Journal of Analytical Toxicology, Vol.

34, January/February 2010

Beating the System: A Study of a Creatinine Assay and

Its Efficacy in Authenticating Human Urine Specimens
Vincent P. Villena
Kern County Regional Criminalistics Laboratory, Toxicology Unit, Bakersfield, California 93301

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Abstract
Creatinine concentration is commonly used to verify the
authenticity of urine specimens submitted for illicit drug screening.
This study evaluated creatinine screening of donor urine specimens
as a tool for detecting substituted and/or tampered specimens. The
study carried out creatinine assay of animal urine, fruit juices, and
urine from creatine-supplemented subjects by a modified version
of the Jaffe reaction. All specimens were analyzed for creatinine
concentration in a chemistry-immuno analyzer. Results showed
that urine specimens from common domestic pets, including cats,
dogs, and horses, have creatinine values similar to normal human
values. Most fruit juices tested contained no detectable creatinine,
and the few that did showed poor “urine” chemical integrity.
Creatine supplementation by donors was found not to provide an
effective means of elevating creatinine concentration in urine
when attempting to flush out water-soluble drugs in the body. Thus,
the assay for creatinine proved useful for the detection of some but
not all adulterated urine specimens.
The serum creatinine test is used in the medical field as an
indicator of renal function (7). In forensic drug screening,
urine creatinine concentration is measured in order to deter-
mine whether a specimen is consistent with normal human
urine or has been diluted (8). One method used to determine
the creatinine concentration in urine specimens is based on the
Jaffe reaction. In this reaction, a red-orange color is formed by
the interaction of alkaline picrate and creatinine (8,9). A color
change in a reaction is simply measured photometrically in a
chemistry analyzer.
This study was designed to determine which specimens pro-
vide a false positive in the assay of creatinine in urine or other
substances that display urinelike properties. Its purpose is to
raise analysts’ awareness to whether specimens were tampered
with or substituted.
Experimental

Introduction Specimen selection and collection

Animal creatinine. Urine specimens were obtained from
common healthy domestic pets, including seven canines, seven
Creatinine (C4H9N3O2) is the metabolic waste product of felines, one equine, and one rodent. Specimens either were ob-
creatine phosphate (C4H10N3O5P) in muscle (1). Creatine phos-
phate (Figure 1), or phosphocreatine, provides continuous
phosphorylation of ADP to ATP in the cyclic production of free
energy used by the body. The reaction remains in near-equi-
librium keeping ADP and ATP almost constant and creating a
buffered system (2). Energy is released during the removal of
the phosphate group from ATP → ADP and PCr → Cr. This
chemical form of energy is used by the body for biological and
physical work (3). The body normally produces creatinine at a
constant rate depending on muscular size and structure. Al-
though the human body synthesizes creatine, the supply is
inadequate to maintain sufficient ATP in the body. Red meat
and fish are rich sources of creatine (4,5). Creatine supple-
ments may also be taken as an alternative or as a complement
(4,5). Harris et al. (6) indicate that the normal daily require-

exogenous sources in order to replace catabolized creatine,

ment from endogenous creatine needs to be supplemented by
which is excreted from the kidneys as creatinine.
* Author to whom correspondence should be addressed. E-mail: vvillena@co.kern.ca.us.
Figure 1. ATP breaks down to ADP and releases free energy. Phospho-
creatine (PCr) provides phosphorylation of ADP to ATP and creates a
cycle of energy production.

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 Journal of Analytical Toxicology, Vol. 34, January/February 2010

tained by direct bladder extraction (performed by a certified
veterinarian) or immediately post-urination into a vial. The
specimens were capped and refrigerated (< 5°C) until ana-
lyzed.
Fruit creatinine. Fruit juice samples (Table I) were selected
based on coloration, which mimics the appearance of a normal
human urine specimen. The possibility that urine specimens
may contain blood was also taken into consideration in
choosing a range of sample hues. Fruit juice samples tested in-
cluded apple, cranberry, grape, orange, pineapple, strawberry,
and mixed fruit. Other drinks with similar coloration were
also tested, including energy drinks (Table II) and margarita
mixers (Table I).
Creatine supplementation. Participants consisted of four male
and four female healthy adults. An initial sample was obtained im-
tions, (Dade Behring, Cupertino, CA) 15.0 mg/dL and 25.0
mg/dL, were analyzed prior to running of samples to determine
accuracy of the instrument and the reagents. Kern County
Regional Crime Laboratory, KCRCL, and Syva standards re-
quire the 15.0 mg/dL control to be within 11.0–18.0 mg/dL,
and 22.0–29.0 mg/dL for the 25.0 mg/dL control. Calibration is
performed if these prerequisites were not met. Additionally, the
chemical integrity of a drug-free urine sample and of a CEDIA®
Sample Check (Microgenics, Fremont, CA) control solution
was also confirmed. Sample Check percentage had to be at
least 80–105% for the drug-free urine sample and 60–75% for
the Cedia Sample Check control solution per KCRCL and man-
ufacturer’s standards for valid assays.

