I
I
Retrospective
AnalysisofSomeL-Methamphetamine/
L-Amphetamine
UrineData*
Cecil L. Hornbeck**
and Robert J. Czarny
Navy Drug Screening Laboratory, San Diego, CA 92134-6900
;
:_Abstract;
L
i
A limited analysis is presented of data accumulated over about
three years for specimens containing either nonracemlc
L-methamphetamine/L-amphetamine or racemic mixtures of the
n and Lstereoisomers. 55 data points for both nonracemic
L-methamphetamine and racemic mixtures show that it is
possible to report a specimen containing L-methamphetamine
positive for illegal o-methamphetamine based on current
guidelines unless chiral assays are performed on selected
methamphetamine positives. Generation of only 45 racemic
specimens from chiral analysis of about 5,000 urine
methamphetamine positives demonstrates the overwhelming
prevalence of nonracemic illegal o-methamphetamine in
Southern California.
Introduction
,
f
I
Most GC/MS confirmation tests are not able to differentiate
between the schedule two drug, D-methamphetamine,
and overthe-counter (OTC) L-methamphetamine.
Even though the immunoassavs currently available for use by both the federally
certified civilian (NLCP) and military (DOD) urine drug testing
laboratories are relatively insensitive to L-methamphetamine,
these same screening tests often give positive results due to the
presence of several other frequently used OTC amines. These
compounds taken in combination
with use of legal Vicks Inhalers containing L-methamphetamine,
could result in a positive
finding for methamphetamine
in many laboratories
not performing a chiral GC/MS assay to separate the two stereoisomers (1). The NLCP requires a level of 200 ng/mL of amphetamine to report a methamphetamine
positive, and therefore, the
levels of L-amphetamine generated by L-methamphetamine
are
also of interest. Only limited data has appeared in the literature
for excretion of the stereoisomers of methamphetamine
and amphetamine (2-4). We are reporting data accumulated over the past
three years from urine specimens which contained either pure t.methamphetamine
and L-amphetamine or a racemic mixture of
the two pairs of stereoisomers,
*The
opLnions expressed
by the authors
are not necesse, nly those of the Department
nor of the Department
of the Navy but are solely the opinions
of the authors.
*"Author
towhomcorrespondence
shouldbeaddressed',
of Defense
Materials
and Methods
ThestandardsD-methamphetamine
hydrochloride,
L-methamphetamine free base, D-amphetamine sulfate, and L-amphetamine
free base were obtained from Sigma Chemical and the internal
standard N-ethylbenzylamine was obtained from Aldrich. The
derivatizing reagent, N-trifluoroacetyl-L-prolyt
chloride (L-TPC),
was purchased from Regis Chemical as a 0.1M solution in chloroform. As analyzed by the vendor, the various lots always contained less than 2% of the D-TPC isomer. The derivatizing
reagent, 4-carbethoxyhexafluorobutyryl
chloride (CB), was obtained from PCPJSCM Specialty Chemicals. The kits for the
two Abuscreen high specificity radioimmunoassays
(RIA) for
amphetamine and methamphetamine were purchased from
Roche Diagnostics.
More recently,
specimens
have been
screened with the Coat-A-Count
methamphetamine
RIA kits
purchased from Diagnostics Products Corporation. The RIA kits
were used according to the manufacturers
instructions (5).
The total concentrations
of methamphetamine
and amphetamine were generated by GC/MS after a liquid-liquid extraction and derivatization with the CB reagent (5,6). Separation
of the L and D-methamphetamine
and amphetamine stereoisomers. to determine the percentages (%) of each. was achieved
using the CB extraction method, but substituting 20 gL of the
chiral denvatizing reagent N-trifiuoroacetyl-L-prolyl
chloride tLTPC) (7) at a concentration of 0.1M in chloroform and N-ethylbenzylamine as the internal standard at a concentration of 2.000
ng/mL. The % of each stereoisomer was calculated by the Cae/MS
software, which divided the L or D peak area of the component to
be determined by the total peak area for both components. The %
of L or D component peak areas from assay of hundreds of nonracemic L or D-methamphetamine and amphetamine standards extracted from water ranged as low as 92% (typically 96-97%),
mostly because of the presence of small quantities of D-TPC in the
L-TPC derivatizing reagent. A racemate was classified as any
specimen that contained an L or D component area between 10 and
90%, and nonracemic L or D was classified as specimens having
a component area greater than 90% of the total for both methamphetamine and amphetamine (5). The concentrations of the individual stereoisomers were calculated by multiplying the % area
times the total concentration determined from the CB assay. To be
reported as positive for methamphetamine,
a specimen had to conrain both a D component greater than 10% of the L and D total and
a D component concentration greater than 500 n_mL. Since the
% D-TPC in the derivatizing reagent was always less than 2%, the
error in calculating the stereoisomer concentration by this method
was consideredto be insignificant(8).
The same GC capillary columns (Hewlett-Packard Ultra 1, 12 m
x 0.2 mm) and GC/MS instruments (Hewlett-Packard 5880
GC/5970A MS) that determined methamphetamineJamphetamine
concentrations were also used for stereoisomer analysis (5,6). The
GC temperature was typically 190°C, and the retention times
were typically about 3.5, 3.75, 4.45, 5.7, and 6.0 minutes for Land
D-amphetamine, intemal standard, and L and D-methamphetamine
respectively. The chiral assay monitored ions 58, 91, 118, and
251 for methamphetamine; 91,118, and237 for amphetamine; and
91 and 134 for the internal standard. Retention times were determined for every assay utilizing two separate standards extracted
from water which contained 2,000 ng/mL of either L-methamphetamine/L-amphetamine
or D-methamphetamine/D-amphetamine. Additional controls were extracted from urine and inctuded a negative control and two positive controls. The two
positive controls contained 50% (L)/50% (D) and 80% (L)/20%
(D) methamphetamine and amphetamine, respectively. The Lmethamphetamine component was expected to be within 45-55%
and 75-85% respectively for the two positive controls (5). All
other parameters and identity criteria were identical to the CB
assay (5,6).
Results and Discussion
The data in Tables I and II were accumulated from the analysis
of about 5,000 urine specimens positive for methamphetamine by
the CB assay over the past three years. However, over the latter
two years of that period, the chiral assay was only performed on
positive specimens which contained concentrations
of amphetamine less than 1,000 ng/mL (5); this represents slightly less
than half of the total methamphetamine positives for the latter
two-year period, and therefore, some racemic specimens may not
have been analyzed. Regardless, because of the relatively small
number of racemic specimens found, it is clear that the Disomer
is overwhelmingly the form of illicit methamphetamine found in
SouthernCalifornia.
