**NISTIR 6468**

**Interlaboratory Studies**

**on the Analysis of Hair**

**for Drugs of Abuse**

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United States Department of Commerce

Technology Administration

> National Institute of Standards and Technology

**ABOUT THE LAW ENFORCEMENT AND CORRECTIONS**

**STANDARDS AND TESTING PROGRAM**

The Law Enforcement and Corrections Standards and Testing Program is
sponsored by the Office of Science and Technology of the National
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to improve the criminal justice system and to disseminate the results to
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The Law Enforcement and Corrections Standards and Testing Program is an
applied research effort that determines the technological needs of
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devices, tests commercially available equipment against those standards,
and disseminates the standards and the test results to criminal justice
agencies nationally and internationally.

The program operates through:

The *Law Enforcement and Corrections Technology Advisory Council*
(LECTAC) consisting of nationally recognized criminal justice
practitioners from Federal, State, and local agencies, which assesses
technological needs and sets priorities for research programs and items
to be evaluated and tested.

The *Office of Law Enforcement Standards* (OLES) at the National
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available equipment.

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**NISTIR 6468**

**Interlaboratory Studies**

**on the Analysis of Hair**

**for Drugs of Abuse**

Michael J. Welch and Lorna T. Sniegoski

Chemical Science and Technology Laboratory

Analytical Chemistry Division

National Institute of Standards and Technology

Gaithersburg, MD 20899

March 2000

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**U.S. DEPARTMENT OF COMMERCE**

**William M. Daley, Secretary**

**TECHNOLOGY ADMINISTRATION**

**Dr. Cheryl Shavers, Under Secretary of Commerce for Technology**

**NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY**

**Raymond G. Kammer, Director**

**National Institute of Justice**

Jeremy Travis

Director

The technical effort to develop this report was conducted

under Interagency Agreement 94-IJ-R-004,

Project No. 97-002/IA.

This report was prepared by the Office of Law Enforcement

Standards (OLES) of the National Institute of Standards

and Technology (NIST) under the direction of

Alim A. Fatah, Program Manager

for Chemical Systems and Materials, and

Kathleen M. Higgins, Director of OLES.

The work resulting from this report was sponsored by the

National Institute of Justice (NIJ), David G. Boyd, Director,

Office of Science and Technology.

**FOREWORD**

The Office of Law Enforcement Standards (OLES) of the National Institute
of Standards and Technology (NIST) furnishes technical support to the
National Institute of Justice (NIJ) program to strengthen law
enforcement and criminal justice in the United States. OLES's function
is to conduct research that will assist law enforcement and criminal
justice agencies in the selection and procurement of quality equipment.

OLES is: (1) Subjecting existing equipment to laboratory testing and
evaluation, and (2) conducting research leading to the development of
several series of documents, including national standards, user guides,
and technical reports.

This document covers research conducted by OLES under the sponsorship of
the National Institute of Justice. Additional reports as well as other
documents are being issued under the OLES program in the areas of
protective clothing and equipment, communications systems, emergency
equipment, investigative aids, security systems, vehicles, weapons, and
analytical techniques and standard reference materials used by the
forensic community.

Technical comments and suggestions concerning this report are invited
from all interested parties. They may be addressed to the Office of Law
Enforcement Standards, National Institute of Standards and Technology,
100 Bureau Drive, Stop 8102, Gaithersburg, MD 20899-8102.

> Kathleen M. Higgins, Director
>
> Office of Law Enforcement Standards

iii

**CONTENTS**

Foreword iii

1\. INTRODUCTION 1

2\. EXPERIMENTAL METHODS 2

3\. RESULTS 4

4\. SUGGESTED READINGS 18

**TABLES**

Table 1. Overall results from interlaboratory Exercises 5, 6, & 7 6

Table 2. Comparison of results by hair type and form for Exercise 5 7

Table 3. Example of results report sent to participants 11

Table 4. Overall results on positive and negative challenges for the
seven exercises 12

