*

abstrac
Sebuah metode yang sederhana, cepat, dan sensitif untuk mendeteksi langsung spektrum yang luas dari
obat-obatan penyalahgunaan dalam darah keseluruhan hemolysed oleh enzim yang dikalikan
immunoassay teknik (EMIT) yang digambarkan. Ekstrak methanollc 1 ml darah keseluruhan langsung
dianalisis dengan tes urin EMIT. ]'heproposed metode sangat sensitif dan dapat mendeteksi konsentrasi
obat di rendah terapi untuk rentang konsentrasi subtherapeutlc untuk semua tes sepuluh yang
digunakan. Tes urin EMIT yang digunakan dalam kajian ini termasuk orang-orang untuk opiat,
amfetamin, metadon, barbiturat, phencyclldine (PCP), methaquslone, propoxyphene, metabolit kokain,
benzodlazeplne metebollte, dan cannablnolds. Metode ini harus paling berguna untuk pemeriksaan
forensik kasus oleh EMIT ketika urin tidak tersedia, seperti dalam kasus gangguan mengemudi.
Introduction

Pada tahun 1972 Rubenstein et al. (1) melaporkan penggunaan teknik immunoassay dikalikan en-zyme
(EMIT) pertama untuk layar-ing urin untuk obat-obatan penyalahgunaan. Sejak saat itu, enzim immunoassay teknik telah pernah meningkatkan penggunaan. Saat ini tersedia EMIT tes untuk
mendeteksi obat-obatan penyalahgunaan dalam urin termasuk amfetamin, barbiturat, kokain metabolit
(benzoylecgonine), benzodiazepin metabolit, pro-poxyphene, methaqualone, phencyclidine, opiat,
metadon, dan cannabinoids. Tes urin ini memiliki hanya terbatas di Toksikologi forensik. Dalam upaya
untuk memperluas kegunaan dari EMIT, air liur (2,3), vitreous humor (4), dan air mata (5,6) telah
dianalisis secara langsung sebagai urin. Itu belum dimungkinkan untuk langsung menganalisis seluruh
darah, empedu, atau jaringan ekstrak oleh EMIT karena latar belakang tinggi absorbansi tingkat (7),
tetapi prosedur EMIT telah berhasil dimodifikasi untuk digunakan dengan darah, empedu dan jaringan
ekstrak (8,9). Prosedur ini melibatkan relatif besar volume (5-10 mL) darah, prosedur ekstraksi panjang
dengan pelarut organik immiscible air, penguapan kekeringan, dan pemulihan dengan pengakuan
buffer. Beberapa berbeda ekstraksi pro-cedures diperlukan untuk melakukan layar komprehensif obat.
.
Until recently, most of the Syva EMIT d.a.u.| (drugs of abuse in urine) assays used a lysozyme-mediated reaction. In 1981, Fletcher et al. (10)
noted that this enzyme reaction could be per-formed in an environment containing as much as 75 percent methanol. Consequently, a methanolic
extract of whole blood could be analyzed directly by radioimmunoassay (RIA) for the presence of tetrahydrocannabinol (THC). Peel and Perrigo
(12) have used an assay mediated by malate dehydrogenase to detect cannabinoids in a methanolic extract of whole blood. In early 1985, Syva
converted seven EMIT d.a.u, assays from the ly-sozyme-mediated reaction to one utilizing glucose-6-phosphate dehydrogenase (13). Currently,
all ten EMIT urine assays utilize glucose-6-phosphate dehydrogenase. This conversion has allowed increased sensitivity with fewer interferences.

Tujuan dari penyelidikan ini adalah pertama untuk menentukan apakah prosedur ekstraksi methanolic
yang digunakan oleh kupas dan Perrigo (12) dapat digunakan dengan dehidrogenase fosfat glukosa 6
tersedia dimediasi tes EMIT. Tes EMIT skr kini sedang tersedia sepuluh ditemukan untuk berfungsi
dengan baik di lingkungan methanolic. Langkah berikutnya adalah untuk mengembangkan metode
yang sederhana, cepat yang memungkinkan semua sepuluh EMIT urin tes digunakan untuk analisis
langsung ekstrak methanolic 1 ml darah. Tujuan akhir dari penyelidikan ini adalah untuk menentukan
batas minimum konsentrasi terdeteksi dengan kepercayaan dari tiga deviasi standar untuk setiap assay
obat. EMIT tes secara tradisional telah dikritik karena tidak sensitif yang RIA (7,14). Ini adalah niat
kita untuk menentukan apakah ekstrak methanolic 1 ml darah dapat diuji oleh semua sepuluh EMIT tes
urin dengan cukup kepekaan untuk mengaktifkan deteksi obat penyalahgunaan pada konsentrasi
rendah atau kantong.