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mediately after awakening in the morning for each participant.
Analysis
Approximately 10.5 g (2 scoops) of creatine monohydrate (Body
The Olympus AU400e was utilized in this study to detect the
Fortress™ Super Creatine High Performance) dissolved in
creatinine concentration of the specimens. The Olympus
480 mL (2 cups) of distilled water was then consumed by the
AU400e is an automated chemistry immunoanalyzer used by
participants. Urine specimens were collected after creatine
KCRCL in screening for drugs of abuse in urine and vitreous
supplementation at least once every hour for up to 8 h. Urina-
samples by enzyme multiplied immunoassay technique
tion was induced by hyper-consumption of distilled water (240
(EMIT). Creatinine tests in urine samples were based on the
mL/h). No other form of fluids was consumed except during
Jaffe reaction. The reagents used to perform this reaction were
lunch. Samples of lunch drinks were obtained and tested for
purchased from Syva (Dade Behring) including reagent 1: 0.2
creatinine content and/or cross-reactivity to the assay. Food
M NaOH with stabilizer and preservative and reagent 2: 25
consumed by the participants during the experimental period
mM picric acid. The creatinine in specimens interacts with al-
consisted of a low-calorie meal that excluded fish, red meat,
kaline picrate to form a reddish-yellow solution over time.
and any other major creatine food source. A baseline test was
The rate of change of the absorbance is measured photomet-
conducted a day prior without creatine consumption by par-
rically 28 times in 18-s intervals. The detector translates this
ticipants, Negative Phase (participants consumed 240 mL of
rate into a quantitative value. The Syva Creatinine Validation
distilled water initially followed by the same 240 mL after
Test is a modified version of the Jaffe method, which considers
every hour for 8 h). Urine specimens were stored in a refriger-
sample appearance and specific gravity as adjuncts in detecting
ated area (< 5°C) and were analyzed in the Olympus™ AU400e
adulteration of urine specimens (8).
for urine creatinine.

Controls
Syva® control samples with known creatinine concentra-
Results and Discussion
Table I. Fruit Juice Appearance, Creatinine Concentrations, and Sample Check
Results In the KCRCL, we regularly screen
urine specimens for illicit drugs and,
Sample Check Creatinine thus, require an effective means of de-
Sample Appearance Color (mg/dL) (%) tecting altered specimens. The reference
values in the KCRCL for creatinine con-
Apple clear amber 6.4 80.5 centrations in human urine specimens
Cranberry clear red 61.5 39.0 are 19.5 mg/dL or higher. The average
Grape clear purple 22.0 66.8 creatinine concentrations for men and
women KCRCL subjects are 180 mg/dL
Margarita mix clear pale yellow 70.5 74.2
and 140 mg/dL, respectively. Creatinine
Orange cloudy yellow-orange 2.9 73.4 concentrations that are less than 19.5
Orange juice, mango, tangerine cloudy yellow w/ 3.4 80.7 mg/dL are labeled suspect. Furthermore,
pale red concentrations that are less than 3.0
Orange juice, lemon, lime, tangerine cloudy yellow 3.9 80.1 mg/dL are not considered human urine.
Pineapple cloudy yellow 4.0 75.2
Other studies support KCRCL’s standards
of acceptable creatinine concentrations.
White grape, blueberry clear yellow –0.1 77.7 The World Health Organization recom-
Strawberry margarita mix clear red 76.4 72.5 mends that a sample be between 30 and
300 mg/dL in determining the validity of