Table 5. Laboratory performance by analyte 16

**FIGURES**

Figure 1. The relationship between mean analyte levels (in ng/mg) in
hair samples

and the ability of laboratories to correctly identify the presence of
the analyte

for exercises 5, 6, and 7 8

Figure 2. The relationship between mean analyte levels (in ng/mg) in
hair samples and the

interlaboratory precision for exercises 5,6, and 7 9

Figure 3. The relationship between reported analyte levels (in ng/mg)
and the number of

false-positives reported. Only data for cocaine (COC), benzoylecgonine
(BZE),

morphine (MOR), and codeine (COD) in exercises 2 through 7 were included
14

Figure 4. The relationship between mean analyte levels (in ng/mg) in
hair samples and

results that were outliers. Only data fo cocaine (COC), benzoylecgonine
(BZE),

morphine (MOR), and codeine (COD) in exercises 2 through 7 were included
15

v

**COMMONLY USED SYMBOLS AND ABBREVIATIONS**

A ampere H henry nm nanometer

ac alternating current h hour No. number

AM amplitude modulation hf high frequency o.d. outside diameter

cd candela Hz hertz (c/s) Ω ohm

cm centimeter i.d. inside diameter p. page

CP chemically pure in inch Pa pascal

c/s cycle per second IR infrared pe probable error

d day J joule pp. pages

dB decibel L lambert ppm parts per million

dc direct current L liter qt quart

°C degree Celsius lb pound rad radian

°F degree Fahrenheit lbf pound‑force rf radio frequency

dia diameter lbf⋅in pound‑force inch rh relative humidity

emf electromotive force lm lumen s second

eq equation ln logarithm (base e) SD standard deviation

F farad log logarithm (base 10) sec. section

fc footcandle M molar SWR standing wave ratio

fig. figure m meter uhf ultrahigh frequency

FM frequency modulation min minute UV ultraviolet

ft foot mm millimeter V volt

ft/s foot per second mph miles per hour vhf very high frequency

g acceleration m/s meter per second W watt

g gram N newton λ wavelength

gr grain N⋅m newton meter wt weight

area=unit^2^ (e.g., ft^2^, in^2^, etc.); volume=unit^3^ (e.g., ft^3^,
m^3^, etc.)

**PREFIXES**

d deci (10^-1^) da deka (10)

c centi (10^-2^) h hecto (10^2^)

m milli (10^-3^) k kilo (10^3^)

μ micro (10^-6^) M mega (10^6^)

n nano (10^-9^) G giga (10^9^)

p pico (10^-12^) T tera (10^12^)

**COMMON CONVERSIONS**

**(See ASTM E380)**

> 0.30480 m =1ft 4.448222 N = lbf

2.54 cm = 1 in 1.355818 J =1 ft⋅lbf

> 0.4535924 kg = 1 lb 0.1129848 N⋅m = lbf⋅in

0.06479891g = 1gr 14.59390 N/m =1 lbf/ft

> 0.9463529 L = 1 qt 6894.757 Pa = 1 lbf/in^2^
>
> 3600000 J = 1 kW⋅hr 1.609344 km/h = mph

Temperature: T~°C~ = (T~°F~-32)×5/9

Temperature: T~°F~ = (T~°C~×9/5)+32

vi

**INTERLABORATORY STUDIES ON THE ANALYSIS**

**OF HAIR FOR DRUGS OF ABUSE**

### Michael J. Welch and Lorna T. Sniegoski, Analytical Chemistry Division

*Chemical Science and Technology Laboratory, National Institute of
Standards and Technology*

1.  **INTRODUCTION**

In 1990, the National Institute of Standards and Technology (NIST) began
research into the analysis of hair for drugs of abuse\[1\][^1]. Included
in that research has been an ongoing study of how well laboratories
could detect and measure drugs of abuse in hair \[2,3,4\]. The study has
included seven exercises in which NIST has sent hair samples to
participants who agreed to analyze them. The purposes of these exercises
were: (1) to evaluate the state-of-the-art for laboratories analyzing
hair for drugs of abuse, (2) to provide laboratories with feedback on
their performance, (3) to investigate the relationship between drug
levels in hair and analytical accuracy, and (4) to compare the efficacy
of various approaches used for hair analysis. The exercises were open to
anyone who wished to participate and were strictly voluntary. No fees
were charged for participation nor were the laboratories reimbursed for
their costs associated with participation. The participants did not know
which hair samples had target analytes in them, but they knew which
compounds might be present.