Materials

Instruments

Sistem Syva MEMANCARKAN digunakan. Ini terdiri dari Stasar-sakit Spectrophotometer (Gilford
instrumen Labs), Syva pipetter diluter (Cavro instrumen ilmiah), EMIT klinis Pro-cessor Model CP5000, dan pompa Gilford 3021 vakum Penerima dan DeVilbiss. Spectrophotometer dioperasikan dalam
modus penyerapan di 340 rim, dan sel microflow ditetapkan pada 30 ~ pipetter-diluter ditetapkan untuk
sedot 50/LL sampel dan untuk memberikan contoh ditambah 250/zL buffer. CP-5000
adalah waktunya untuk mengukur absorbances pada 15 dan 95 s dan cal-culate absorbansi perbedaan
(2tA).
The methanolic supernatant was filtered with a 0.45-#m disposable filter assembly (Gelman Acrodisc, Product No. 4184) and a l-cm ~
tuberculin disposable syringe (Becton Dickinson & Co. Ltd.).

Reagents

Berikut sepuluh memancarkan d.a.u , assay kit yang dibeli dari Perusahaan Syva dan dipersiapkan
sebagai diarahkan : ampheta - tambang , barbiturat , benzodiazepin metabolit , cannabinoid ,
kokain metabolit , metadon , methaqualone , opiat , Phen - cyclidine , dan propoxyphene . Reagen
A untuk setiap assay con - terkandung antibodi , substrat ( glukosa - 6 - fosfat ) , dan koenzim
( nicotinamide adenin dinukleotida ( NAD + ) ) di 0.055M Tris - HCI , pH 5.2 . Reagen B terkandung
turunan obat berlabel dengan dehidrogenase glukosa - 6 - fosfat dan 0,05 % natrium azida di
0.055M Tris - HCI , pH 8,0 . Buffer konsentrat terkandung 0.825M Tris - HCl buffer pH 8,0 dan 0,05
% natrium azida . Metanol dibeli dari Caledon dan kelas HPLC .
The following drugs or metabolites (obtained from the De-partment of Health and Welfare, Ottawa) were prepared in ethanol at a concentration
of approximately 1 mg/mL calculated as the free base or acid: dextroamphetamine, secobarbital,

EXTRACTION

PROCEDURE

1 M.L WHOLE

BLOOD

+
2 ML M E T H A N O L

VORTEX

VIG O R O U SLY

3 MIN.

CENTRIFUGE 5

REMOVE

MIN. ( 3 0 0 0 RPM)

S U P E R N ATAN T

( A P P R O X 1 . 0 ----* 1 . 5 ML)

F I L T E R WITH

ANALYZE

SAMPLE

0 . 4 5 MICRON

D I RE CTLY

SUFFICIENT

IN DUPLICATE

OF

50 /tl WHOLE

FOR

USING

BLOOD

FILTER

BY

EMIT

i0 SEPARATE

THE

PER

ASSAYS

EQUIVALENT

ANALYSIS

oxazepam, tetrahydrocannabinol (THC), benzoylecgonine, metadon, methaqualone, morfin,

phencyclidine, dan pro-poxyphene. Setiap larutan stok diencerkan 1:10 dengan air. Darah (10 ml)
dibubuhi kuantitatif dengan masing-masing larutan stok air pada rentang konsentrasi dari
subterapeutik ke beracun, termasuk kosong. Satu set obat darah standar solu-tions mengandung
obat berikut: dextroamphetamine, ben-zoylecgonine, metadon, morfin, dan secobarbital (cormenanggapi Syva A solusi kalibrator). set standar obat darah terkandung methaqualone,
oxazepam, phencyclidine, dan propoxyphene (sesuai dengan solusi kalibrator Syva B). Standar
darah cannabinoid dibuat dari larutan stok etbanolic dari tetrahydrocannabinol baru disiapkan
pada concemratJon dari 1 mg / mL. Darah, yang telah dibekukan, darah sapi yang mengandung
1% sodium fluoride dan 0,25% kalium oksalat. Semua reagen dan standar, berpendingin untuk
penyimpanan, diizinkan untuk menyeimbangkan selama minimal 2 jam pada suhu kamar sebelum
digunakan.
Procedure
One milliliter of blood was added to a 13-mL plastic centri-fuge tube and 2 mL of methanol was added by streaming down the inside of the
tube. The mixture was vigorously vortexed for 3-5 min and then centrifuged at 3000 rpm for 5 min at - 20~ The clear supernatant (1.0-1.5 mL)
was filtered with a 0.45-#m filter attached to a l - cm' tuberculin syringe. As much sample as possible was removed from the filter assembly by
purging