40
Journal of Analytical Toxicology, Vol. 34, January/February 2010

a urine specimen (10). The U.S. Department of Transporta-
tion defined an acceptable urine specimen for the screening of
selected drugs of abuse to have a creatinine concentration of no
less than 5 mg/dL, a value less conservative than KCRCL. This
low value was presumed to include the majority of all people
with no serious medical situation (11).
The Sample Check Assay allows determination of any adul-
terants in a urine specimen (e.g., detergents, bleach, vinegar,
tea, etc.). Successful adulterants can produce a false-negative
result for drugs of abuse by reducing the signal produced by
immunoassays, thereby avoiding detection. The CEDIA Sample
Check Assay is based on the fragmentation and reassociation of
bacterial enzyme β-galactosidase. The inactive enzyme
fragments spontaneously reassociate to form an active enzyme
that generates a color change, which is measured photo-
metrically. Any interference in the reassociation of the β-galac-
tosidase enzyme fragments results in the reduction in assay
signals (12).
Animal creatinine
The objective of this first part of the research was to deter-
mine whether animal urine, obtained from common house-

Table II. Energy Drinks Appearance, Creatinine Concentrations, and Sample Check Results

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Creatinine Sample Check
Sample Fruit Content Appearance Color (mg/dL) (%)

1 Amp Energy orange clear yellow 6.5 87.0

2 Amp Energy Sugar Free – clear green-yellow 0.0 93.5
3 Amp Energy Overdrive – clear red 12.9 85.6
4 Clone Energy Drink – clear yellow 2.1 75.7
5 Full Throttle Energy Drink – clear light yellow 23.2 87.3
6 Full Throttle Fury Energy Drink – clear orange 14.0 86.7
7 Full Throttle Nature is One Bad Mother apple, pear, blueberry clear light red 3.5 75.7
8 Glaceau Vitamin Energy (Dragon Fruit) – clear light pink 62.8 91.3
9 Glaceau Vitamin Energy (Fruit Punch) – clear pink 57.1 91.7
10 Hooters Energy Drink – clear orange 6.1 85.9
11 Jeff Gordon 24 Energy Drink – cloudy orange 31.3 85.7
12 Lost Big Gun Energy Drink – clear yellow 3.3 81.3
13 Lost Five-0 Energy + Juice Drink apple, pear, orange cloudy yellow 3.4 85.7
14 Lost Perfect 10 Energy Drink – clear yellow 0.8 87.1
15 Monster Energy Drink Energy Energy – clear dark yellow 1.3 73.0
16 Monster Energy Drink Energy Assault – clear dark red/brown 1.4 76.4
17 Monster Energy Drink Khaos – cloudy yellow-orange 3.6 85.1
18 Monster Energy Drink Lo-Carb Energy – clear dark yellow 0.6 90.0
19 Nitro2Go The High Energy Drink – clear dark yellow 0.8 91.9
20 No Fear Energy Drink – clear dark red 5.7 77.7
21 NOS High Performance Energy Drink passion fruit cloudy light yellow 4.4 87.0
22 Red Bull – clear yellow 3.2 77.1
23 Red Bull Sugar Free – clear yellow 0.5 90.4
24 Redline The Ultimate Energy Rush (Green Apple) – clear colorless 0.3 92.3
25 Redline The Ultimate Energy Rush (Triple Berry) – clear pink 0.4 91.0
26 Rip It Energy Fuel Power – clear light yellow 42.1 86.7
27 Rip It Energy Fuel Power Citrus X – cloudy yellow 42.0 85.4
28 Rip It Energy Fuel Power Lime Wrecker – clear pale yellow 38.6 86.1
29 Rip It Energy Fuel Power Sugar Free – clear light yellow 0.0 95.0
30 Rockstar Energy Drink – clear bright yellow 1.7 73.8
31 Rockstar Energy Drink Punched – clear dark red 3.6 75.4
32 Rockstar Energy Drink Sugar Free – clear bright yellow 0.0 92.2
33 Rockstar Juice Energy + Guava apple, pear, guava clear light red 3.8 85.5
34 Rockstar Juiced Energy + Juiced Passion apple, pear, mango, passion fruit, orange cloudy yellow 4.1 83.7
35 Rockstar Juiced Pomegranate apple, pear, pomegranate clear dark red 1.7 73.8
36 Rockstar Zero Carb Energy Drink – clear red –0.3 71.7
37 Sobe Adrenaline Rush Red Label – cloudy light yellow 0.3 93.0
38 Sobe Adrenaline Rush Blue Label – cloudy light yellow 35.9 84.4
39 Sobe Essential Energy Berry Pomegranate grape, raspberry, pomegranate clear purple-red 57.1 87.2
40 Sobe No Fear Gold – clear dark yellow 5.3 80.7
41 Sobe No Fear Super Energy Supplement – clear light red 7.6 78.9
42 Sobe Sugar Free No Fear – clear light red 0.5 87.8
43 Unbound Energy Drink – clear dark yellow 3.0 71.9
44 Vasodex Black Pearl High Energy Libido Matrix – clear pale yellow 0.4 103.0