A controlled study with three volunteers using Vicks Inhalers
was performed previously in another laboratory (4). This study
demonstrated L-methamphetamme urine concentrations of up to
6,000 ng/mL. In our report only limited intbrmation is available on
the circumstances of L-methamphetamine ingestion for the 10 Lmethamphetamine data points in Table I. Four of the urine specimens were collected from two male laboratory employees who
were using Vicks Inhalers to relieve nasal congestion. They
achieved levels of L-methamphetamine similar to the controlled
study with the highest levetjust under 5,000 ng/mL for Employee
1. This individual weighed 112 lbs. and had been using the inhaler
for 4 consecutive days while inhaling 20-40 times/day before
achieving an initial L-methamphetamine level of 3123 ng/mL.
Continued use of the inhaler at the same approximate rate for two
more days gave a second L-methamphetamine level of 4,952
ng/mL. Employee 2 weighed about 125 lbs. and stated that he had
inhaledabout12timesovera 12-hourperiodandthenvoided12
hours after the last inhalation. This sample had an L-methamphetamine concentration of 3,835 ng/mL. After the fkst specimen was collected, he inhaled 4 more times over the next 12
hours. A urine specimen was collected about 9 hours after the last
inhalation and this specimen contained a level of L-metham? dt
phetamine of 951 ng/mL. None of these specimens were positive
by the murine methamphetamine RIA screening procedure used by
this laboratory (5). A fifth specimen collected routinely under
DoD guidelines did screen positive with the methamphetamine
RiA. This specimen contained an L-methamphetamine concentration of 875 ng/mL by GC/MS. An interview through a responsible third party revealed only that the male donor had been using
pseudoephedrine and a Vicks Inhaler prior to urine collection.
The only information available on the 5 remaining specimens in
Table I is that they were all collected under the DoD guidelines and
all screened positive with an RIA test designed to detect amphetamine instead of methamphetamine.
Because the amphetamine RIA has a low specificity for L-methamphetamine, a
positive test above the 1,000-ng/mL cutoff concentration in at
least four of these remaining five specimens must have been
caused by something other than amphetamine. Although one specimen concentration may be a result of abuse of L-methamphetamine (27,540 ng/mL), without a chiral assay to determine the
presence of L-methamphetamine, all 6 of the specimens collected
under DoD guidelines would have generated a positive report for
illegal D-methamphetamine. However, 3 of these 6 specimens
would not have been reported as positive by certified civilian laboratories because of the current NLCP requirement for the presence of at least 200 ng/mL of amphetamine (Table I).
The 45 racemate data points in Table II were included in this report to yield more information about the urine excretion of L-amphetamine. The percentage concentration of L-amphetamine compared to L-methamphetamine averaged 8.0% in the 32 racemic
specimens containing measureable L-amphetamine. This average is
similar to the 5.5% L-amphetamine percentage concentration in the
8 pure L-methamphetamine specimens containing measureable L-amphetamme in Table I.There was a 0.90 linear correlation between %
L-amphetamine and % D-amphetamine in Table II and the concentration of D-amphetamine relative to D-methamphetamine averaged
32% or 4 times greater than the concentration of L-amphetamine telative to L-methamphetamine seen above. The same calculation for the
% mamphetamine concentration in over 2,200 murine positives containing nonracemic D-methamphetamine up to a concentration of
12,000 ng/mL revealed a similarly high value of 22%. Even when a
racemic specimen contained much greater concentrations of Lmethamphetamine
than D-methamphetamine,
theamountof D-amphetamme present was always proportionately much greater then LTable I. Nonracemic L-Methamphetamine/
L-Amphetamine Data
Concentration
(ng/mL)
t-Meth
L-Amp
% L-Amp4
27,540
10,236
7,879
4,9521
4,244
2,332
356
707
216
73
179
98
<50
116
7.8
3.4
8.2
4.2
1.7
4.5
3.0
10.9
3,8352
3,123
_
1,963
9512
8753
< 50
,.2Specimens
from[aborato_/employees
1and2.
34 Donor
hadbeen
using
pseudoephedrine
andaVicks
Inhaler.
i %L-am!3
= [_.-amo/(c-meth
+ t.-amp) i x 100
'
-
f,
Journalof AnalyticalToxicology,
Vol.17,January/February1993
Table II. Raaemate
controlled study (4,5). Nevertheless, the data in
Tables I and II indicate that at an L-metham-
Data
phetamine concentration below 12,000 ng/mL,
there were still 22 of 55 specimens (40%)
Concentration(ng/ml)
% L-Meth*
,
L-Meth
L-Amp
0-Meth
D-Amp
65
80
46
41
62
59
57
74
54
78
67
62
83
68
71
51
70
72
69
64
63
59
82
61
37,679
27,399
26,697
17,076
15,609
10,410
10,104
9,448
9,297
7,727
6,915
6,375
6,267
6,253
4,827
4,689
4,571
4,077
4,003
3,882
3,786
3,577
3,250
3,111
503
2,496
674
680
463
227
250
2,722
234
887
422
178
2,715
290
223
396
461
227
316
142
234
209
1,079
<50
20,288
6,850
31,341
24,573
9,567
7,234
7,622
3,319
7,919
2,179
3,406
3,907
1,283
2,943
1,971
4,506
1,959
1,586
1,798
2,183
2,224
2,485
714
1,989
1,360
5,067
3,289
4,985
1,743
529
713
2,949
1,324
1,276
986
532
2,312
744
603
2,245
1,186
530
671
302
633
1,022
2,917
112
1.3
8.3
2.5
3.8
2.9
2.1
2.4
22.4
2.5
0.3
5.8
2.7
30.2
4.4
4.4
7.8
9.2
5.3
7.3
3.5
5.8
5.5
24.9
--
6.3
42.5
9.5
16.9
18.2
6.8
8.6
47.0
14.3
36.9
22.4
12.0
64.3
20.2
23.4
33.3
37.7
25.0
27.2
12.2
22.2
29.1
80.3
5.3
a positive for methamphetamine by the NLCP
standards. Further examination of Tables I and
II will show how many of these specimens
have concentrations of L-amphetamine above
200 ng/mL at other levels of L-methamphetamine.
