Effect of Color and Curvature on the
Concentration of Morphine in Hair Analysis
Thomas M. Mieczkowski
Professor
Department of Criminology
University of South Florida
St. Petersburg, Florida
Introduction.......Sample Frame.......Purpose of the Study.......Delimitation
Data
Analysis Methods......Hypotheses.......
Data
Analysis: Morphine Concentration and Hair Color.......
Discussion:
Morphine Concentration and Hair Color.......
Data
Analysis: Morphine Concentration and Hair Curvature.......
Descriptive
Data.......Discussion:
Morphine Concentration and Hair Curvature
Discriminant
Analysis.......Summary
and Conclusion.......References
Introduction
Hair analysis for psychoactive
drugs has become a progressively more common method for ascertaining
toxicological evidence of drug use in forensic cases. Recently
the Federal Food and Drug Administration gave approval for the
use of hair analysis for determining heroin use. Currently a
large number of commercial laboratories offer hair analysis services
for psychoactive drugs, and a large number of corporations, some
government agencies, and a number of criminal justice agencies
use hair testing to identify potential drug abuse.
There has been concern that several factors
associated with human hair may cause variation in the retention
and recovery of specific analytes when using hair as a test matrix
for toxicological analysis (Kidwell 1992; Skopp et al. 1997).
Among the most important variables are different coloration and
coloration patterns associated with hair and, to a lesser extent,
the degree of coiling, curvature, or curling variations associated
with human hair (Joseph et al. 1996; Knight et al. 1996). Coiling
or curvature of hair has not been specifically discussed, but
it is often used as the primary surrogate measure for race or
ethnicity identification that has been noted as a potential confounder
of hair analysis interpretation (Joseph et al. 1996). Researchers
who have suggested an ethnic or race bias in hair analysis have
de facto used kinkiness of hair as a race marker (Henderson et
al. 1998). There has been an ongoing discussion in the literature
about the importance of these factors. A number of published
articles suggest that this putative systematic variation may
not be important in hair analysis interpretation (Hoffman 1999;
Kelly et al. 2000; Mieczkowski and Newel 1993, 2000b).
Two opiates widely used in medical practice
as well as by abusers are codeine and morphine. Some studies
have concluded that the incorporation of these opiates into hair
is influenced by the melanin content of the hair. Research looking
at opiates in human and animal populations, for example, has
suggested that melanin binding accounts for higher concentrations
of codeine and morphine in darkly pigmented hair (Rollins et
al.1996; Wilkins et al. 2000). Kronstrand et al. (1999) published
data on codeine recovery from hair as a function of hair melanin
concentration and reported a strong correlation between codeine
concentration and melanin concentration. However, there have
been some interesting effects and variations noted in these reports.
The strength of the contribution may be dependent on the specific
opiate under scrutiny.
Pigmentation appears to play an important
role in codeine retention in hair, for example, but a diminished
role in morphine retention. Rollins (1995) observed, for instance,
that given constant concentrations in the plasma of codeine and
morphine, morphine appears to incorporate at approximately one
half the rate as codeine. He suggested that a variety of factors
including the variations in molecular properties of morphine
in comparison to those of codeine might be responsible for this
difference (Rollins 1995). Rothe et al. (1997) reported, consistent
with Rollins, that morphine and 6-acetylmorphine were near-equivalence
in concentration between pigmented and nonpigmented hair when
comparing hair collected from the same individuals taking controlled
doses of morphine. Wilkins et al. (2000) reported findings indicating
the difficulty of positing pigmentation as a simple predictor
of codeine concentration. Variations in codeine concentrations
recovered from uniformly black hair (samples taken from several
racial and ethnic groups) showed dramatic variations in concentration
even though subjects were given equivalent controlled doses of
codeine. The variations in concentration approached 300%, indicating
the likelihood that factors other than pigmentation of hair play
a major role in influencing concentration values. In their analysis,
Mieczkowski and Newel (2000b) evaluated the outcome of a series
of studies assessing hair assay values for opiates and hair color
(Goldberger et al. 1991; Kintz et al. 1998). They reported that
there was no statistically significant relationship between either
parent drug or metabolites and hair color.