A total of 26 laboratories participated in one or more exercises. Only a
single laboratory participated in all seven of the exercises, while
seven participated in only one. The participants included 15
laboratories from the U.S. and 11 from other countries, mostly in
Western Europe. Law enforcement organizations, commercial drug testing
laboratories, and university and government research laboratories made
up most of the participants. The laboratories were promised
confidentiality. The results from a given laboratory were known only to
themselves and to the NIST staff involved with the study. After each
exercise, each participating laboratory in that exercise was sent a
report in which its results were compared with the overall mean and
standard deviation of the group and with the NIST results. Erroneous
results were noted. These fell into three categories: reports of finding
target analytes when they were not present in the samples
(false-positives), failing to find target analytes that were present
(false-negatives), and reporting quantitative results that greatly
disagreed with the study mean values (outliers).

For each exercise, six to eight samples of hair were sent to each
participant. Each sample was one of three types: (1) drug-free hair, (2)
drug-free hair into which NIST soaked target compounds, and (3) hair
from known drug users provided by the participants. Although hair

from drug users would seem to be most appropriate for these studies,
there are several factors that limited the use of such hair. NIST does
not perform drug tests on hair of individuals, and therefore, has no
direct access to hair from drug users. The amount of hair required to
enable each participant to test for all of the target compounds is very
large relative to what is typically collected in drug testing
situations. The distribution of drugs in the hair of a drug user may not
be uniform. Finally, there may be other compounds present in such hair
that would interfere with some of the analytical methods being used,
thus adding an uncontrollable variable to the study. Therefore, NIST
elected to use clean, drug-free hair into which selected target
compounds were soaked for many of the samples in the exercises.

The first two exercises included cocaine (COC), benzoylecgonine (BZE),
and morphine (MOR) as the target analytes, and codeine (COD) was added
for the third exercise. In subsequent exercises additional analytes were
added. Participants in the seventh exercise had to test for

10 analytes including 6-monoacetylmorphine (6-MAM), cocaethylene (CE),
amphetamine (AMP), methamphetamine (MET), phencyclidine (PCP), and
tetrahydrocannabinol (THC) in addition to the four original analytes.
Levels of the target analytes in the recent exercises have generally
been much lower than they were in the initial exercises, thus providing
a greater challenge for the laboratories.

2.  **EXPERIMENTAL METHODS**

    1.  **Selection Process**

**2.1.1 Recruitment of Participants**

Letters of invitation to participate were sent to laboratories known to
be investigating hair analysis for drugs of abuse. Some of the
participants spread the word to their colleagues, thus increasing the
number of participants. All laboratories that responded positively for a
particular exercise were sent samples.

**2.1.2 Determination of Which Analytes to Include in the Exercise**

The analytes included were based upon feedback from participants in
previous exercises and on capabilities at NIST for accurate measurements
for a particular analyte.

2.  **Hair Samples**

**2.2.1 Preparation of Hair Samples with the Target Analytes at
Realistic Levels**

NIST has no direct source of drug users' hair. Occasionally, reasonably
large hair samples from individual drug users were donated to NIST for
use in the exercises. These hair samples were washed and cut into small
segments. For most exercises, the quantity of hair required for each

laboratory to perform all of their tests was relatively large compared
to what is typically available from a drug user. Consequently, many of
the samples included in the exercises were drug-free hair samples
obtained from volunteers at NIST and treated with solutions of target
compounds. After considerable trial and error, conditions were developed
that permitted incorporation of realistic levels of the various analytes
in these hair samples. Generally, the analytes were dissolved at
appropriate concentrations in solutions of dimethylsulfoxide (DMSO) with
or without water added. For most of the exercises, the hair was cut into
short segments

(0.3 cm to 0.7 cm typically) and considerable effort was expended to mix
the hair in a given sample to improve homogeneity. However, for two
exercises, the samples included hair that was cryogenically ground to a
fine powder. The resulting material is very homogeneous and easy to work
with, but is not as realistic or challenging as analyzing hair segments.
Each exercise also included one or two unspiked samples from drug-free
individuals.

