Transcript کارگاه آزمایشهای تشخیص مواد مخدر و روان گردان

IN THE NAME OF GOD
‫وزارت بهداشت درمان و آموزش پزشكي‬
‫اداره كل آزمايشگاه مرجع سالمت‬
‫سید علی ناظری‬
‫کارشناس ارشد سم شناس ی‬
‫‪Seyed Ali Nazeri , Toxicology M.S.‬‬
‫‪[email protected]‬‬
‫‪021-81452381‬‬
‫‪021-66752515‬‬
‫‪09121960690‬‬
‫كارگاه‬
‫آزمایشهای تشخیص مواد مخدرو روان گردان‬
‫‪ 31‬اردیبهشت ‪94‬‬
‫دانشگاه علوم پزشکی شهید صدوقی یزد‬
‫عناوین مورد بحث‬
‫‪ ‬رویکرد جدید آزمایشهای تشخیص مواد مخدرو روانگردان‬
‫‪ ‬بررس ي ن انييواه ناونييل ژييای بیو ييو یکی ( ادرار‪،‬عرق‪،‬بييزاق‪ ،‬ييون و ‪ )...‬مييورد اسييبراده بييرای آزمييای مييواد مخييدرو روان‬
‫گردان‬
‫‪ ‬مدت زمان تشخيص مواد مخدرو روان گردان درناونل ادرار‬
‫‪ ‬بررس ی ‪( Cross-reactivity‬واکنشهای مبقاطع ‪ ،‬تدا الت دارویی)‬
‫‪ ‬اصول ناونل گیری و شناسایی انواه مواد مدا لل گردرآزمایشهای تشخیص مصرف مواد مخدرو روان گردان‬
‫‪ ‬روشهای تشخیص مواد مخدرو روان گردان‬
‫‪‬‬
‫‪ -‬روش غربالی‬
‫‪‬‬
‫‪ -‬کروماتو گرافی الیل نازک )‪(TLC‬‬
‫‪‬‬
‫‪TLC Troubleshooting -‬‬
‫ ترسیرنبايج آزمايشهاي تشخيص مواد مخدرو روان گردان ( نبایج مثبت آمربامین و مت آمربامین )‬‫‪‬‬
‫‪ ‬کنترل کیری و اعبباربخش ی روش آزمای‬
RECOMMENDED METHODS FOR THE IDENTIFICATION AND ANALYSIS OF
AMPHETAMINE, METHAMPHETAMINE and ...
United Nations Office on Drugs and Crime ,Vienna
UNITED NATIONS
New York, 2006
Clarck `s Analysis of Drug and Poison
Pharmaceutical Press , Third Edition , Published 2004
RAPID ON-SITE SCREENING OF DRUGS OF ABUSE
A summary of commercially available products and their applications:
guidance for the selection of suitable products
United Nations Office on Drugs and Crime
‫مقدمل‬
‫ها ت س‬
‫هدیدس ته‬
‫هاس یه‬
‫هاد س‪ 23‬الیحه‬
‫هاستن ه مادس ه‬
‫‪ ‬به‬
‫ر کبینستهرتم س هدتدس رهدرسدسردتماهردتنسدس‬
‫قدتمینس صدبس ت عس یریصس صلح سمظا سدسنهایرس‬
‫صدبا ستلنا سن ادس بار سسبهاس هدتدس رهدرس‬
‫ریان ست هدریسصددرسگهدتی سدهد ستد یهادسبهاسس‬
‫ههدتدس رههدرسسدسردتنسگههردتنسسبههاسس مظههدرس‬
‫تن ردت ‪،‬سکنبسدکهار‪،‬ست ددت‪ ،،‬نهررسبهاسرهار‪،‬س‬