Sample Frame
To further assess the possible influence of
pigmentation on the concentration of morphine in hair, data was
presented on the basis of a random selection of 95 morphine-positive
cases retrieved from a database produced by a major hair analysis
laboratory (Psychemedics Corporation, Culver City, California).
These 95 cases were selected from approximately 6,000 cases that
had a positive assay for some drug. These 6,000 cases were selected
from a sampling frame of 80,000 cases submitted to the laboratory
for analysis of the presence of five illicit psychoactive drugs
(cocaine, cannabinoids, opiates, amphetamines, and PCP). The
samples were sent as part of a preemployment or employment drug
screening. The only inclusion criteria used in constituting the
sample frame were that cases included a confirmed positive hair
assay for morphine with a concentration value of 0.2 ng/mg of
sample mass. It is important to emphasize that it is very rare
for persons in preemployment and employment categories to test
and confirm positive for morphine. Furthermore, when stratifying
the data further by hair color, the number of cases available
for analysis can become extremely small. This is further exacerbated
by the confounding effect that hair coloration is distributed
unevenly, with natural red and natural light blond hair being
relatively scarce.
The testing methods used by the Psychemedics
Corporation are based on the protocols developed by Baumgartner
(Baumgartner 1995; Baumgartner et al. 1995; Baumgartner and Hill
1996). This protocol includes a complex wash-and-wash analysis,
enzymatic digestion of the washed hair, radioimmunoassay screening
of the digest, and GC/MS or GC/MS/MS confirmation of any radioimmunoassay-positive
result. For details of the chemical analysis protocol, please
consult the cited publications.
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of the page
Purpose of the Study
The study consists of the presentation and
analysis of data for the 95 selected cases. The analysis is directed
at two purposes. The first is to determine whether there is a
statistically significant relationship between the hair color
categorization and the morphine concentration reported for each
sample. The second is to determine whether there is a relationship
between the hair curvature or curliness and the morphine concentration
reported for each sample. Technical personnel of the testing
laboratory, using internal categorization standards, performed
the assessments and assigned the characterizations of the hair
by color and curvature. Curvature was assessed by visual examination
of the sample laid on a glass surface. Hair was classified as
curved if it exceeded values of 02 on the Bailey and Schleibe
(1985) curvature measurement scale, with most hair so classified
being above 05 on the scale. An internally developed color schema
derived from hair cosmetic industry color comparison charts was
used to assess sample color on a categorical basis. Color classification
was a five-fold schema: black, brown, blond, red, and gray/gray
mixed.
Delimitation
This study has several notable delimitations.
First, the number of samples is relatively small, especially
for light-colored hair. Red hair is entirely absent. Although
samples were taken from a very large initial database, the rate
of opiate-positive samples is generally very low in preemployment
populations. The representation here is a reflection of the real
distribution of these cases in the general employment-seeking
population. This becomes further exacerbated by the subdivision
of these cases into hair color categories. Samples that could
be determined to be dyed, bleached, or tinted or colored cosmetically
were excluded. The resultant number of persons with blond and
red hair that occurs naturally is low in the general population.
The appearance of these colors to the casual observer in everyday
experience gives the impression that these color categories should
be more frequent, but this is because a substantial number of
individuals modify their natural hair color through cosmetic
processes. Consequently, the data offered do not permit any statistical
inference because of the convenience sampling method.
Additionally, the study is delimited by not
knowing the actual dosages consumed, and it cannot be excluded
from possibility that the results here are a measurement of dose
preference differences, which correspond to hair color. However,
that presumption is no more or less defensible than the assumption
that in a large preemployment population, the dosage ranges throughout
many cases is likely to resemble a Gaussian distribution, which
is the assumption made here. Ideally, of course, one would like
to conduct a controlled-dose experiment. This, however, presents
some problems as well. Perhaps most notably, the ability to conduct
such an experiment on a very large scale and to conduct it throughout
a wide-dose range.
The data and analysis offered here are designed
to suggest what may be possible and reasonable in considering
the hypotheses.