**2.2.2 Analysis of the Samples to Ensure That Appropriate Levels Were
Achieved**

NIST used gas chromatography/mass spectrometry (GC/MS) methods with
isotope dilution to measure the concentrations of the target analytes in
the hair samples. If a hair material that had been soaked in a solution
containing one or more target analytes did not have appropriate levels,
the hair was soaked further. If the concentration was too low, the hair
was soaked longer or in a higher concentration solution to raise the
levels. If the concentration was too high, the hair was soaked in a
drug-free solution of DMSO to extract some of the analyte. Once
appropriate concentrations were achieved, more samples from the batch
were analyzed by GC/MS to better measure the mean concentrations of the
analytes.

**2.2.3 Preparation and Shipment of Samples to Participants**

Fortunately, the hair materials are stable at room temperature for at
least several weeks, so special precautions in shipping are not
necessary. Each hair sample was placed in a small glass, screw-capped
vial and carefully packed to prevent damage during shipment.

3.  **Data Assessment**

**2.3.1 Compilation and Interpretation of Results**

With up to 14 participants per exercise, six to eight samples per lab,
and up to 10 target compounds, a large quantity of data was generated.
The results were entered into a large spreadsheet with a column for each
participant. Each sample had a section of rows, with each row assigned
to a target compound. With this approach it was possible to sum the
positives and negatives reported for each analyte in each sample and to
calculate means and standard deviations. The laboratories provided
information about the methods they used, thus permitting tests for
correlation between methods used and results. For two of the exercises,
a control material, NIST Reference Material (RM) 8448 Drugs of Abuse in
Human Hair Segments, was

sent along with the NIST assigned values, and the participants were
asked to analyze the control along with the unknowns and report their
results on the control. From these data, performance on the control
material could be correlated with performance on the unknowns. Another
aspect of the data to be investigated was the relationship between the
level of analyte in the hair and analytical performance. Such a
relationship is important when a laboratory establishes its cutoff
levels.

**2.3.2 Providing Feedback to Each Laboratory on its Performance Versus
the Group**

After the results were compiled and analyzed for each exercise, the
participants were sent a report that showed how they performed versus
the group as a whole. For each analyte present in a sample, information
was provided on the mean, range, and standard deviation as well as the
number of positive and negative responses and the number of outliers.
For the analytes not present in a sample, the numbers of positive and
negative responses were recorded. Each participant received an
individualized report that included its results compared with the
summary results, but did not have the individual results from the other
participants.

3.  **RESULTS**

    1.  **Results from the Three Most Recent Exercises**

The first four exercises have been described in detail previously
\[2,3,4\]. A summary of the last three exercises, 5, 6, and 7 is shown
in table 1. For exercises 5 and 6, samples included drug users' hair,
soaked hair, and drug-free hair. The analytes for each exercise are
listed along with the overall performance of the participants for each
exercise. Three of the samples in exercise 5 were cryogenically powdered
and three were in the form of segments. For exercises 6 and 7, all of
the samples were in the form of segments. For exercise 7, only soaked
hair and drug-free hair were included. For the positive challenges, the
ability of the laboratories to detect the analytes correlates with the
mean levels in the hair. Figure 1 shows how the percentage of correctly
identified analytes increases with the mean level. For analyte levels
below 0.5 ng/mg of hair, positive results were reported only 68 percent
of the time, while for levels above 2 ng/mg, positive results were
reported 100 percent of the time. In contrast, quantitative results do
not show any significant correlation with concentration, as shown in
figure 2, where the relationship between the interlaboratory
coefficients of variation (CVs) and concentration is plotted. As with
previous exercises, the quantitative results exhibit considerable
scatter*.* These CVs are calculated after outliers are excluded from the
calculations. For these studies, outliers were defined as any positive
result that was more than a factor of three different from the mean. In
these exercises, slightly fewer than half of the participants reported
one or more results that were considered to be outliers, with most of
these having one or two outliers in an exercise. In exercise 7, one
laboratory accounted for five outliers out of a total of 12. Most of the
outliers reported are low results. Laboratories with above average
incidence of low results that were outliers were

advised to carefully examine their procedures, particularly their
extractions, to see if their recoveries were low.