‫ت کیدر‪،‬سترذپردتماسکنب‪،‬س یاغلسآ تد‪،‬سس ه ینس‬
‫د‪ ...‬باسدهد سد تر سبهدتی سگذتی اسید ستن ‪.‬‬
‫‪ ‬مظار سبرسد لکردسآ اییاایهایستمتا سدیمهد س‬
‫تینسآ اییها‪،‬سسیا لس‪ :‬آ اییهاایهایسیهبکاس‬
‫بهدتیهه سدتمیههاایهاوسدلههد سپ یهه‪ ،‬سسکیههدر‪،‬س‬
‫آ اییاایهایسمیردیستم ظا ‪،‬سدس‪ ....‬باسدهد س‬
‫تینسد تر راماسگذتی اسید ستن س‪.‬‬
‫دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس‬
‫ردرسدسردتنسگردتن‬
‫‪‬‬
‫‪‬‬
‫دسبورا عال آزمای مواد مخدرو روان گردان براساس بند ‪ 5‬مصوبات جلسل ‪ 126‬سباد‬
‫مب ييارزه ب ييا م ييواد مخ ييدرریاس ييت جاه ييوری ک ييل ب ييل توش ييی ریاس ييت محت ييرم جاه ييوری درت يياری‬
‫‪ 90/2/6‬بييا امءييا وزیييرمحتييرم کشييور و دبیييرکييل محتييرم سييباد مبييارزه بييا مييواد مخييدربييل کلیييل‬
‫دسبگاژهای ذیربط ازجالل وزارت بهداشت ابالغ شده است‪:‬‬
‫بي ييراسي يياس ایي يين دسي ييبورا عال ابالمي ييی ‪ ،‬ان ي ييام آزمي ييای تشي ييخیص مصي ييرف مي ييواد مخي ييدرو‬
‫روانگ ييردان توس ييط آزمایش ييگاژهای مربوط ييل ب ييل ص ييورت ی ييک تکلی يين ييانونی درآم ييده اس يت و‬
‫دسييبگاژهای اجرایييی موظرنييد نسييبت بييل جاييع آوری و ارسييال الصييل آميياری نبييایج آزمایشييها‪،‬‬
‫ژر‪ 3‬ماه یک باربل دبیر انل سباد مبارزه با مواد مخدرریاست جاهوری ا دام ناایند‪.‬‬
‫دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس‬
‫ردرسدسردتنسگردتن‬
‫دراين دسبور ا عال‪:‬‬
‫‪ ‬آزمایشگاه ‪ :‬بل عنوان آزمایشگاه تشخیص مواد مخدرو روانگردان شنا بل می شود‪.‬‬
‫‪ ‬سييو مصييرف مييواد‪ :‬شييامل مصييرف کلیييل مييواد مخييدرو روانگردانييی واژييد بييود کييل در ييانون‬
‫مانوه می باشد‪.‬‬
‫‪ ‬ناونل مناسب برای آزمای ‪ :‬درآزمایشيگاژهای تشيخیص ميواد مخيدرو روانگيردان بيل منظيور‬
‫ازدواج ‪ ،‬اس ييبخدام‪ ،‬پ ش ييل وري و‪ ...‬فق ييط ناون ييل ادرار)‪ )Urine‬واژ ييد ب ييود و ازا ي س ييایر‬
‫ناونل ژای بیو و یک مثل ون‪ ،‬عرق‪ ،‬مو و‪ ...‬ود داری شود‪.‬‬
‫نحوه ان ام آزمایشهای تشخیص مواد مخدرو روانگردان‬
‫مطييابا ایيين دسييبورا عال کييل نجی ييل مصييوبات کایبييل فمييی سييباد مبييارزه بييا مييواد مخييدرو نیي‬