In summary, there is sufficient evidence that
82
59
73
62
25
43
51
41
39
23
75
34
29
62
17
20
69
17
23
34
27
2,888
2,578
2,433
1,849
1,652
1,843
1,629
1,510
1,471
1,426
1,416
1,300
963
859
807
751
603
602
356
351
241
634
1,792
900
1,134
4,957
2,179
1,566
2,172
2,301
4,775
472
2,524
2,357
526
3,940
3,006
271
2,939
1,193
681
652
1,191
90
607
694
876
885
2,372
755
882
2,563
392
165
813
230
3,920
1,678
455
475
275
1,100
851
17.5
-10.9
4.4
-9.3
16.6
-7.5
-9.8
--10.2
--14.4
-----
65.3
4.8
40.3
38.0
15.0
28.9
60.2
25.8
27.7
34.9
45.4
6.1
25.6
30.4
49.9
35.8
62.7
13.9
18.7
61.8
56.6
References
613
<50
299
86
<50
168
324
<50
120
<50
153
<50
<50
98
<50
<50
93
<50
<50
<50
<50
% L-Ampt % o-Amp**
· % L-meth= [L-meth/(L-meth+ o-meth)] x 100
t % L-amp = [L-amp/(L-meth+ L-amp)] x 100
** % D-amp= [o-arnp/(o-meth + o-amp)] x 100
amphetamine. The data indicates that either the metabolism of Lmethamphetamine
to L-amphetamine or the excretion of L-amphetamineitself is generally much lessefficient than that for the cpmparable D isomers. While excretion of these amines is affected by
urinary pH, no pH data was available for these specimens,
A guideline for possible methamphetamine
urine concentrations generated by "normal" use of a Vicks Inhaler has not been
,
formally established. To avoid reporting legal use of an inhaler as
illegal D-methamphetamine,
the chiral assay was required for
specimens having a methamphetamine
concentration
below
12,000 ng/mL. This concentration was arbitrarily set at twice the
L-methamphetm'nineconcentration of 6,000 ng/mL observed in the
having a concentration
of L-amphetamine
greater than the 200 ng/mL required to report
a combination of cold medications or other
OTC amines and legal use of L-methamphetamine in aninhalercan cause a positive report
for illegal use of D-methamphetamine.
Even
though the NLCP requirement for amphetamine
concentration
at or above 200
ng/mL may not have been originally intended
to prevent L-methamphetaminepositives, it
appears prudentto require a chiralanalysison
all urine specimens with methamphetamine
concentrations
mined value.
below some yet to be deter-
1. M.D. Solomon and J.A. Wright. False-positive
for (+)-methamphetamine. C/in. Chem. 23:
1504 (1977)
2. A.H. Beckett and M. Rowland. Urinary excretion kinetics of methylamphetamine in man.
J. Pharrn. Pharmacol. 17 Suppl: 109S--114S
(1965).
3. L. Gunne. The urinary output of d- and I-amphetamine in man. Biochem. PharmacoL 16:
863-69 (1967).
4. R.L. Fitzgerald, J.M. Ramps, S.C. Bogema,
and A. Poklis. Resolution of methamphetamine stereoisomersin udne drug testing:
urinary excretion of R(-)-methamphetamme
following use of nasal inhalers. J. Anal. Toxicol. 12:255-59 (1988).
5. Navy Drug Screening Laboratory Operating
Procedures (SOP) Manual. Washington,
D.C., Bureau of Medicine, U.S. Navy,Jan. 15,
1990.
6. R.J. Czarny and C.L. Hornbeck. Ouantitation
of methamphetamine and amphetamine in
urine by capillary GC/MS Part II. Derivatization
with 4-carbethoxyhexafiuorobutyryl chloride.
J. Anal. Toxicol. 13:257-62 (1989).
7. J.H.Uu, W.W. Ku, J.T.Tsay,M.P.Fitzgerald,and S. Klm. Approaches
to drug sample differentiation. III. A comparative study of the use of
chiral and achiral capillary column gas chromatography/mass spectmmetry for the determination of methamphetamine enantiomers
and possible impurities. J. Forensic Sci. 27:39---48 (1982).
8. S.M. Hayes, R.H. Liu,W. Tsang,M.G.Legendre, R.J. Bemi, D.J. Pillion, S. Barnes, and M.H. Ho. Enantiomedc composition analysis of
amphetamine and methamphetamine by chiral phase high-performance liquid chromatography-mass spectrometry.J. Chrornatogr.
398:239-46 (1987).
Manuscript receivedOctober 18, 1991;
revision received February 1O, 1992.
journal oTAnalytical rox:cology, vol. 17, January/Februar! !993
I
I
I
Fluorescence
Polarization
Immunoassay
Detection
of
Amphetamine, Methamphetamine, and Illicit Amphetamine
Analogues*
John T. Cody
ClinicalInvestigationDirectorate,WilfordHall Medical Center, LacklandAFB, TX 78236-5300
Robert Schwarzhoff
Drug TestingDivision,ArmstrongLaboratory,Brooks AFB, TX 78235-5000
I Abstract
expansion of drug testing in the workplace, the validity of the
testingprocess
andits abilityto detectanddeterdrugabuseisan
The Abbott Diagnostics Amphetamine/MethamphetamineII
and AmphetamineClass reagentswere evaluated on the
Abbott TDx for cross-reactivity to amphetamineand
methamphetaminestereoisomers,several of their metabolites,
and various illicit analogues, including 2-methoxyamphet-
important issue. The analysis of large numbers of samplesby
testing laboratories has spurred the use of relatively specific iramunoassay testing procedures which give quantitative results.
Thus, time-consuming gaschromatographic (GC) analysisand
nonquantitative thin-layer chromatographic (TLC) procedures
have,for the most part, beenreplacedby immunoassaysystems.
Chemical modification of a drug to alter its effect while still
maintaining its primary function is a common medicinal technique used in the search for new drugs. This same process has led
amine, 4-hydroxymethamphetamine,
2,5-dimethoxy-
amphetamine(DMA),4-bromo-2,5-dimethoxyamphetamine
(DOB),4-bromo-2,5-dimethoxy-¢-phenethylamine
(BDMPEA),
3,4,5-trimethoxyamphetamine{TMA),3,4-methyleneclioxyamphetamine (MDA),N,N-dimethyl-3,4-methylenedioxyamphetamine, N-hydroxy-3,4-methylenedioxyamphetamine
(/V-OHMDA),3,4-methylenedioxymethamphetamine
(MDMA),
3,4*methylenedioxyethylamphetamine
(MDEA),2,5-dimethoxy4-ethylamphetamine(DOE),2,5-dimethoxy-4-methylamphetamine (DOM),and mescalinein concentrations ranging from
100to 100,000ng/mL.Resultsdemonstrate the utility of this
assayfor detectionof severalof the above compounds;
unfortunately many are still not detectable.Significant
differenceswere observed betweenthe Amphetamine/
Methamphetamine
II and Amphetamine Class reagents,
particularly regarding their cross-reectivity to over-the-counter
medications. Detection of the drugs amphetamine,
methamphetamine,and the illicit analoguesis not enhanced
with the AmphetamineClass reagents,and unless detection of
the over-the-countercompounds is of interest, these reagents
are a poor choice compared to the Amphetamine/
MethamphetamineII reagents.Cross-reactivity of some of the
illicit analoguesis such that the assay can reliably be used for
the routine screening of these compounds,
Introduction
The use and abuse of amphetamines has undergone a resurgence in the United States during the last few years, particularly
with the advent of methamphetamine in the form of "ice." The
production and use of chemical analogues to amphetamine and
methamphetamine has compounded the problem for some years,
The amount and type of analogues have changed, but their presence has remained a constant concern. With the relatively recent
· The
views
expressed
of the Department
in this article
of Defense
are those
or other
of the authors
Departments
and
of the U.S.
do not
reflect
Government.
the
official
policy
to production of compounds for the expressed purpose of
evading law enforcement efforts to control drug abuse. An example of this chemical manipulation is the production of what
have generally beentermed "designer drugs" to circumvent the
laws and allow individuals to create, possess, and sell abusable
drugs that are,becauseof the modifications, no longer covered
by the Controlled SubstancesAct. These manipulations have
posed problems for law enforcement officials, analytical toxicologists, and chemists.