The incidence of false-positives was very low in the last three
exercises. There were 12 instances of a particular drug being reported
in a sample when it was not present. For exercises 5, 6, and 7, 18
laboratories participated in one or more exercises and three
laboratories accounted for all of the false-positives. Two of these had
false-positives in exercise 5 while the third had false-positives in all
three exercises, including all of the false-positives in exercise 7.
Thus, the most recent data suggest that most laboratories are quite
careful, but there are a few that must improve performance if they are
to use hair analysis in their work. The particular laboratory with the
high error rate was advised to carefully check their results to
determine if they were misinterpreting interferences as the target
compounds. If the target compounds were actually present in the
chromatograms, the laboratory must have had a serious problem with cross
contamination, and was advised to take appropriate action.

Both drug users' hair and fortified (soaked) hair were included in
exercise 5. A comparison of the results is shown in table 2. All of the
missed positives for the soaked samples were for analytes whose mean
concentrations were below 0.5 ng/mg as were half of the missed positives

***Table 1. Overall results from interlaboratory Exercises 5, 6, & 7***

*[EXERCISE]{.underline}*

[5]{.underline} [6]{.underline} [7]{.underline}

Number of Participants 14 12 12

Number of Samples 6 6 6

Analytes COC COC COC

BZE BZE BZE

CE CE CE

MOR MOR MOR

COD COD COD

MAM MAM MAM

AMP AMP

MET MET

THC

PCP

[Positive Challenges]{.underline} [5]{.underline} [6]{.underline}
[7]{.underline}

[*Number* *% *]{.underline} [*Number* *%*]{.underline} [*Number*
*%*]{.underline}

Positive Responses 197 87.6 143 84.3 100 92.6

Negative Responses 28 12.4 27 15.7 8 7.4

Outliers 11 5.6 10 7.0 11 11.0

[Negative Challenges]{.underline} [5]{.underline} [6]{.underline}
[7]{.underline}

[*Number* *%*]{.underline} [*Number* *%*]{.underline} [*Number*
*%*]{.underline}

Positive Responses 4 1.6 2 0.7 6 1.4

Negative Responses 245 98.4 282 99.3 432 98.6

Table 2. Comparison of results by hair type and form for Exercise 5

  ----------------- ----------------- ----------------- -----------------
                    \% Positives      \% Negatives      

                    correctly         correctly         

  Hair type         identified        identified        \% Outliers

  Drug users'       90.6              97.7              8

  Fortified         84.9              100               3

  Drug-free                           98.7              

                                                        

  Hair form                                             

  Segments          98.7              99.4              2

  Powder            81.5              96.7              9
  ----------------- ----------------- ----------------- -----------------

on drug users' hair. This agrees with what was described above for the
correlation between mean levels and the ability of the laboratories to
detect the analytes. Otherwise, the performance was a little better for
the soaked samples.

Exercise 5 also included both hair segments and powdered hair. These
results are also summarized in table 2. Previous experience with these
two forms suggested that results should be significantly better for the
powdered hair. Analytes should be easier to extract from powdered hair
and powdered hair samples should be more homogeneous than hair segment
samples. However, for exercise 5, results were actually better for the
hair segments. The average level of analytes in the hair segments was
considerably higher than the average in the powdered hair. Thus one
might expect fewer misses on positive challenges, but the results on the
hair segments also had fewer false-positives and fewer outliers. Perhaps
the fact that the laboratories have more experience with hair segments
than with powdered hair accounts for the difference.

Exercise 6 was the first for which amphetamine (AMP) and methamphetamine
(MET) were included and eight laboratories attempted to detect and
measure each of them. Results were much better than had been seen for
other analytes. There were no false-positives reported, no
false-negatives, and only one outlier for the two samples that had these
compounds present. The CVs averaged about 20 percent for each of these
analytes.

Exercise 7 was the first exercise for which phencyclidine (PCP) and
tetrahydrocannabinol (THC) were included and, again, eight laboratories
attempted to detect and measure each of these analytes. Qualitatively,
the results were quite good with only one missed positive between the
two positive PCP samples and one missed positive for the one positive
THC sample. Quantitatively, the interlaboratory CVs were quite large but
there were no outliers.