‫مطابا با اسبانداردژای بین املللی می باشد روش صحی برای تشخیص ميواد مخيدرو روان‬
‫گييردان بييل ایيين ترت ييب اسييت کييل اببييدا بييا رعایييت کلیييل نکييات مربييو بييل هیييل ناونيل صييحی‬
‫(جل ييوگیری ازتقلبه ييای رای ييج)‪ ،‬ابب ييدا ای يين آزمایش ييها ب ييا روش ژ ييای غرب ييا گری س ييریع (روش ييهای‬
‫مطلييوو و ابييل بييول ازنظييرکنتييرل کیرييی کييل بييل تاییييد وزارت بهداشييت رسييیده باشييد) ان ييام‬
‫شده سپس‬
‫ً‬
‫نبایج مثبت با روش کروماتو گرافی تایید شوند‪( .‬ترجیحا درژاان آزمایشگاه‪).‬‬
‫گرچل روش مرجع برای تشخیص ميواد مخيدرروش ‪ GC/MS‬ميی باشيد وليی محيدودی های‬
‫ایيين روش اسييبراده ازآن را محييدود مييی کنييد و درزييال زا ييرازروشييهای کروميياتوگرافی‬
‫سياده تير مثيل کروميياتو گرافيی الیيل نيازک )‪ (TLC‬بييرای آزمایشيهای روزانيل اسيبراده مييی‬
‫شود‪( .‬ا ببيل بيا رعایيت کلیيل نکيات فميی مربيو بيل مرازيل ان يام آزميای و نکيات مربيو‬
‫بل ترسیرنبایج)‪.‬‬
‫برخی ازمحدودی های روشهایی نظیر‪ GC/MS,GC‬بل شرح زیراست‪:‬‬
‫‪ ‬این س سباها یلی گران ژسجند ‪.‬‬
‫‪ ‬اسبراده ازمواد مشيبا سيازو ميواد شيیایایی بيا درجيل ليوز بياال ژزینيل آزمایشيها را بياال ميی‬
‫برد‪.‬‬
‫‪ -3 ‬نیييازبييل افييراد مبخصييص داشييبل تييا توانييایی کيياربييا ایيين دسييبگاژها و ترسييیرطیرهييای بدسييت‬
‫آمده را داشبل باشد‪.‬‬
‫‪ ‬نیازمند دمات پس ازفروش وی می باشد‪.‬‬
‫‪ ‬عدم دسترس ی و امکان پ یرنبودن اسبراده آنها درنقا دور دست‪.‬‬
‫نگهداري سوابق و مستندات انجام آزمايشها‬
‫سوابا ان ام آزمایشها بصورت صحی بایگانی و نگل داري شود ‪:‬‬
‫‪ ‬عرف سما اسیاسدسدرردتنه هاوسآ های س هدتدس‬
‫ردرسدسردتنسگردتنسباس د سددسنال‪.‬‬
‫‪ ‬دف رسثب سم ایجس مر سآ های س هدتدس رهدرسدس‬
‫ردتنسگردتنسباس د سناسنال‪.‬‬
‫‪ ‬دف رثب سم ایج سآ اییهاو س اییهدو( ‪،‬رد ها دس‬
‫گرتف ) دتدس ردرسدسردتنسگردتن باس هد سد س‬
‫نال‪.‬‬
‫هایجس مره سدس ثبه س‬
‫هاسم ه‬
‫هدتباستل‪ ،‬ردمی‪،‬ه س(‪،‬لیه‬
‫‪ ‬نه‬
‫آ‬
‫)‬
‫‪‬‬
‫اییهاوس ذ‪،‬درسدسدرصدر ست ‪،‬انستن‪،‬نس عرف سما اسیهاس‬
‫ادت ستلع رماهدتروسیدد‪.‬‬
‫پلی هاو ‪ TLC‬ربدطسباسم یتاسآ اییهاس د سیكس‬
‫نالسباسصدر س مظ سدسبرچنبس د سید سبهاسذ‪،‬هرس‬
Specimen (matrix)
Specimen (matrix)