Data Analysis Methods
The independent variables in this analysis
are the color category of the hair sample and the curly/straight
dichotomous category for the hair sample. The dependent variable
is the concentration value of morphine in the hair sample. The
assessment of the case data is done by univariate ANOVA for color
categorizations and by independent sample t-test for hair
curvature. Each independentdependent variable relationship
is also assessed by discriminant analysis. Multiple statistical
methods are used as indicated to provide a more comprehensive
assessment of the possible relationship. The criteria used to
determine statistical significance is p = .05 for all
methods. The analysis of this data set was done with SPSS v 10.0.
The particulars of the data file follow in Table 1.
Table 1. Structure of the Data
File
Variable/Name |
Type |
Value labels |
ID: Case identifier |
Nominal |
None |
HRCOLOR: Hair color |
Nominal |
1 = Black
2 = Brown
3 = Dark brown
4 = Blond
5 = Gray |
HRSHAPE:
Straight/curly hair |
Nominal |
1 = Straight
2 = Curly |
MORPHINE:
Morphine concentration
(ng/10 mg) |
Ratio |
|
Hypotheses
The hypotheses for the analysis are specified
as follows:
Hypothesis 1
The null hypothesis: There is
no significant relationship between the concentration of morphine
recovered from each hair sample and the color categorization.
The alternative hypothesis: Hair samples that are darker
in color will have a higher concentration value than those that
are lighter in color.
Hypothesis 2
The null hypothesis: There is
no significant relationship between the concentration of morphine
recovered from each hair sample and the curvature categorization.
The alternative hypothesis: Hair samples that have discernible
physical curvature will have statistically significant differences
in morphine concentration when compared to those that lack curvature
(straight).
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Data
Analysis: Morphine Concentration and Hair Color
Table 2 reports the mean value (M)
and standard deviation (SD) for the recovered morphine
for each hair color category.
Table 2. Mean Concentration of
Morphine by Hair Color
Hair color |
Mean morphine concentration
(ng/10 mg) |
Standard deviation |
Number of samples
(N = 95) |
Black |
10.69 |
8.32 |
42 |
Brown |
15.02 |
10.17 |
13 |
Dark brown |
13.40 |
9.10 |
32 |
Blond |
7.50 |
3.80 |
5 |
Gray |
10.10 |
5.31 |
3 |
Overall mean |
12.01 |
8.71 |
|
Figure 1 presents the box-and-whisker
plot of morphine concentration by hair color. The horizontal
bars in the box represent the group mean, the box boundaries
represents the 25th and 75th percentile values, and the whiskers
delineate the cases within 1.5 "hspreads" (box lengths)
of the mean.
Table 3 contains the outcome of a one-way
(univariate) analysis of variance (ANOVA) for all color combinations
contrasting the groups by morphine concentration. ANOVA seeks
to compare the degree of within-category variance to between-group
variance to assess the possible significance of difference by
the independent variable category set. Table 3 indicates that
there is no significant effect for hair color for morphine concentration.
Table 3. Analysis of Variance,
Hair Color (IV) and Morphine Concentration (DV), All Hair Colors
|
df |
SS |
MS |
F |
Morphine in hair by hair color |
Between groups |
Combined |
4 |
364.479 |
91.120 |
1.213*** |
Linearity |
1 |
11.657 |
11.657 |
.155*** |
Deviation from linearity |
3 |
352.822 |
117.607 |
1.566*** |
Within groups |
|
90 |
6761.154 |
75.124 |
|
Total |
|
94 |
7125.633 |
|
|
*p = .311. **p = .695. ***p
= .203.
Further analysis by Tukey's honestly significant
difference (HSD) method (also known as the Tukey a procedure),
which permits complete pair-by-pair comparisons for all possible
color combinations and their contrasts, does not show significance
for any color combination.
Discussion:
Morphine Concentration and Hair Color
Statistical analysis by univariate ANOVA of
these 95 cases supports the following observations regarding
color and morphine concentrations.