Performing relatively simple chemical modifications to illicit
drugs can yield compounds that are no longer on the list of controlled substances. The Controlled Substance Analogue Act I 1t
has changed _he essentially comptete freedom from iegai consequences, but the detection of these drugs is still a factor in their
identification and deterrence.
With the vast amount of drug testing currently being administeredin theU.S., thepotential for detectionof illegal drug usehas
significantly increased. This increased testing has a clear deterrent
effect on some individuals. When faced with the possibility of detection of drug use with consequences that are detrimental to job
or career, some individuals may choose to avoid the use of drugs.
Another option is to use drags that are not detected by the system.
This switching from drug to drug may include changing drug
class or using a drug within the same class that is not detected by
the testing system. The use of amphetamine analogues is therefore
of concern because testing for amphetamines in drug testing programs is generally limited to amphetamine and/or methamphetamine. Therefore, an abuser who switches from methamphetamine to MDMA has little to fear from pre-employment or
even random drag testing programs.
Analysisof samplesfrom individuals who haveconsumedsome
of the controlled substance analogues may or may not reveal the
Journal of Analytical
toxicology,
Vol. 17, January/PeDruary
1993
presence of the drug, depending on the analytical procedures used.
Unfortunately, many of the analogues are not detected. Because immunoassay is a common procedure for detection of drugs of abuse
in urine, a group of amphetamine analogues were studied, along
with amphetamine and methamphetamine in their various forms, to
determine their cross-reactivity with the fluorescence polarization
immunoassay (FPIA) reagents from Abbott Diagnostics.
,
Materials and Methods
Amphetamine analogues were obtained from the following
sources: 2-methoxyamphetamine, 4-hydroxymethamphetamine,
2,5-dimethoxyamphetamine
(DMA), 4-bromo-2,5-dimethoxyamphetamine (DOB), 4-bromo-2,5-dimethoxy-13-phenethylamine
(BDMPEA), 3,4,5-trimethoxyamphetamine
(TMA), 3,4methylenedioxyamphetamine
(MDA), N,N-dimethyl-3,4methylenedioxyamphetamine, and N-hydroxy-3,4-methylenedioxyamphetamine (N-OHMDA)were obtained from the National
Institute on Drag Abuse (NIDA). 3,4-Methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine
(MDEA), 2,5-dimethoxy4-ethylamphetamine,
2,5-dimethoxy-4methylamphetamine (DOM), and 3,4,5-trirnethoxyphenethylamine
(mescaline) were obtained from Alltech. The Dand [ amphetamine
and the Dand Lmethamphetamine were from Sigma. Amphetamine
Class and Amphetamine/Methamphetamine II reagents were obrainedfrom Abbott Diagnostics.
Test compounds were prepared as ethanolic solutions and
stored at 4°C. Aliquots of these drug solutions were added to negative urine to give final concentrations of 200 to 100,000 ng/mL,
depending on their respective reactivity. Fluorescence polarization
immunoassay analysis was performed on the samples using the
Amphetamine Class and Amphetamine/Methamphetamine
II
reagents from Abbott Diagnostics. Prepared positive and negative
controls were used to compare the cross-reactivity of the analogue
compounds to the drug for which the assay antibody was targeted
(D,L-amphetamine for the Amphetamine Class reagents and D-amphetamine for the Amphetamine/Methamphetamine
II assay
reagents). Standard curves, made up of calibrators at 0, 500,
10013,2000, 4000, and 6000 ng/mL for the Amphetamine Class
reagents and 0, 150, 300, 1000, 3000, and 8000 ng/mL for the
Amphetamine/Methamphetamine II assay, were prepared and
verified analyzing one positive and one negative control with
each ring of 20 (or fewer) specimens tested. Each concentration
of the various amphetamine analogues was analyzed in duplicate
and the average measured concentration of the replicates was
used to determine percent cross-reactivity.
Results and Discussion
,
Results of the analysis of samples with the Amphetamine Class
reagents and Amphetamine/Methamphetamine II reagents are
shown in Tables I and II, respectively. Discussion of comparative
cross-reactivityis based on the results obtained at a 1000-ng/mL analyte concentration. From a structural standpoint, both systems
showed reduced cross-reactivity for compounds that have a substitution on the amine nitrogen. An exception to this is methamphetamine, which has a higher cross-reactivity
to the Amphetamine
Class reagents than does amphetamine. The stereochemistry of
the compound is also an important factor in its cross-reactivity. The
Amphetamine/Methamphetamine II reagents show greater cross-
reactivity with 9,L-amphetamine than with D or L individually. The
Amphetamine Class reagents also show greater cross-reactivity
with D,L-amphetamine than with D or L amphetamine alone. This
observation was also seen with earlier amphetamine reagents from
Roche Diagnostics (2) but is not now seen with the Roche amphetamine or methamphetamine reagents. For the Amphetamine
Class reagents, the same is true for methamphetamine, while the
Amphetamine/Methamphetamine II reagents show greater reactivity toward D-methamphetamine than to L or D,L isomers. The
Amphetamine/Methamphetamine II reagents show essentially
equal reactivity for D-amphetamine and D-methamphetamine,while
the Amphetamine Class reagents showed slightly higher crossreactivity for D-methamphetamine. The presence of groups on the
aromatic ring has varying degrees of influence on cross-reactivity,
' depending on the number and size of the substituents. The addition
of a methylenedioxy substituent does not significantly impede
binding with the antibody. The presence of methoxy groups on the
ring does decrease binding however, with the single substitution
showing less effect than two or three methoxy groups. The
methylenedioxy group reacts far more readily with the Amphetamine/Methamphetamine
II reagents than with the Amphetamine Class reagents. MDA, for example, shows 10-fold
higher cross-reactivity with the Amphetamine/Methamphetamine
II reagents compared to the Amphetamine Class reagents. For
MDMA and MDEA, the Amphetamine/Methamphetamine II
reagents show 4-fold and approximately 125% cross-reactivity respectively compared to Amphetamine Class reagents, with the
MDMA behaving essentially the same as methamphetamine when
analyzed with the Amphetamine/Methamphetamine II reagents.
The cross-reactivity of the Amphetamine/Methamphetamine II
reagents to 4-hydroxymethamphetamine
is significantly greater
than that of the Amphetamine Class reagents. Because 4-hydroxymethamphetamine is a metabolite of methamphetamine, it would
be expected that the detection of methamphetamine use would be
extended because a higher response would be expected for samples
obtained from drug users. While both reagents reacted with MDA,
MDMA, and MDEA, the Amphetamine Class reagents' response
was low enough to make it of little practical value when cornpared with the response of Amphetamine/Methamphetamine II
reagents for those drugs. Furthermore, the ability to detect 2methoxyamphetamine, 2,5-dimethoxyamphetamine, 4-bromo2.5-dimethoxyamphetamine,
trimethoxyamphetamine, N.Ndimethyl-3,4-methylenedioxyamphetamine.