There are a variety of biological specimens suitable for
drug screening, including urine, sweat and saliva, blood and
hair/nails. The choice of a specimen for analysis depends on :
the purpose of the testing,
and may be affected by :
concerns about sample collection, transport, handling, and
assurance of sample integrity between the collection site and
point of analysis.
Most commonly, focus on the detection of illicit drugs in
urine.
Specimen (matrix)
Urine:
However,urine samples can be relatively easily
substituted, or adulterated.
 Among the most popular manipulations is dilution of
the sample, for example, by excessive drinking or use
of diuretics, or simply by adding water.
 Furthermore, the patterns of drug excretion are
dependent on the pH value of the urine and are thus
influenced by diet. Deliberate changes of the pH of the
urine can be effected by the addition of pH-modifying
agents (e.g.,vinegar,ascorbic acid, lemon juice).
Specimen (matrix)


Similarly, addition of oxidizing (sodium hypochlorite)
and surface-active (e.g.,detergents,soap) agents, certain
medicaments , and even sweeteners (saccharin) or table
salt (sodium chloride), may also lead to false results.
Therefore, to ensure the integrity of the sample it may
be necessary to observe directly the collection of urine,
i.e., urine testing can be invasive of privacy.
Specimen (matrix)


There is no clear relationship between dose and urine
concentration.
If the purpose of drug testing is to relate concentrations
to impairment or other toxicological / pharmacological
responses, blood is generally the specimen of choice, as
blood drug concentrations are most closely related to
concentrations at receptor sites.
Comparison of biological specimens for
drug testing
Table 1:
Comparison of biological specimens for drug testing with on-site screening devices
Note that this comparison does not consider applications of the specimen in other than ON-SITE situations. It does,
therefore, not consider sweat testing in the form of sweat patches, which (because worn for several days) are a cumulative
measure of drug excretion, thus allowing to monitor drug intake for a period of days to weeks
Criteria for
Urine
Comparison
History of use/
Long history of use;
experiences
Uniform testing criteria
with specimens
established
Sweat
Relatively new approach;
Relatively new approach;
performance testing under
performance testing under
development; more R&D
development; more R&D
required
required
Easy;
Sample acquisition (note:
practicalities of sampling
depend on the facilities at
Saliva
Easy
Easy,
but existing collection
(but less practical, for
but sensitivity might
devices may still need
example, at the roasided)
be a problem
improving for practical use in
the sampling sites)
on-site situations
Privacy
Invasive
Target analyte
Metabolite(s)
Analyte concentration
High
Non-invasive
Non-invasive
Parent drug
Parent drug
(and metabolites)
(and metabolites)
Low
Low
Comparison of biological specimens for
drug testing
Table 1:
Comparison of biological specimens for drug testing with on-site screening devices
Note that this comparison does not consider applications of the specimen in other than ON-SITE situations. It does,
therefore, not consider sweat testing in the form of sweat patches, which (because worn for several days) are a cumulative
measure of drug excretion, thus allowing to monitor drug intake for a period of days to weeks
Criteria for
Comparison
Detection time
Urine
Relatively long
Adulteration,
Substitution and
Easily adulterated /
substituted
Contamination
Result
Sweat
Relatively short
Adulteration possible but
difficult; external
contamination possible
Saliva
Relatively short
Adulteration possible, but
difficult
Indicates prior exposure in
“Current-status” / “real
“Current-status” / “real
past few days
time”results
time”results
DRUG DETECTION TIMES IN
URINE
DRUG DETECTION TIMES IN URINE
DRUG DETECTION TIMES IN URINE
Table 2: The following table should be used as a guide only (individual differences in the metabolism and
excretion of drugs and their metabolites may affect specific detection times).
DRUG type *
INFREQUENT
FREQUENT
CHRONIC
USER
USER
USER
Amphetamine
1-3 days
2-6 days
Several weeks
Methamphetamine
1-3 days
2-6 days
Several weeks
Morphine
12-48 hours
2-6 days
Up to several weeks
Codeine
1-3 days
2-5 days
Up to several weeks
Cannabis products
2-5 days
4-14 days
Up to 2-3 months
Cocaine
12-48 hours
1-4 days
Up to several weeks
Methadone
1-4 days
2-10 days
Up to several weeks
Note: Drug detection times actually refer to the urinary metabolites of most of the drugs listed.
Source: 1-Step Detect Associates
Amphetamine
Racemic Desoxynorephedrin:
After a larg dos in urine for several days.
30%
90%
74%
1-4%
dose extracted unchange in urine after 24 h.
dose extracted in urine after 3-4 days.
increased in urinary pH acidic (unchange).
decreased in urinary pH alkalin.
Hypuric acid,Benzoic acid,Hydroxyamphetamin,
Benzoylglocoronid, 4- Hydroxyamphetamine,norephedrine,...
Half life in plasma : 4-8 h.
Metamphetamine
Desoxyephedrine,Crank,Crystal,Ice,Meth,Speed,
Norodine ,‫ شیشه‬.
%70 dose extracted in urine in 24h.
43 % extracted in urine unchange.
15 % 4- Hydroxymetamphetamine .
5 % Amphetamine. (20-30%)
Urinary pH alkalin reduced 2% dose.
Half life in plasma : 9 h
Amph, Meth, MDMA
Morphine
Opium: 10-12 % Morphine
0.5 % Codeine
90 % dose extracted in urine in 24h.
10 % Free.
65-70 % Conjogate ; M3G, M6G
Normorphine,...
increased in urinary pH acidic. (Free)
decreased in urinary pH alkalin.(Conjogate)