Rank order of mean concentrations of morphine
by hair color from high to low is as follows:
1. Brown
2. Dark Brown
3. Black
4. Gray
5. Blond
Although there appears to be a concentration
gradient, which decreases from darker to lighter hair, the one-way
ANOVA is not significant for morphine concentration and hair
color category, F(90, 4) = 1.213, p = 0.311. The
R2 value for this relationship is 0.002 and
the h2
value is 0.051. If the nominal values for hair color are transformed
into a dichotomous contrast (black, dark brown in contrast to
blond/gray), there is not a significant difference in morphine
concentration, F(93, 1) = 1.446, p = 0.232, R2
= .015. These values indicate that color contributes negligibly
to variations in concentration values and is not a function of
a dilution effect by the number of dark categories.
For the color contrasts, the Levene Test for
homogeneity of variance is not significant, F(90, 4) =
1.941, p = 0.110, indicating that the groups have homogeneous
variances. Subsequent examination by Tukey's HSD analysis shows
no significant morphine concentration effect for any color combination,
nor are there any homogeneous concentration subsets by color.
Data
Analysis: Morphine Concentration and Hair Curvature
The same analytic approach can be applied
to assessing the potential that gross hair morphology as straight
or curled hair may have a relationship to morphine concentration
in hair. The only required modification is that the curly/straight
variable is dichotomous, and thus instead of a one-way ANOVA
(which is used for three or more group comparisons), a t-test
to assess the significance of the hypothesized relationship can
be applied.
Table 4. Mean Concentration Values
of
Morphine for Straight and Curled Hair
Curvature morphology |
Mean morphine concentration
(ng/10 mg) |
Standard
deviation |
Number of samples
(N = 95) |
Straight |
12.26 |
8.79 |
76 |
Curled |
11.02 |
8.54 |
19 |
Total |
12.01 |
8.71 |
|
Descriptive Data
|
Figure 2.
Box-and-whisker plot shows a comparison of curly and straight
hair according to morphine concentration in hair. Click for enlarged
image. |
Of the 95 cases under analysis, 76 are
classified as straight, and 19 are classified as curled. All
cases have a confirmed positive hair assay for morphine. Table
4 presents the mean values and associated standard deviations
for the two categories.
Figure
2 is a box-and-whisker plot of this same contrast.
Table 5 presents the results of a t-test
contrasting the straight and curled hair samples by morphine
concentration. The Levene Test indicates that the criterion of
equality of group variance is met and that the t value
does not attain significance. Assessment of this relationship
by a general linear model indicates that the R2
value is 0.003.
Table 5. Results of t-test
for Mean Difference in Morphine Concentration by Hair Curvature
|
t-test for equality
of means |
t |
df |
Mean difference |
Morphine in hair (ng/10 mg) |
.554 |
93 |
1.2408 |
p
= .581, two-tailed.
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Discussion:
Morphine Concentration and Hair Curvature
Statistical analysis by t-test and
univariate ANOVA of these 95 cases supports the following observations
regarding hair curvature and morphine concentration. Neither
one-way ANOVA nor an independent sample t-test is significant
for morphine concentration and hair curvature, t = 0.554,
df = 93; F(93, 1) = .306. The R2
value for this relationship is 0.003. These values indicate that
hair curvature or curliness does not contribute in a meaningful
way to variations in morphine concentration values.
Discriminant Analysis
Last, a discriminant analysis on this data
was performed. Discriminant analysis is a technique using aspects
of multivariate ANOVA and regression. It permits the analysis
of a quantitative predictor variable (or a series of quantitative
predictor variables) and the ability of these variables to successfully
classify outcomes or states. In this case the predictor variable
is morphine concentration. The approach of discriminant analysis
is such that it tests the following concept: If the concentration
of morphine for any particular sample is known, that sample is
assigned to a predicted group membership. The predicted memberships
are compared to the empirically known group membership, and the
utility of the relationship between predictor and category is
assessed on the basis of agreement between predictive outcome
and known outcome.
