accounted for >80% of the variation in hair codeine
concentration regardless of the method used for melanin determination
or whether one used the hair codeine:AUC ratio. This is important in a
clinical setting when monitoring compliance because the AUC would not
be available.
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Ozeki et al. (27) found that the
A650/A500
ratio (see Fig. 1
) provides information on the melanin composition of
the hair. They postulated
A650/A500
ratios for typical hair colors as follows: red hair, 0.12–0.14; blond
hair, 0.15–0.2; and dark brown/black hair, 0.30–0.32. Our results
showed greater variance and overlap between the different hair colors
but with a clear tendency for increased
A650/A500
with increased eumelanin contents.
We gave an equal dose of codeine to all subjects, but the AUC values for codeine were between 500 and 1538 (ng · h/g) with a mean of 854 ng · h/g. The hair concentrations of codeine in samples from weeks 2–4 ranged from 0.027 to 0.57 ng/mg, which is consistent with the concentrations found by Rollins et al. (15) after a single oral intake of codeine. The variations in plasma concentrations or AUC, however, cannot explain the 20-fold variation in the concentration of codeine in hair. Considering a hair growth rate of ~1.0 cm/month (1), the results should be similar in all of the samples from one individual because the part of hair containing the codeine will remain within the 2-cm segment throughout the study. The concentrations in the samples obtained after 1 week were lower than in the remaining samples (or below the threshold), which could be explained by the fact that some hair grown during the first week still remained beneath the skin.
As shown in Figs. 6
and 7
, we found an excellent correlation between
codeine in hair and the amount of pigment. It is worth mentioning that
Tracqui et al. (23) concluded that "the idea of using hair
analysis to ascertain whether a patient has taken his treatment exactly
as prescribed, clearly appears to be inapplicable". However, our data
strongly suggest that normalization of drug amounts in hair for melanin
or eumelanin content makes therapeutic compliance monitoring
worthwhile.
We conclude that codeine is readily detected in hair after a single oral dose of 100 mg, in both light blond and dark-haired individuals, for at least 4 weeks after administration. The codeine concentration in hair strongly correlates with the melanin content of the hair. We suggest that normalization for melanin should always be included in the quantitative evaluation of codeine concentrations in hair. Our data also show that normalization for AUC in plasma is not necessary, which is important in the clinical application, for which AUC usually is unavailable.
The methods for determining total melanin and PTCA described here are suitable for routine measurements of hair pigmentation. The use of hair analysis to monitor compliance in a clinical setting should be further investigated using the melanin analyses described. Studies on the relationship between melanin and drugs other than codeine should also be performed to investigate the role of pigmentation on drug incorporation into hair.
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Acknowledgments |
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References |
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, subject 1; 
, subject 2; 
, subject 3; X, subject 4;
*, subject 5; •, subject 6; +, subject 7; –, subject 8; —,
subject 9. A sample drawn at 24 h after administration (not
shown) was also included in the estimation of plasma AUC for
codeine.