Methadone
2-ethylidene 1,5,dimethyl 3,3 diphenylpyrrolin (EDDP)
2- (ethyl-5-dimethyl-3,3 diphenyl -1-pyrrolin (EMDP)
60% after 24h urine
33% unchange , 43% EDDP , EMDP
Half life: 10-25h (15h) , 13-55h (30h)

Tramadol
O-monodesmethyltramadol
90% after 3 days Urine
30% Unchange
Half life : 6 h (6-7)

Buprenorphine
Buprenorphine
Norbuprenorphine
Half life : 1.2 – 7.2 h
N-O-didesmethyltamadol
Cross-reactivity
Cross-reactivity

Drugs and
drug metabolites with significant
structural similarities to the target analyte may crossreact with target
analyte-specific antibodies,
producing false positive results.
 It is important to know that some immunoassays are
class-specific only.
They cannot be used therefore to identify,
specifically, individual drugs within a class.
Examples
are
tests
for
amphetamines,
benzodiazepines, barbiturates, and opiates.
Cross-reactivity
Manufacturers test for cross-reactivity by spiking test
samples and documenting test results. This is the
information that is found in package inserts.
However, the cross-reactivity lists provided by most
manufacturers have been found to be far from
cmplete.
Moreover, since these data are not obtained from
actual ingestion of drug, the reported concentrations
are not necessarily physiologic and may not give
information about possible interference of drug
metabolites.
Cross-reactivity
It is also highly possible that some crossreacting
compounds may not have been tested, and are therefore
not listed.
Lists of compounds, which have been tested and found
not to cross-react, are therefore equally informative
to obtain a more comprehensive picture of possible
interferences.
Cross-reactivity
Aamphetamine-type Stimulants (ATS):

Because of the large number of closely related
substances, including ecstasy-type analogues (such
as MDMA,MDA,MDE,etc.
 Although amphetamine class tests(Amphetamine
and Metamphetamine ) are usually designed to
cross-react with ecstasy and other illicit ATS.
Cross-reactivity

degrees of cross-reactivity may vary considerably
from one substance to another. Because of direct
cross-reactivity of some of their ingredients, or
because their main urinary metabolites are the target
drugs tested.
 For amphetamine tests, examples include certain
nasal decongestants and anorectics
e.g.,ephedrine,phenylpropanolamine,
phentermine),and the anti-parkinsonian drug
selegiline, which is metabolized to amphetamine.
 Benzphetamin...
Cross-reactivity










Amphetamin Test:
Methylenedioxyamphetamine (MDMA)
Phentermine
Benzphetamin
Ephedrine
Pseudoephedrine
Selegiline
Deprenyl
Methamphetamine
L-Methamphethamine (Vick’s inhaler)

Phenylpropanolamine
 Phenylephrine
 Ranitidine
 Tyramine , ….
 Metamphetamine Test:
 Methylenedioxymethamphetamine (MDMA)
 Amphetamine
 Ephedrine
 Phenyletylamine
 Chloroquine
 …
Cross-reactivity
Opiate-type tests:
Most opiate assays are designed to detect morphine.
Heroin use causes a positive opiate test result
because its predominant urinary metabolite is
morphine.

A number of cough suppressants, such as codeine,
some analgesics, and morphine agonists and
antagonists cross-react to a high extent with opiatetype tests.
Cross-reactivity
False positive results may also be produced from the
ingestion of food containing poppy seeds.
In contrast, the ability to detect the use of synthetic
opioids, such as hydromorphone, hydrocodone,
oxycodone and oxymorphone varies among
immunoassays from different manufacturers.
Cross-reactivity


Opiate,Opioids tests:
Morphine
 Codeine
 Poppy seed
 Dextromethorphn
 Diphenhidramine
 Hydrocodone
 Hydromorphon
 Nalorphine
 Procaine
 Tebaine
 Naloxane
Regulatory Process
Evolution of Workplace testing
Mandatory Guideline For Drug Testing:
Health and Human Services (HHS)
Which became known as the:
National Institute For Drug Abuse (NIDA)
Today sa The:
Substance Abuse and Mental Health Services
Administration (SAMHSA)
and
United Nation on Drug and Crim (UNODC)
1994 Cannabioids, 1999 Morphine
Cut-off value / Concentration