INSTRUMENT
WHOLE

TEMPERATURE :
WAVELENGTH:
DELA Y

TIME :

SETTINGS
BLOOD

30"C
340

nm

15 SEC.

URINE

30"C
340

nm

15 SEC .

MEASURE TIME

80 SEC.

ABSORPTION

MODE

3 0 SEC .
CONCENTRATIO N

PROCEDURE (SAME AS URINE ASSAYS) 50 ~ul SAMPLE + 250 ,all BUFFER

ADD

50

,ul REAGENT

A + 2 5 0 )al BUFFER

1
EQUILIBRATE

30 SECONDS

ADD

50

2 5 0 ~ul BUFFER

).11 REAGENT B +

1
IMMEDIATELY ASPIRATE INT0 FLOW CELL REAGENT A: ANTIBODY+ SUBSTRATE - eNAD+

REAGENT B:

DRUG -

ENZYME COMPLEX

with air. After equilibration to room temperature, the filtered supernatant was analyzed directly by EMIT. This procedure is outlined in
Figure I. With the pipetter-diluter, 50 #L of metha-nolic supernatant was added to 250 #L of buffer solution and mixed in a 2-mL
disposable beaker. Fifty microliters of reagent A (antibody, substrate, and coenzyme) and 250 #L of buffer were added to the beaker.
After a 30-s equilibration, 50 #L o f reagent B (drug-enzyme complex) and 250 #L of buffer were added to the beaker. The contents of the
beaker were immediately aspirated into the flow cell of the spectrophotometer, and ab-sorbance readings were taken automatically at 15
and 95 s. This procedure is outlined in Figure 2.

Results

and

Discussion

The standard curves for each of the ten assays are shown in Figures 3 to 12. Each sample was analyzed in duplicate, and
range shown for THC - COOH, Syva reports that the response o f THC is less than that o f THC - COOH in the cannabinoid assay (15).
It is estimated that 20 ng/mL of THC - COOH will give the same response as 70 ng/mL of THC. In this procedure the minimum
detectable blood concentration of THC would be approximately 40 ng/mL THC. This is a relatively high con-centration of THC, but in
terms of relative enzyme response it corresponds to approximately 12 ng/m L THC - COOH, to which the antibody is most sensitive. The
metabolite THC-COOH reportedly appears in blood at higher concentrations for a number of hours after smoking marijuana, whereas
THC disappears relatively quickly (16).

Fletcher (10) observed that the lysozyme-mediated assays could tolerate up to 75 percent methanol, Peel and Perrigo (l 2) showed that
the cannabinoid assay using malate dehydrogenase was also tolerant of elevated methanol concentrations. It was obvious from the
standard curves shown in Figures 3-12 that

the glucose-6-phosphate dehydrogenase mediated reaction was also tolerant o f methanol at a concentration o f approximately 66
percent. It should be noted that the final concentration of methanol in the sample cuvette would be approximately 3.7~ by volume. As
might be expected, the rate of reaction has been decreased, as noted by the change in absorbance per minute.

For urine analysis, Syva recommends a maximum allowable difference between replicates of 6 milliabsorbance (mA) units in the
concentration mode, which incorporates an amplifica-tion factor of 2.667 relative to absorbance data. Thus the max-imum allowable
difference in the absorbance mode would be 6 divided by 2.667, or 2.25 mA units. Presumably this cor-responds to 1 standard deviation.
The recommended difference between the negative and low calibrators of 18 m A units in the concentration mode would correspond to
6.75 m A units in the absorbance mode. This value presumably corresponds to 3 standard deviations.
The proposed method using methanolic extracts of whole blood is performed in the absorbance mode rather than the con-centration mode. Our
results have shown that replicate analyses of methanolic extracts can achieve a difference of 2 mA units. This is best illustrated in Table 1, where
20 samples of outdated whole blood obtained from the Red Cross (presumably drug-free) showed a standard deviation in nine assays as a mean
value of 1.63 mA units. Three standard deviations would be 4.9 mA units. This data corresponds very well to the recommended values of 2.25
mA units and 6.75 mA units for urine analysis.

The minimum detection limit for each assay was determined to be the highest absorbance value for the negative calibrator plus 6 mA units (3
standard deviations). The minimum detec-table drug concentration for each assay is shown in Table II. All of the levels shown correspond to low
or subtherapeutic drug concentrations. These levels correspond to absolute minimum detection limits and may be somewhat low for routine toxicological drug screening. For routine work, the authors have adopted a cutoff of 10 mA units (approximately 5 standard deviations), rather than 6
mA units.