As an example of the data reported back to the participants, the results
for each hair sample in exercise 7 are summarized in table 3. The data
in the "Your Results" column are not real, but is typical of what was
observed. This column would have the results from the particular
participant to whom the table was being sent. Outliers were counted as
positives but were omitted from the calculation of the mean if they were
more than a factor of three from the mean. The column headed by \"mean
w/o outliers\" contains the mean results after these outliers were
excluded. Thus each participant could compare its results with the
overall mean and the NIST results to determine how good the agreement
was. Although amphetamine and methamphetamine were included in the list
of analytes to test for, they were not present in any of the samples.
There were no false-positives among the nine laboratories that tested
for amphetamine and methamphetamine. These results are omitted from the
table to conserve space.

**3.2 Composite Results From all Seven Exercises**

To date, seven exercises have been completed. A summary of results on
the seven exercises is shown in table 4. There has been no trend in the
percentage of correct responses over the seven

***Table 3. Example of results report sent to participants***

<table style="width:100%;">
<colgroup>
<col style="width: 8%" />
<col style="width: 1%" />
<col style="width: 4%" />
<col style="width: 4%" />
<col style="width: 3%" />
<col style="width: 6%" />
<col style="width: 1%" />
<col style="width: 10%" />
<col style="width: 11%" />
<col style="width: 9%" />
<col style="width: 9%" />
<col style="width: 10%" />
<col style="width: 9%" />
<col style="width: 9%" />
</colgroup>
<tbody>
<tr class="odd">
<td colspan="7"><h1 id="drugs-in-hair-rr-7">Drugs in Hair RR 7</h1></td>
<td colspan="2"><h1 id="overall-lab-results">Overall Lab
Results</h1></td>
<td colspan="3"><h1 id="positive-specimens">Positive Specimens</h1></td>
<td colspan="2"><h1 id="negative-specimens">Negative Specimens</h1></td>
</tr>
<tr class="even">
<td></td>
<td colspan="2"></td>
<td colspan="2"><h1 id="your">Your</h1></td>
<td colspan="2"><h1 id="nist">NIST</h1></td>
<td><strong>Mean</strong></td>
<td><strong>Std Dev</strong></td>
<td>Positives</td>
<td>Negatives</td>
<td>Number of</td>
<td>Positives</td>
<td>Negatives</td>
</tr>
<tr class="odd">
<td>Sample</td>
<td colspan="2">Drug</td>
<td colspan="2"><strong>Results</strong></td>
<td colspan="2"><strong>Results</strong></td>
<td>w/o outliers</td>
<td>w/o outliers</td>
<td>Reported</td>
<td>Reported</td>
<td>3X outliers</td>
<td>Reported</td>
<td>Reported</td>
</tr>
<tr class="even">
<td>1</td>
<td colspan="2">COC</td>
<td colspan="2">3.2</td>
<td colspan="2">2.95</td>
<td>2.9</td>
<td>2.1</td>
<td>10</td>
<td>0</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2">0.3</td>
<td colspan="2">0.44</td>
<td>1.1</td>
<td>0.1</td>
<td>9</td>
<td>1</td>
<td>3</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"><strong>0</strong></td>
<td colspan="2">0.32</td>
<td>0.3</td>
<td>0.3</td>
<td>7</td>
<td>1</td>
<td>2</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>11</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="even">
<td>2</td>
<td colspan="2">COC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>8</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>11</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2">X</td>
<td colspan="2">2.81</td>
<td>1.6</td>
<td>1.1</td>
<td>6</td>
<td>1</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="even">
<td>3</td>
<td colspan="2">COC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>8</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>11</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2">2.5</td>
<td colspan="2">4.86</td>
<td>3.7</td>
<td>2.1</td>
<td>7</td>
<td>0</td>
<td>0</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>4</td>
<td colspan="2">COC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>8</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>11</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="even">
<td>5</td>
<td colspan="2">COC</td>
<td colspan="2">6.2</td>
<td colspan="2">2.09</td>
<td>1.8</td>
<td>0.4</td>
<td>10</td>
<td>0</td>
<td>2</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2">0.4</td>
<td colspan="2">0.22</td>
<td>0.5</td>
<td>0.2</td>
<td>10</td>
<td>0</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"><strong>0</strong></td>
<td colspan="2">tr</td>
<td>0.1</td>
<td>0.1</td>
<td>6</td>
<td>2</td>
<td>3</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>11</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2">0.8</td>
<td colspan="2">1</td>
<td>0.7</td>
<td>0.5</td>
<td>6</td>
<td>1</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td>6</td>
<td colspan="2">COC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>10</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">BZE</td>
<td colspan="2"><em>0.5</em></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>1</td>
<td>9</td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">CE</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>8</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MOR</td>
<td colspan="2">4.4</td>
<td colspan="2">6.9</td>
<td>3.6</td>
<td>1.3</td>
<td>11</td>
<td>0</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">COD</td>
<td colspan="2">5.2</td>
<td colspan="2">5.54</td>
<td>3.7</td>
<td>2.4</td>
<td>10</td>
<td>0</td>
<td>1</td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">MAM</td>
<td colspan="2">0.6</td>
<td colspan="2">0.81</td>
<td>0.4</td>
<td>0.2</td>
<td>8</td>
<td>2</td>
<td>4</td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td></td>
<td colspan="2">THC</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="odd">
<td></td>
<td colspan="2">PCP</td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td>0</td>
<td>7</td>
</tr>
<tr class="even">
<td colspan="3">Pos. on Neg. Challenges</td>
<td colspan="2">1</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td colspan="3">Neg. on Pos. Challenges</td>
<td colspan="2">2</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td colspan="3">3x Outliers</td>
<td colspan="2">2</td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="odd">
<td colspan="3"></td>
<td colspan="2"></td>
<td colspan="2"></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
<td></td>
</tr>
<tr class="even">
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="8"></td>
</tr>
<tr class="odd">
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="8"></td>
</tr>
<tr class="even">
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="2"></td>
<td colspan="8"></td>
</tr>
</tbody>
</table>