N-hydroxy-3.4methyienedioxyamphetamme, 2,5-d/methoxy-¢-ethylamphetamme.
2,5-dimethoxy-4-methylamphetamme,
and mescaline with the Amphetamine Class reagents is virtually non-existent. In the case of the
Amphetamine/Methamphetamine II reagents, these compounds
have substantially higher cross-reactivity; however the likelihood
of getting a positive response (at or above 1,000ng/mL) is unlikely
in most cases because of low cross-reactivity. The measured response for a sample can, however, be used to help support the detection Of the compounds using another method, both as an indicator of the utility of further testing and as a necessity to help
fulfill the forensic requirement for two different testing principles
for reporting positives.
Amphetamine/Methamphetamine Il reagents showed approximately the same cross-reactivity to MDA as the Amphetamine vadioimmunoassay (RIA) reagents from Diagnostic Products Corporation (DPC), but greater than that seen with Roche reagents (3,4).
The methamphetamine
reagents from Roche were similar to the reactivity seen with the Amphetamme/Methamphetamine Il reagents,
but both were substantially less than that of the DPC reagents.
MDEA, on the other hand, was very similar for the DPC and Am-
Journal ot ^nalytmal Iox_co_ogy,Vol. l/, ,January/t-eoruary ] _;_
Table
I. Amphetamine
Class
Reagents*
Concentration(ng/mL)
Drug
200
4OO
566
760
1,000
1,566
2,0O0
5,006
10,600
25,666
3,4-Methylenedioxyamphetamine(MBA)
53
26.26%
91
22.72%
114
22.85%
125
16,69%
145
14.49%
175
11.64%
208
10.39%
342
6.84%
475
4.75%
NT
NA
NT
NA
NT
NA
3,4,-Methylenedioxymethamphetamine
(MDMA)
93
46.54%
150
37.43%
189
33.75%
199
26.50%
245
24.48%
304
20.25%
317
15.85%
464
9.28%
741
7.41%
NT
NA
NT
NA
NT
NA
3,4-Meth¥1enedioxyethyl- 146
amphetamine(MDEA) 72.95%
221
55.18%
251
50.11%
273
38.44%
322
32.17%
365
24.36%
404
20.21%
630
12.59%
905
9.05%
1755
7.02%
2832
5.66%
4505
4.50%
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
36
0.72%
65
0.65%
148
0.59%
294
0.59%
615
0.61%
2,5-DimethoxyamphetamineNT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
30
0.59%
38
0.38%
56
0.22%
72
0.14%
79
0.08%
N-Hydroxy-3,4-methylene- NT
dioxyamphetamine
NA
(NOH-MDA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
49
0.98%
81
0.81%
127
0.51%
191
0.38%
NT
NA
54
27.05%
76
18.95%
72
14.38%
85
11.33%
86
8.61%
136
9.05%
159
7.97%
289
5.77%
469
4.69%
NT
NA
NT
NA
NT
NA
2,5-Oimethoxy-4-methylamphetamine(DOM)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
27
0.55%
29
0.29%
46
0.19%
83
0.17%
NT
NA
3,4,5-Trimethoxyphenethylamine(mescaline)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
19
0.08%
32
0,06%
NT
NA
2,5-Dimethox,/-4-ethylamphetamine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
30
0.59%
38
0.38%
56
0,22%
72
0.14%
NT
NA
4-Sromo-2,5-dimethoxy_-phenethylamine
(60MPEA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
27
0.05%
37
0.04%
4-8romo-2,5-dimethoxyamphetamine(DOB)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
24
0.48%
34
0.34%
63
0.25%
92
0.18%
NT
NA
N,N-Oimethyl-3,4-methytene-NT
dioxyamphetamine
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
79
0.79%
250
1.00%
524
1.05%
NT
NA
3,4,5-Trimeth0xyamphetamine(TMA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
21
0.41%
30
0.30%
38
0.15%
60
0.12%
NT
NA
D-Amphetamine
233
116.27%
444
111.12%
522
104.43%
734
97.84%
910
91.02%
1163
77.53%
1488
74.38%
2980
59.60%
5250
52.50%
NT
NA
NT
NA
NT
NA
L-Amphetamine
132
86.25%
238
59.41%
304
60.82%
443
59.01%
611
61.12%
838
55.86%
1571
78.57%
2886
57.72%
HI
ND
NT
NA
NT
NA
NT
NA
o-Methamphetamine
414
207.09%
675
168.69%
790
157.96%
1033
137.80%
1234
123.38%
1610
107.31%
1883
94.16%
3309
66.18%
5161
51.61%
NT
NA
NT
NA
NT
NA
L-Methamphetamine
139
69.72%
325
81.33%
533
106.63%
765
101.94%
1148
114,81%
1746
116.39%
2316
115.80%
HI
ND
HI
ND
NT
NA
NT
NA
NT
NA
D,L-Methamphetamine
375
187.74%
787
196.70%
1009
201.72%
1480
197.37%
1693
199.26%
2791
186.09%
3849
192.43%
HI
ND
HI
ND
NT
NA
NT
NA
NT
NA
Phenylpropanolamine
NT
NA
NT
NA
NT
NA
NT
NA
132
13.22%
NT
NA
NT
NA
427
8.54%
721
7.21%
1517
6.07%
3697
7.39%
HI
ND
Pseudoephedrine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
17
0.83%
102
2.04%
237
2.37%
658
2.63%
1498
3.00%
3466
3.47%
Ephedrine
NT
NA
NT
NA
NT
NA
NT
NA
263
26,27%
NT
NA
NT
NA
631
12,62%
943
9.43%
1476
5.91%
2252
4.50%
3853
3.85%
using
the following
2-Methoxyamphetamine
4-Hydroxymethamphetamine
· The
first
row
of numbers
for
a particular
drug
indicates
concentration
formula:
100 x (Measured
amount)
/ (Actual
concentration),
NT = Not tested;
NA = Not applicable;
ND = Not determined;
measured
in ng/mL
The
second
Sensitivity
of the assay
is reported
to be
HI = Greater
than
highest
calibrator.