Table 6. Classification Resultsa
of Discriminant Analysis of Morphine Concentration and Hair Color
Hair color |
Predicted group membership |
Black |
Brown |
Dark
brown |
Blond |
Gray |
n |
% |
n |
% |
n |
% |
n |
% |
n |
% |
Black
(N = 42) |
1 |
2.4 |
11 |
26.2 |
3 |
7.1 |
23 |
54.8 |
4 |
9.5 |
Brown
(N = 13) |
1 |
7.7 |
6 |
46.2 |
0 |
.0 |
5 |
38.5 |
1 |
7.7 |
Dark brown
(N = 32) |
2 |
6.3 |
14 |
43.8 |
0 |
.0 |
11 |
34.4 |
5 |
15.6 |
Blond
(N = 5) |
0 |
.0 |
0 |
.0 |
1 |
20.0 |
3 |
60.0 |
1 |
20.0 |
Gray
(N = 3) |
1 |
33.3 |
1 |
33.3 |
0 |
.0 |
1 |
33.3 |
0 |
.0 |
a10.5% of original grouped cases
correctly classified.
A discriminant analysis was performed for
hair color categorizations and for curvature characterizations
relying on morphine concentration as the predictor variable.
Analysis assumptions used were the following: Equiprobability
was assigned for all groups for prior probabilities for the analysis,
and the covariance matrix used was a within-groups design. The
outcomes are presented here as classification tables, which indicate
the degree of success of the classification on the basis of a
predicted group membership versus actual group membership. Tables
6 and 7 show by count and percentage the coincidence of predicted
and original group membership, using morphine concentration as
the predictor variable.
Table 7. Classification Resultsa
for Discriminant Analysis of Morphine Concentration and Hair Curvature
Hair curvature |
Predicted group membership |
Straight |
Curly |
n |
% |
n |
% |
Straight
(N = 76) |
30 |
39.5 |
46 |
60.5 |
Curly
(N = 19) |
7 |
36.8 |
12 |
63.2 |
a44.2% of original group cases
correctly classified.
In cases of color-versus-curvature categorization,
the selected variable was less effective as a predictor relative
to random chance assignments. For hair color prediction, the
rate of correct classification was approximately 10%, about half
of the value of correct predictions that would result from random
chance. For curvature, the value is approximately 44%, slightly
less than the 50% value that would be true of random assignment.
These outcomes are consistent with the t-test and ANOVA
outcomes of the previous analysis and reinforce the findings
that neither hair color or hair curvature appears to be significantly
related to morphine concentration.
Summary and Conclusion
Analysis of the samples does not reveal a
statistically significant effect at p = .05 between hair
color or hair curvature and the concentration of morphine recovered
by chemical analysis. For multigroup comparisons (color) or dichotomous
comparisons (curvature) there is a very low value for R2,
indicating that for this particular drug these variables do not
have significant explanatory power associated with variations
in the value of the drug's concentration. A second assessment
using discriminant analysis is consistent with the outcomes for
the first set of ANOVA and t-test results. Classification
results show that for color and curvature the assignment performance
is degraded over random chance when using either independent
variable as a predictor. The hypotheses identified are thus not
supported by these findings. These results may or may not hold
true when evaluating other important opiates and their concentration
in hair.
The data here suggest that the relationship
between color, curvature, and morphine concentration may not
be a meaningful effect. But the sample size and the assumption
of approximate dose equivalencies, as noted, seriously delimit
the study. A controlled-dose, large-scale clinical experiment
would be the most fruitful approach to definitively evaluating
the presence and degree of these possible effects and better
assessing the hypotheses specified in this study. The use of
controlled-dose experiments is an unlikely approach because of
human subjects and ethical constraints. Compiling and analyzing
a larger database, which would result in a larger representation
in the scarcer color categories, is a more pragmatic approach.
Furthermore, the dosage equivalency assumption becomes more plausible
with an increase in analytic N. This effort is currently
underway and may produce a clearer picture of the results of
the study presented here.
References
Bailey, J. and Schleibe, S. The precision
of the average curvature measurement in human head hair. In:
Proceedings of the International Symposium on Forensic Hair
Comparisons. Federal Bureau of Investigation, U.S. Government
Printing Office, Washington, DC, 1985.
Baumgartner, W. A. Hair analysis method. U.S.
Patent 5,466,579 (1995).
Baumgartner, W. A., Cheng, C., Donahue, T.,
Hayes, G., Hill, V., and Scholtz, H. Forensic drug testing by
mass spectrometric analysis of hair. In: Forensic Application
of Mass Spectrometry. I. Yinon, ed. CRC Press, Boca Raton,
Florida, 1995, pp. 6194.