Table 4. Overall results on positive and negative challenges for the
seven exercises

Positive Challenges

**Responses**

*[Exercise]{.underline}* *[Positive]{.underline}*
*[Negative]{.underline}* *[% correct]{.underline}*

1 46 3 93.9

2 106 25 80.9

3 173 11 94.0

4 158 23 87.3

5 197 28 87.6

6 145 27 84.3

# 7 100 8 92.6

Overall 925 125 88.1

Negative Challenges

**Responses**

*[Exercise]{.underline}* *[Positive]{.underline} [Negative]{.underline}
[% correct]{.underline}*

1 0 63 100.0

2 5 72 93.5

3 5 179 97.3

4 22 323 93.6

5 4 245 98.4

6 2 282 99.3

# 7 6 432 98.6

Overall 44 1596 97.3

exercises. However, the challenges for the laboratories have generally
become more difficult

over the course of this study. The first exercise included only three
analytes and these were present at very high concentrations. In
subsequent exercises, more analytes were added and the analyte levels
have decreased. In the most recent exercise, laboratories could test for
up to 10 analytes and almost half of the mean concentrations were under
1 ng/mg.

The most important factors in good performance on these exercises are
experience and adherence to good quality assurance procedures. This was
clearly illustrated in exercise 4, when the laboratories analyzed NIST
RM 8448 as a control \[4\]. The results showed a strong correlation
between good performance on the reference material and good performance
on the unknowns.

In general, the laboratories have performed at about the same level for
all of the analytes. Table 5 shows the overall performance of the
laboratories for the four compounds included in the largest number of
exercises: cocaine, benzoylecgonine, morphine, and codeine. The results
for exercises 2 through 7 were included in this table; exercise 1 was
not, because cocaine and benzoylecgonine data were combined for this
exercise to better compare results from different methods. The data in
table 5 demonstrate that the overall performance is very similar for the
different analytes.

As shown in table 5, the frequency of false-positives is also not
related to the analytes. Figure 3 shows the combined results for COC,
BZE, MOR, and COD for the number of false-positives as a function of the
level that was reported. If the laboratories use a cutoff value, some of
the false-positives seen in this study would probably not be reported as
positives in actual drug testing situations. More than half of the
false-positives reported levels below 1 ng/mg, so a cutoff at this level
would reduce the number of false-positives by more than a factor of two.
Most of the participants have not reported any false-positives. An
average of 18 percent of the participants in a particular exercise have
reported one or more false-positives, ranging from 0 percent for
exercise 1 to 36 percent for exercise 4.

As shown in figure 4, there is no correlation between concentration and
the rate of outliers. These results demonstrate that many of the
laboratories have serious problems with their quantitative analysis not
related to the limit of quantitation.