The data reported above was obtained with the flow cell temperature set at 30~ and a measure time of 80 s. Preliminary studies were conducted
in the hope of lowering the minimum detection limits. One approach was to increase the temperature to 37~ It was found that the rate of
absorbance change did increase with the increase in temperature, and the absolute dif-ference in absorbance was increased by an increase in
measure time longer than 80 s. However, the relative change in absor-bance did not change significantly. Therefore, in order to main-tain the
same level of precision and for practical considerations, the reaction conditions were not altered.

By using 1 mL of blood and 2 mL of methanol, the superna-tant volume was between 1 and 2 mL, depending on the hemat-ocrit. The
methanolic extract was then analyzed by ten different assays. Each assay required 50 #L of extract--100/zL if done in duplicate. Since the
maximum allowable difference between replicates was only 2 mA units, it was occasionally necessary to sample in triplicate. If it was necessary
to sample each of the ten assays in triplicate, the volume of methanolic extract
required would be 1.5 mL (10 assays x 3 samples x 50#L per sample). The supernatant volume may, however, be only ap-proximately 1 mL.
Fortunately it has been the authors' ex-perience that if a sample yields an absorbance value less than or equal to that of the negative calibrator,
then only one analysis is required. Since most of the ten assays for any given blood sample will be negative, there should be more than enough
supernatant for all 10 assays.

Occasionally it was found that blood with a very high hemat-ocrit, such as heart blood, could yield an extract that was still pale red, indicating
that not all of the hemoglobin had been precipitated. Usually these samples could still be analyzed directly, even though the initial absorbance
values were some-what elevated. If the analyst wants to remove the remaining hemoglobin, two procedures can be attempted. The first is to
freeze the extract causing additional precipitation which can then be filtered off. Alternatively, additional methanol (0.5 mL) can be added, the
sample revortexed and centrifuged. This addi-tional procedure will result in a minor dilution relative to the standards. A number of actual forensic
case blood samples and selected spiked blood samples were analyzed by the proposed method.
Five postmortem blood samples were analyzed by the amphet-amine assay. The data is summarized in Table III. Case 4 was negative, while
cases 1 and 2 showed a very weak positive response. Cases 3 and 5, which were very putrid, showed a strong positive response. The EMIT
amphetamine assay is known to cross-react with a broad spectrum of phenethylamines, many of which are putrefactants (7,16). All five actual
cases examined had been previously analyzed by thin-layer chroma-tography (TLC), gas chromatography with nitrogen specific detection
(GC/NPD), gas chromatography/mass spectrometry (GC/MS), and EMIT on urine samples where possible. All five cases were found to contain
no detectable amphetamine related drugs. Thus, with postmortem samples a higher minimum cutoff is recommended for the amphetamine assay-perhaps corres-ponding to 0.20 or 0.30 #g/mL amphetamine. Due to the high cross-reactivity of this assay to putrefactants, a number of false
positive cases can be expected. Antemortem samples, with no

blanks accompany each assay. The therapeutic drug concen-tration range is indicated in each figure. For the barbiturate assay, the
therapeutic range of butalbital is shown, rather than that of secobarbital, as it is one of the most potent barbiturates. For the cocaine
metabolite assay, the therapeutic range of co-caine is shown. The cannabinoid assay standard curve was prepared with
tetrahydrocannabinol (THC), because the car-boxylic acid metabolite (THC-COOH) was not available. The therapeutic range shown in
Figure 6 corresponds to the estimated

Summary

Data menunjukkan penerapan memperluas d.a.u EMIT, tes untuk analisis seluruh hemolysed
darah. Ini dapat dilakukan dengan langsung menganalisis ekstrak methanolic seluruh darah.
Keuntungan dari metode ini meliputi:
1. membentang darah assay untuk lebih toxicologically signifikan spesimen, yaitu seluruh
hemolysed EMIT, yang biasanya tersedia dalam kasus forensik.
2. prosedur ini sangat sederhana dan cepat.
3. kontrol pH sebelum ekstraksi tidak diperlukan.
4. hanya satu tahap yang dihasilkan dengan percepatan protein, karena metanol dengan air.
Dengan demikian, obat-obatan asam, netral dan dasar semua secara bersamaan diekstrak ke
supernantant metanol-plasma.
5. ukuran sampel kecil (1 mL) dapat digunakan untuk analisis duplikat 10 tes terpisah.
6. konsentrasi terdeteksi minimum sesuai dengan konsentrasi rendah atau kantong untuk obatobatan target di setiap tes.
7. metode dapat digunakan untuk semiquantitation obat-obatan di seluruh darah. Satu harus
diingat, bagaimanapun, bahwa teknik immuno-assay dapat bereaksi silang dengan metabolit obat
analog.