row
100
of numbers
ng/mL;
values
is percent
less
than
cross-reactivity,
100
are
reported
calculated
only
56,660 166,600
for comparison
purposes,
Journal of Analytical Toxicology,Vol. 17, January/February 1993
Table
II. Amphetamine/Methamphetamine
II Reagents*
Concentration(ng/mL)
Drug
200
400
500
750
1,000
1,500
2,000
5,000
10,000
25,000
56,000
100,000
3,4-Methylenedioxyamphetamine(MDA)
272
136.00%
571
142.75%
708
141.60%
1227
163.60%
1478
147.80%
2411
160.73%
3406
170.30%
HI
ND
HI
ND
NT
NA
NT
NA
NT
NA
3,4,-Methylenedioxymethamphetamine
(MDMA)
183
91.50%
367
91.75%
452
90.40%
734
97.87%
969
96.90%
1499
99.93%
2043
102.15%
5196
103.92%
HI
ND
NT
NA
fit
NA
NT
NA
3,4-Methylenedioxyethyl- 133
amphetamine(MDEA) 66.50%
231
57.75%
261
52.20%
395
52.67%
427
42,70%
576
45.07%
791
39.55%
1616
32.32%
3056
30.56%
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
134
26.80%
NT
NA
300
30.00_o
500
33.33%
643
32.15%
1674
33.48%
3457
34.57%
NT
NA
NT
NA
fit
NA
2,5-OimethoxyamphetamineNT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
326
6.52%
525
5.25%
1199
4.80%
2057
4.11%
HI
ND
N-Hydroxy-3,4-methylenedioxyamphetamine
(NOH-MDA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
135
2.70%
227
2.27%
438
1.75%
663
1.33%
1000
1.00%
4-Hydroxymethamphetamine
NT
NA
NT
NA
397
79.40%
NT
NA
787
78.70%
NT
NA
2076
103.80%
4451
89.02%
7292
72.92%
NT
NA
NT
NA
NT
NA
2,5-Dimethoxy-4-methylamphetamine(DOM)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
210
4.20%
356
3.58%
673
2.69%
1125
2.25%
NT
NA
3,4,5-Trimethoxyphenethylamine(mescaline)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
2
0.02%
4
0.02%
18
0.04%
NT
NA
2,5-Oimethoxy-4-ethylamphetamine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
197
3.94%
315
3.15%
573
2.29%
874
1.75%
NT
NA
4-Bromo-2,5-dimethoxy[_-phenethylamine
(BDMPEA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
119
0.12%
4-Bromo-2,5-dimethoxyamphetamine(DOB)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
254
5.08%
401
4.01%
690
2.76%
1068
2.14%
1800
1.80%
N,N-Dimethyl-3,4-methylene-NT
diox,/amphetamine
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
38
0.38%
124
0.50%
430
0.86%
NT
NA
3,4,5-Trimethoxyamphetamine(TMA)
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
172
3.44%
323
3.23%
685
2.74%
1028
2.06%
2040
2.04%
L-Amohetamine
121
60.50%
227
56.75%
269
53.80%
414
55.20%
569
56.90%
794
52.93%
1025
51.25%
3001
60.02%
6626
66.26%
NT
NA
NT
NA
NT
NA
O,L-Amphetamine
240
120.00%
546
136.50%
728
145.60%
1160
154.67%
1651
1.65.10%
2983
198.87%
4204
210.20%
HI
ND
HI
NO
NT
NA
NT
NA
NT
NA
D-Methamphetamine
215
108.00%
430
107.50%
543
108.60%
756
101.07%
978
97.80%
1479
98.60%
1968
98.40%
4278
85.56%
HI
NO
NT
NA
NT
NA
NT
NA
L-Methamphetamine
NT
NA
NT
NA
NT
NA
NT
NA
72
7.20%
103
6.87%
144
7.20%
461
9.22%
1039
10.39%
NT
NA
NT
NA
NT
NA
O,L-Methamphetamine
115
57.50%
256
64.00%
321
64.20%
457
60.93%
623
62.30%
964
64.27%
1219
60.95%
3229
64.58%
6554
65.54%
NT
NA
NT
NA
NT
NA
Phenylpropanolamine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
72
0.07%
Pseudoephedrine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
0
.00%
Ephedrine
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
NT
NA
1
.00%
2-Methoxyamphetamine
_.
* Thefirst rowof numbersfor a particulardrugindicatesconcentration
measuredin ng/mL.Thesecondrowof numbersis percentcroas-raactlvity,
calculatedusingthefollowing
formula:100x (Measuredamount)/ (Actualconcentration).
Sensitivity
of theassayis reportedto be100ng/mL;valuesleasthan100are reportedonlyfor comparisonpuq3oses.
NT= Nottested;NA= Notapplicable;ND = Notdetermined;
HI = Greaterthanhighestcalibrator.
I
Journal of Analyticai Toxicology, Vol. 17, January/February
phetamine/Methamphetamine II reagents, and both were far above
the cross-reactivity seen with the Roche assay (2,4). The 4-hydroxymethamphetamine cross-reactivity for the Amphetamine/Methamphetamine II reagents was greater than either the DPC and Roche
reagents although all showed significant cross-reactivity. In virtually
every case, RIA reagents performed better than the FPIA Amphetamine Class reagents. This was true not only in the ability to
identify illicit analogues, but also in the decreased cross-reactivity
with over-the-counter medications. The Amphetamine/Methamphetamine I/reagents have one significant advantage over the RIA
reagents; a single test using the former reagents will detect the presence of amphetamine or methamphetamine, whereas this is currenfly accomplished only by conducting different tests with the RIA
systems.
Studies have been reported elsewhere for MDA, MDMA, and
MDEA using Syva EMIT (5,6) and Abbott TDx (5), and for
MDA and MDMA using the Abbott TDx system (7) and radioimmunoassay (3,4). The ability of each of these systems to
identify the analogues varies, but all show some significant limitations. There is now sufficient data to properly evaluate results
from these systems to assess the presence of the analogues,
While no immunoassay result should by itself be considered
unequivocal proof, use of these tests in conjunction with another chemically different method (i.e. GC/MS) should be considered scientifically and forensically acceptable,
The availability of reagents with higher specificity (Amphetamine/Methamphetamine II) has the significant advantage of decreasing the number of"positive" results from common phenethylamines seen with the Amphetamine Class reagents. Phentermine
remains the only commonly encountered medication that has
substantial cross-reactivity with the Amphetamine/Methamphetamine II reagents (8). It also has the advantage of increased crossreactivity to some of the more common illicit analogues like
MDA, MDMA, and MDEA (although the MDEA values are relatively close with both reagent systems), thereby allowing the
identification of analogues without loss of the ability to identify
the primary analytes. This allows those interested in screening for
these compounds to do so rapidly and easily, and they are not
likely to cause significant added confirmation workload for labs
that do not perform confLrmationfor thesedrugs.
i
I
Positive immunoassay results for amphetamines are not absolutely specific for the drug and can be caused by a number of
other phenethylamines. Positive results may also be caused by
the presence of an amphetaImne analogue. When the use of an
amphetamine analogue is suspected, it is valuable to evaluate
negative (below the established cutoff) immunoassay results
with regard to the cross-reactivity
data. Because these cornpoundshave a high abusepotential, it is important to keep the
cross-reactivity of thesecompoundsin mind.
When a positive sample from the AmphetaminefMethamphetamine II assay shows no indication of amphetamine or
methamphetamine by GC/MS, one should consider the possible
presence of an amphetamine analogue. Investigation of the
sample for one or more of these compounds should be undertaken using someother methodology which is capableof identifying the specific compounds.