For both isolating the analytes from the hair matrix and for the
analysis of the extracts, no single analytical approach has been found
to be clearly superior. Participants in these studies have used three
principal approaches, acid extraction, methanol extraction, and enzyme
digestion for removing drugs from the hair matrix. For each of these
approaches, some laboratories have consistently performed well, while
others have been less successful, although error rates, particularly
very low quantitative data, are significantly higher for laboratories
using methanol extractions. For analysis of the extracts, GC/MS has been
the approach used by most of the laboratories, with again a wide range
in the quality of results received. A few laboratories have used other
techniques for measurement. Good results were obtained by one laboratory

####   {#section-2 .unnumbered}

####   {#section-3 .unnumbered}

#### Table 5. Laboratory performance by analyte

[COC BZE MOR COD]{.underline}

Correct Positives 88.3 86.3 89.4 88.1

Correct Negatives 96.4 96.6 96.1 98.8

\% Outliers 14.0 11.2 9.3 9.3

performing tandem mass spectrometry (MS/MS) after extraction of analytes
from the hair and further processing of the extracts. In contrast, the
worst performance observed in any exercise involved a laboratory
experimenting with direct MS/MS analysis of intact hair. Liquid
chromatography with electrochemical detection has also been used with
mixed results.

**3.2 Future Work**

Participation in a proficiency testing program is an important part of a
laboratory's effort to evaluate its performance and to demonstrate its
commitment to monitoring and improving the quality of its work. This
study has served as a means for laboratories to assess the quality of
their analyses. However, the focus was not on proficiency testing, but
rather to assess the overall quality of laboratory measurements of drugs
of abuse in hair. Laboratories and their customers would benefit from a
formal proficiency testing program, involving at least two regularly
scheduled exercises per year. Such a program should be a full-time
activity for an organization with experience in proficiency testing
programs for drug testing. NIST is not the organization to operate such
a program, but could provide quality assurance for the program.

An important aspect of quality assurance that would fit with NIST
strengths and interests would be to develop new Standard Reference
Materials (SRMs) for drugs of abuse in hair that laboratories could use
to evaluate the accuracy of their methods. Several years ago, NIST
developed two reference materials (RM 8448 and 8449) that were human
hair into which cocaine, benzoylecgonine, morphine, and codeine were
soaked \[5\]. Concentrations were determined by GC/MS. As noted
previously, good performance on control materials such as these RMs
correlates well with performance on unknown specimens \[4\]. These
materials are now out of stock and resources have not been available for
a renewal of these materials. The next generation will be SRMs
containing more analytes that are certified using two independent
analytical methods. These reference materials will help laboratories
assess and demonstrate the accuracy of their methods and help assure
public confidence in forensic measurements.

**4. SUGGESTED READINGS**

\[1\] Welch, M.J., L.T. Sniegoski, C.C. Allgood, and M. Habram, Hair
Analysis for Drugs of Abuse: Evaluation of Analytical Methods,
Environmental Issues, and Development of Reference Materials, *J. Anal.
Tox.,* 17 (1993) 389-396.

\[2\] Welch, M.J., L.T. Sniegoski, and C.C. Allgood, Interlaboratory
Comparison Studies on the Analysis of Hair for Drugs of Abuse, *Forensic
Sci. Int.,* 63 (1993) 295-303.

\[3\] Welch, M.J. and L.T. Sniegoski, \"Interlaboratory Studies on the
Analysis of Hair for Drugs of Abuse - Results from Three Exercises,\"
*Hair Testing for Drugs of Abuse: International Research on Standards
and Technology, NIH Publication No. 95-3727* (1995) 133-147.

\[4\] Sniegoski, L.T. and M.J. Welch, \"Interlaboratory Studies on the
Analysis of Hair for Drugs of Abuse: Results from the Fourth Exercise,\"
*J. Anal. Tox.* 20 (1996) 242-247.

\[5\] Welch, M.J., P. Ellerbe, S.S.-C. Tai, R.G. Christensen, L.T.
Sniegoski, L.C. Sander, and C.S. Phinney, \"NIST Reference Materials to
Support Accuracy in Drug Testing,\" *Fresenius J. Anal. Chem.*, 350
(1995) 4730-4734.

#### 

[^1]: Numbers in brackets refer to suggested readings in section 4.