Baumgartner, W. A. and Hill, V. Hair analysis
for organic analytes: Methodology, reliability issues and field
studies. In: Drug Testing in Hair. P. Kintz, ed. CRC Press,
Boca Raton, Florida, 1996, pp. 223265.
Goldberger, B., Caplan, Y., Maguire, T., and
Cone, E. Testing human hair for drugs of abuse: Identification
of heroin and 6-acetylmorphine as indicators of heroin use, Journal
of Analytical Toxicology (1991) 155:226232.
Henderson, G., Harkey, M., Zhou, C., Jones,
R., and Jacob, P. Incorporation of isotopically labeled cocaine
into human hair: Race as a factor, Journal of Analytical Toxicology
(1998) 22:156164.
Hoffman, B. H. Analysis of race effects on
drug test results, Journal of Occupational and Environmental
Medicine (1999) 41:612614.
Joseph, R., Tsung-Ping, S., and Cone, E. In
vitro binding studies of drugs to hair: Influence of melanin
and lipids on cocaine binding to Caucasoid and Africoid hair,
Journal of Analytical Toxicology (1996) 20:338344.
Kelly, R. C., Mieczkowski, T., Sweeney, S.,
and Bourland, J. Hair analysis for drugs of abuse: Hair color
and race differentials or systematic differences in drug preferences,
Forensic Science International (2000) 107:6386.
Kidwell, D. A. Hair analysis: Techniques and
potential problems. In: Recent Developments in Therapeutic
Monitoring and Clinical Toxicology. I. Sunshine, ed. Marcel
Dekker, New York, 1992, pp. 555563.
Kintz, P., Bundeli, P., Brenneisen, R., and
Ludes, B. Dose-concentration relationships in hair from subjects
in a controlled heroin-maintenance program, Journal of Analytical
Toxicology (1998) 22:231235.
Knight, J. M., Eliopoulos, C., Klein, J.,
Greenwald, M., and Koren, G. Passive smoking in children: Racial
differences in systematic exposure to cocaine by hair and urine
analysis, Chest (1996) 109:446450.
Kronstrand, R., Forstberg-Peterson, S., Kagedal,
B., Ahlner, J., and Larson, G. Codeine concentration in hair
after oral administration is dependent on melanin content, Clinical
Chemistry (1999) 45:14851494.
Mieczkowski, T. and Newel, R. An analysis
of the racial bias controversy in the use of hair assays. In:
Drug Testing Technology. T. Mieczkowski, ed. CRC Press,
Boca Raton, Florida, 2000a, pp. 313348.
Mieczkowski, T. and Newel, R. An evaluation
of patterns of racial bias in hair assays for cocaine: Black
and white arrestees compared, Forensic Science International
(1993) 63:8598.
Mieczkowski, T. and Newel, R. A statistical
examination of hair color as a potential biasing factor in hair
analysis, Forensic Science International (2000b) 107:1338.
Rollins, D. E. Disposition of codeine into
hair. In: Proceedings of the International Conference and
Workshop for Hair Analysis in Forensic Toxicology. R. deZeeuw,
I. Hosani, and S. Al Munthiri, eds. Ministry of the Interior,
Abu Dhabi, United Arab Emirates, 1995, pp. 118135.
Rollins, D. E., Wilkins, D. G., and Krueger,
G. G. Codeine disposition in human hair after single and multiple
doses, European Journal of Clinical Pharmacology (1996)
50:391397.
Rothe, R., Pragst, F., Hunger, J., and Thor,
S. Effect of pigmentation on the drug deposition in hair of grey-haired
subjects, Forensic Science International (1997) 84:5360.
Skopp, G., Potsch, L., and Moellet, M. On
cosmetically treated hair: Aspects and pitfalls of interpretation,
Forensic Science International (1997) 84:4352.
Wilkins, D., Rollins, D., Slawson, M., and
Augsburger, M. Pharmacokinetic Studies of the Disposition
of Drugs of Abuse Into Human Hair. Presented at the California
Association of Toxicologists, Napa, California, 2000.
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