Conclusion
Of theamphetamineanaloguestested,severalshowedsubstanrial cross-reactivitywith the FPIA reagents.In the case of suspected use of an amphetamine analogue other than those which
1_9:.f,
showed substantial cross-reactivity, it must be realized that a posirive FPIA result would not be expected. Therefore, a negative immunoassay result does not indicate that there was no amphetamine
analogue present. Several compounds did cross react at high concentrations, although they did not yield positive results with either
set of reagents. Particularly in suspect cases, samples with the indication of presence of amphetamines should be tested for the illicit
amphetamine analogues. The use of the Amphetamine/Methamphetamine II reagents had several significant advantages over the Amphetamine Class reagents. The cross-reactivity with methamphetamine, which is the more commonly abused drag, is increased
with the Amphetamine/Methamphetamine II reagents, as is the hydroxy metabolite of methamphetamine. The cross-reactivity to the
L-isomer
ofthedrugislowenoughtofairlywellassurethelackof
positive results from the normal use of a Vick's inhaler.
The analysis of samples for the presence of amphetamines and
amphetamine analogues is a difficult process, and no single immunoassay holds a clear advantage over the other commercially
available reagents. Knowledge and application of cross-reactivity
data of the assay systems, however, can make the task of interpretation of analytical results and the reconciliation of immunoassay and GC/MS results easier. As was seen with immunoassay systems from other manufacturers, the lack of
substantial cross-reactivity by the FPIA reagents to some of the
amphetamine analogues evaluated in this study limits the utility of
this methodology in testing for the presence of these analogues.
Acknowledgments
Thanks to the NIDA for several amphetamine analogues used
in this study, to Ms Liserio of the Forensic Investigations branch
for assistance in processing samples, and Quality Control personnel for assistance in preparation of standard solutions.
References
1. ControlleO
SubstanceAnalogue
Act of 1985Reportofthe
Committeeon the
JudiciaryU S Senate21
Nov 85 and Designer
DrugEnforcementAct of 1986Report to Accompany H.R. 5246
19 Sep 86.
2. J.T. Co0y and R. Schwarzhoff. Unpublishedresults.
3. J.T.Cody.Cross-reactivityofamphetamineanalogues withRoche
Abuscreen radioimmunoassay reagents, d. Anal. 7-oxicoL14:
150-53 (1990).
4. J.T. Cody. Detection of D,L-amphetamine, D,L-methamphetamine
and illicit amphetamineanaloguesusing DiagnosticProducts
Corporation'sAmphetamineand Methamphetamineradioimmunoassay.
J. Anal. Toxicol.14:321-24 (1990).
5. W. Ruangyuttikarn
and D.E.Moody.Comparisonof threecommercial amphetamine immunoassays for detection of methamphetamine,methylenedioxyamphetamine,
methylenedioxymethamphetamine,and methylenedioxyethylamphetamine,
d. Anal.
Toxicol.12:229-33 (1988).
6. W.L. Heam,G. Hime,and W. Andolla.Recognizingecstasy:
Adamand Eve, the MOAderivatives--analytical
profiles.Presentedat Societyof ForensicToxicologists
Meeting,Reno,1986.
7. J.M. Ramos,R.L. Fitzgerald,and A. Poklis.MDMAand MDA
crossreactivityobservedwithAbbottTDxAmphetamine/Methamphetaminereagents.C/in.Chem.34(5):991(1988).
8. SystemAssaysManual,AbbottLaboratories,
NorthChicago,IL,
1989.
ManusciptreceivedOctober4, 1991;
revisionreceivedApril20, 1992.
I
I
Evaluation
oftheSyvaETS®PlusUrineDrugandSerum
EthanolAnalyzer
Saeed
A. Jortani*
and Alphonse
Poklis
Department of Pathology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0597
Abstract
!
The Syva ETS® is an automated system designed for analysis
of drugs of abuse in urine. Recently, the Syva Company has
upgraded this system to the ETS Plus, which is capable of
analyzing ethanol in urine, serum, or plasma. We evaluated the
new ETS Plus for urine screening of barbiturates,
benzodiazepines, cocaine metabolite, opiates, and
t
as a new carousel cover, minimize sample ethyl alcohol evaporation because ethyl alcohol samples are processed first.
The purpose of this study was to compare the performance of
the new ETS Plus system software version 4.04 to that of the
ETS system software version 3.03 in terms of calibrator separation values and patient results. Also, the ability of the new software to accurately quantitate serum ethyl alcohol and simultaneously calculate urine drug-of-abuse results when samples were
phencyclidine. Results of 505 patient sample assays obtained
by ETS Plus were compared with those of ETS. There were
only four discrepant results which had absorbance rates close
to the Iow calibrator cutoff values. The within-run precision of
the ETS® Plus Ethyl Alcohol Assay yielded a CV of 2.6% at a
target value of 1.00 g/L (1.06 -+0.03 g/L, n = 32) and a CV of
3.2% at a target value of 0.40 g/L (0.42 _+0.01 g/L, n = 30). The
linear regression
analysis of 30 patient serum ethanol results
by the ETS Plus and by gas chromatography yielded y = 0.939x
processed in random access mode was evaluated. Results of the
patient serum ethanol analysis by the ETS Plus were compared
with those obtained by a direct-injection gas chromatographic
method (3).
+ 0.03 g/L. The software modifications in the new ETS Plus
allow the accurate quantitation
of ethanol in serum and reliable
detection of drugs of abuse within the same batch.
Materials and Methods
Syva ETS system with software version 3.3 and
Plus system with version 4.04 sotlware were obtained
Company. All reagents, calibrators, and controls for
assays for barbiturates,
benzodiazepines,
cocaine
Syva ETS
from Syva
Emit d.a.u.
metabolite
The Syva ETS ® system is a dedicated analyzer for screening
14 drugs of abuse in urine. The analyzer uses Syva Emit ®
[benzoylecgonine),
opiates, phencyclidine,
and the ETS Plus
Ethyl Alcohol Assay were also obtained from Syva Company.
Emit d.a.u, low calibrators (A and B) containing the following
were used to calibrate the drugs-of-abuse
assays: 0.2 gg/mL
secobarbital
and oxazepam;
0.3 gg/mL benzoylecgonine,
d.a.u. TM immunoassays (1). Edinboro et al. (2) have previously
demonstrated the ETS system's advantages of random access
methadone, and morphine; and 0.025 gg/mL phencyclidine. The
Emit d.a.u, reagents consisted of Emit antibody/substrate
reagent
mode, large sample load, real-time data processing, and the
ability to run six-drug assays on a single sample in six minutes
A, enzyme reagent B, and Emit drug assay buffer concentrate.
Serum ethanol controls of 0.70 g/L and 1.92 g/L were obtained
or less.