41
 Journal of Analytical Toxicology, Vol. 34, January/February 2010

hold pets, could be successfully substituted for human subject
urine specimens for drug screening. Because animal urine
has the same physical appearance and viscosity as human
urine, visual inspection is insufficient to rule it out as substi-
tuted samples. As shown in Table III, the animal urine speci-
mens tested showed concentration similar to normal human
creatinine values except for that of the guinea pig. Because an-
imal muscles bear the same basic physiological functions as
that of human muscles, creatinine production may be ex-
pected in the energy synthesis for any mammals. The re-
sulting low value of creatinine for the guinea pig may also be
explained by the small muscle mass of this species of animal.
The value measured for the guinea pig may be, therefore, ap-
propriate for small animal urine creatinine. The odor of urine
specimens can be used to differentiate animal urine speci-
A total of 41 different brands and/or flavors of energy drinks
were tested for creatinine (Table II) in this study. A number of
samples reacted to the assay and showed normal human range
values. Even though the creatinine concentration was lower
than the average human creatinine concentration, the num-
bers were still considered by the KCRCL’s standards to be
normal. Among the energy drinks that had normal human
range creatinine values, the only one that contained fruit
juice was Sobe Essential Energy Berry Pomegranate. The cre-
atinine level could have been due to its grape juice content.
Grape juice was tested for creatinine and showed a value of
22.0 mg/dL but failed the Sample Check (66.8%). All the sam-
ples, except Sobe Berry Pomegranate, passed the Sample
Check criterion.
This section of the study showed that certain fruit juices,

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mens from that of human urine specimens. However, unless such as grape, strawberry, and cranberry react to the creatinine
an analyst is familiar with the odor of various animal species’ assay and provide values that are acceptable to human creati-
urine specimens, this still may prove to be an obstacle in de- nine concentration. However, the additional Sample Check
termining falsified samples. test eliminates these fruit juices as substitutes to human urine
specimens. Some energy drinks (highlighted in Table II), how-
Fruit creatinine ever, provide false-positives in both tests, regardless of fruit
The objective of this second part of the research was to de- juice content.
termine if fruit juices might be substituted for a subject’s urine
specimen. Results in Table I show that cranberry juice and Creatine supplementation
margarita mixes demonstrate concentrations of creatinine The objective of this part of the study was to determine
ranging from 60 to 80 mg/dL similar to human urine. Cran- whether creatine supplementation could increase the amount
berry juice, however, failed to pass the sample check with a of creatinine in human urine specimens even after hyper-hy-
value of 39.0%. dration. People undergoing mandated drug screening some-
times consume a large volume of water prior to testing in an
attempt to wash away the presence of illicit water-soluble drugs
Table III. Animal Creatinine Concentrations and Sample
Check Results in the body. The tests showed that over the course of an 8-h pe-
riod the creatinine concentration declined consistently without
Sample Check Creatinine any significant elevation even in participants who took creatine
(%) (mg/dL) (Figures 2A–2B). The creatinine concentration among the
male participants was higher than those of the female coun-
Dogs terparts with (Figure 3B) or without (Figure 3A) creatine sup-
Chihuahua 81.8 194.9 plementation. This may be explained by the muscle mass dif-
Chi Mix 51.8 117.1 ference between genders. Because men generally possess
Chow 85.6 93.5 higher muscle mass, the creatine pool is much larger, and
Dachshund 81.8 284.0 thus creatinine production is higher. During the negative
Doberman 77.8 111.6 phase, only 240 mL of water was consumed. On the day of the
Great Dane 85.9 196.9 creatine supplementation, the creatine mixture was dissolved
Pointer 81.5 86.6 in 480 mL of water because of its partial insolubility charac-
teristics. This higher fluid intake triggered a more frequent uri-
Cats
nation and thus lower creatinine concentrations. Another
Domestic Short Hair 35.0 336.7
factor to consider is each individual’s ability to process or digest
Domestic Short Hair 90.0 75.3
creatine. Data from male subjects showed that creatinine ele-
Domestic Short Hair 87.7 66.2
vation were higher at the near end of the sample collection.
Himalayan 22.4 194.5
Himalayan 21.9 142.4
Renal function varies between individuals and may explain the
Siamese 81.5 161.7 shift on some subjects’ creatinine production. A Q-Test elimi-
Siamese 77.9 56.1 nated all the data from one subject based on a 95% confidence
level. Overall, creatinine supplementation did not serve to
Horse mask attempted hyper-hydration, and hyper-hydration at the
Paint 91.9 104.0 levels of water our participants consumed was easily detected
by the creatinine assay used. In a related study, Ropero-Miller
Guinea Pig et al. (13) supported the results in this study, suggesting that
Guinea pig 74.1 0.4 oral creatinine supplementation did not have an effect on com-
monly used urine integrity tests.

42
Journal of Analytical Toxicology, Vol. 34, January/February 2010

Conclusions not result in dramatic elevation of urine creatinine levels as hy-

The creatinine assays illustrated creatinine concentration
from different domestic animal urine specimens that were
comparable to normal human creatinine except that of the
guinea pig. Factors, including breed, size, and gender, did not
significantly affect the concentration of creatinine in the ani-
mals’ urine. The rodent sample was found to be negligible due
to its difficult collection. Therefore, it is highly unlikely to be
utilized as a substitute specimen for drug screening. Other
than small animals, domestic animal urine in general may be
a successful substitute for use by donors with intentions of
cheating in their drug test. Hence, this situation must be made
aware to all analysts testing urine specimens for drugs of abuse.
An alternative, albeit unpleasant, solution to identify animal
pothesized. Instead, data collected were within the expected
values from normal healthy humans. Consumption of a high
amount of creatine, therefore, is insufficient to elevate creati-
nine while attempting to dilute drug contents by flushing ex-
cessive urine in the system.
Overall, this study evaluated potential methods for tam-
pering/substituting samples as well as methods for the detec-
tion of such actions. In this study, we found that the assay for
creatinine cannot differentiate animal urine from human
urine, and therefore, an analyst must be wary of any specimens
with noticeably unusual odor. Some fruit juices also may ap-
pear like urine color-wise, yet the odor and results of the assay
prevent fruit juices from being a viable substitute specimen in
an illicit drug screening. Margarita mixes and some energy

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urine substitutions from a drug screen is an odor test. Animal drinks, on the other hand, did test positive for creatinine. Lab
urine gives off a strong pungent odor that
is differentiable to that of human urine.
This test, of course, is subjective. Only
those who are familiar may be able to cor-
rectly differentiate between urine speci-
mens of various animal species.
Performing a sniff test on samples to
detect fruit juice substitution is fortu-
nately not as foreboding. Fruit juices are
easily identifiable in samples from an odor
test, but their physical appearances are de-
ceiving. Luckily, in this study, most fruit
juices were found to have suspiciously low
to no creatinine concentrations. Cranberry
juice was found to give normal creatinine
values, but its sample check failed to show
good chemical integrity. Margarita mixes,
however, were found to have normal crea-
tinine levels and their Sample Checks are
only borderline suspect. More information
is needed to determine the cause for these
results. Laboratory analysts need to be
aware of the potential to substitute with
cranberry juice and/or margarita mixes
and note unusual colors or odors of all
urine samples so that they can be marked
as suspect. In regards to the energy drinks
data, some of the brands evaluated did
show normal creatinine concentration.
However, actual creatinine content may
not be the reason for a positive response.
Cross-reactivity in the assay may explain
why they showed normal creatinine con-
centrations. With such low creatinine
values and noticeable colors in the energy
drink samples however, it may still be pos-
sible for trained analysts to detect these
samples and mark them as suspect. In ad-
dition, the odor was clearly recognizable in
most cases. Figure 2. Average creatinine concentrations of female subjects’ urine samples (A) and male subjects’ urine
In the third part of the research, inges- samples (B).
tion of creatine by study volunteers did

43
 Journal of Analytical Toxicology, Vol. 34, January/February 2010

nalistics Laboratory staff. The author

would also like to extend his appreciation
to Robert Kane for providing the energy
drink samples he concurrently utilized in
his experiment and Bakersfield Veterinary
Hospital for donating animal urine spec-
imens. In addition, the author thanks the
reviewers of this manuscript, including
Ruth Dickover, Ph.D. of the KCRCL.
Finally, the author thanks the KCRCL su-
pervisors who authorized and supported
this study.

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The author would like to acknowledge the participants and Manuscript received December 3, 2008;
contributors of this research—Kern County Regional Crimi- revision received March 31, 2009.

44