Syva Company has recently upgraded the ETS system to the
new ETS Plus system. Among the features of the upgraded
system are the new 4.04 software version and additional testing
capabilities for quantitation of ethyl alcohol in urine, serum,
from SigmaChemicalCo.The ETSPlusethylalcoholassayutilizes a single-point
calibration
of 1.00 g/L for quantitative
ethanol determinations requiring 300 gL of serum or urine; The
ETS Plus automatically mixes 7.5 gL of sample, with 6.0 gL of
reagent A, 6.0 gL of reagent B, and 187.5 gL of Emit Drug
and plasma. Special features built into the new software, as well
assay buffer. This mixture is incubated for 15 s and the rate of increase in NADH absorbance at 340 nm is monitored for 30 s. The
·Author
towhom
correslx)nclonco should
boaddressed.
Emit d.a.u, and the ETS Plus Ethyl Alcohol
Introduction
!:l_nrorh lotion tr_hr_toeonvintal of _clitorial cc_nt_antc,f thiq italJrnal iq r_rr_hibit_'"l withollt
m jhliqh*ar'_ Dorrrli_sirm
Assays were per-
-_'_
Table I. Drug Assay Calibrator Values
formed asrecommendedby themanufacturer (1).
All gas chromatographic ethanol determinations
wereperformed
ona Shimadzu 14-A GC (Shimadzu Co., Kyoto,
A.ETS_system,Softwareversion3.03
'
Anaiyte
n
Barbiturate
Benzodiazepine
Cocaine
Opiates
PCP
Negto Low(aA)
LowtoMed (aA)
Minimum Mean± SD
Minimum Mean±SD
12
13
13
12
11
>24
>22
>24
>22
>24
75_+9
88± 9
72_+8
145±11
132± 19
>52
>52
>52
>44
>56
239_+49
231-+41
300_+11
308±44
342_+48
B.ETS
®Plussystem,Softwareversion4.04
Analyte
n
Negto Low(z_)
LowtoMed(_d[)
Minimum Mean+SD
Minimum Mean±SD
Barbiturate
Benzodiazepine
Cocaine
12
13
13
>24
>22
>24
101+34
103_+30
90+_22
>52
>52
>52
274±63
222±44
300_+40
Opiates
PCP
12
11
>22
>24
174_+39
165±27
>44
>56
309±65
372+_42
Table II. Patient Urine Drugs-of-Abuse
Screening
Numberof
Assays
Analyte
Barbiturate
Benzodiazephine*
Cocaine*
Opiate
Phencyclidine
Total
Results
ETS
PositiveResult
100
99
107
100
99
505
ETSPlus
PositiveResult
19
24
39
25
0
107
19
23
36
25
0
103
Japan) equipped with a 3-m x 2-mm i.d.
Carbopack B/5% Carbowax ® glass
column (3). The flame ionization detector response was recorded with an HP
3396A integrator (Hewlett-Packard). In a
clean test tube, 0. i0 mL of serum or
urine was mixed with 0.90 mL of 2.34
g/L 1-propanol internal standard solution. After vortexing for 20 s, 1 gL of the
mixture was injected into the gas chromatograph. GC conditions were as follows: helium carrier gas flow, 20
mL/min; injector temperature, 200°C;
column, 75°C; and detector, 230°C.
Under these conditions the retention
times of ethanol and l-propanol were 3.0
and 6.0 minutes, respectively.
All reagents, calibrators, and controls
were stored at 4°C. Samples used in this
study were obtained from patients seen at
the Medical College of Virginia Hospitals
during a 20-week period. Drug screens
performed on patient samples had been
requested by the attending physicians.
Results and Discussion
'Discrepant
sampleswhichgavepositiveresultsby ETSandnegativeby ETSPlus.
Rates
in all four cases
were close
Table I shows the mean calibrator separation values obtained for each drug-ofabuse assay on the ETS (A) and the ETS
Plus CB)systems. The rmnimum calibrator
to cutoff values.
I
eaonve
o
4.0
runbysubtracting
theabsorbance
ofthe
negative
calibrator
fromthatofthelow
calibrator, and the absorbance of the low
calibrator
fromthatof themedium
cali-
3.5
"'
_
_'
I
_
3.0
_'5
t
_
brator°
2.o
1.5
,m 1.o
.5
o
.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
SERUMETOH(g/L),GASCHROMATOGRAPHY
Figure
1.Correlation
of patient
serumethanolresults
obtained
byETSPlusintherandom
access
mode
versusgaschromatography,
r2=0.992.
32
A
positive
result
was
defined
as
a
response equal to or greater than the absorbance rate of the low calibrator. The
calibrator
separation
values
obtained
in
thisstudybybothsoftware
versions
were
wellabove
theminimum
separation
values
required by the manufacturer (1).
Patient drugs-of-abuse urine screening
results by ETS and the ETS Plus systems
are shown in Table II. Results obtained by
the ETS with version 3.03 software and
the ETS Plus with version 4.04 software
were in agreement in 501 out of 505 assays
(99.2%). None of the patient samples assayed for phencyclidine
during this study gave positive results. Discrepant results were obtained with only four samples. One benzodiazepine and three
cocaine assays were positive on the ETS and negative on the
ETS Plus. The rates obtained by both systems for these four sam-
liably screen urine for drugs of abuse by Emit d.a.u, immuneassays while accurately performing serum ethanol quantitation.
pies were close to their calibrator cutoff values. The absorbance
rates of ETS and ETS Plus and their calibrator cutoff differences
for the discrepant benzodiazepine urine sample were +14 and
-7, respectively. The absorbance rates and the calibrator cutoff
differences for the three discrepant cocaine urine samples were
+25, +22, and +4 for ETS, and -8, -13, and --6 for ETS Plus, respectively.
The within-run precision of the ETS Plus Ethyl Alcohol Assay
yielded CVs of 2.6% at a target value of 1.00 g/L (1.06 + 0.03
Acknowledgments
g/L, n = 32) and 3.2% at a target valueof 0.40 g/L (0.42 __.
0.01
g/L, n = 30). The linear regression analysis of 30 patient serum
samples analyzed for ethanol by the ETS Plus and gas chromatography yielded y = 0.939x + 0.03 g/L, r2= 0.992 (Figure 1).
These ethanol determinations were performed in the random access mode with urine drug assays on the same carousel.
The results of this study demonstrate that revisions of the ETS
software to ETS Plus version 4.04 have retained the ability to re-
The authors thank Syva Company for providing the reagents
and software version 4.04 for this study.
References
1. SyvaETS®PlusSystemOperatorsGuide.Syva(a SyntexCo.),
PaloAlto,California(1990).
2. L.E.Edinbom,K.V.Hall,and A.Poklis.Evaluationof the ETS®and
ADxTM urine drug screening immunoassay analyzers, d. Anal.
Toxicol.13:232-34 (1989).
3. A.S. Curry,G.W. Walker, and G.S. Simpson. Determination of
ethanolin blood by gas chromatography.
Analyst91:742-43
(1966).
Manuscript received January 30, 1992;
revision received May 4, 1992.
33