The cut-off value of an assay is the specific
concentration of a drug , or drug metabolite, in the
sample that is chosen as a limit to distinguish a
presumptive positive from a negative test result.
 Samples with concentrations at or above the cut-off
level are considered presumptive positive and
results below are considered negative.
 In this connection, it should be remembered that a
clear correlation between the cut-off concentration
and the level of impairment has not been
established for any of the sample specimens.
Cut-off value / concentration
Moreover, any attempt to correlate test results with
impairment has to carefully consider these
pharmacokinetic / metabolic aspects for individual
drugs and how they are reflected in concentration
profiles in different sample specimens.
Usually established based on epidemiological
information, i.e., they reflect, to a certain extent, the
prevalence of, and the importance attached to the
abuse of certain drugs or drug classes in different
countries.
Workplace drug screening cut-off in urine
(ng/ml)
Table 3:
Workplace drug screening cut-offs in urine (ng/m(
Drug type
Cut-off in urine (ng/mL)
Amphetamines
500
Metamphetamine ***
500
Opiate
300
Methadone or metabolites
300
Cocaine metabolites
300
Cannabis metabolites
50
Methods
Immunochromatography
Chromatography
‫مرور روشهاي متداول آزمايشگاهي تشخيص مواد مخدر و روان گردان‬
‫‪Drug Screening Tests - A‬‬
‫•‬
‫ایمونوکروماتوگرافی )‪(Immonochromatography‬‬
‫•‬
‫ایمونواسي )‪(Immonoassay‬‬
‫•‬
‫ایمونوفلورسانت )‪(Immonofloresent‬‬
‫•‬
‫رادیوایمونواسي)‪(RIA‬‬
‫•‬
‫االیزا )‪(EIA‬‬
‫•‬
‫‪EMIT‬‬
‫•‬
‫روشهاي شیمیایي ‪ :‬ماركي – فرود ‪…,‬‬
Principles of on-site immunoassay devices



Direct competition technology****
Displacement technology
Trapping technology
Principles of on-site immunoassay devices
Immunochromatographic methods
Most of these tests are based on competitive technology. In
these assays, drug (or drug metabolite) that may be present in
the test sample competes with a drug conjugate for antibody
binding sites.
Purpose and intended use of on-site drug
screening






Currently, on-site testing for drugs of abuse is being carried
out, to varying extents, in the following applications:
• Workplace surveillance programmes (including preemployment, random /periodic, “reasonable suspicion”, postaccident, and return-to-duty/follow-up testing), including
testing of military and personnel in other safety-sensitive jobs.
• Roadside drug testing programmes,
• Crime investigations and legal proceedings,
• Drug treatment and rehabilitation programmes,
• Parole/probation programmes (including so-called “drug
courts”),
• Hospital emergency.
Analytical performance characteristics of onsite screening devices
The performance of on-site screening devices is usually
assessed in terms of sensitivity, which is the
percentage of true positive, and specificity or
percentage of true negative results.
These analytical measures therefore indicate the ability
of the on-site device to identify, at a given cut-off
concentration, those samples that truly contain the
target analyte (sensitivity) or are truly drug-free
(specificity )
Analytical performance characteristics of onsite screening devices
In analytical performance studies, sensitivity and
specificity are expressed as percentage of results
confirmed by another method such as gas
chromatography-mass spectrometry (GC/MS).
The specimens identified as positive by the on-site
screening device but later confirmed as negative are
called false positive.
And specimens identified as negative by the device but
confirmed positive are called false negative.
Analytical performance characteristics of
on-site screening devices
The overall performance of a test is often expressed by
the term efficiency (sometimes called accuracy),
which is defined as the percentage of all true (correct)
results, whether positive or negative (equation 3).
High efficiency is desired when both false positives and
false negatives can have equally serious consequences
for the tested individual.
Analytical performance characteristics of onsite screening devices
Sensitivity = TP (TP + FN )x 100
Specificity = TN (TN + FP)x 100
Efficiency = (TP + TN) N x 100
The following analytical criteria are
recommended for a good screening test.
Sensitivity ≥ 90%
 Specificity ≥ 90%
 Efficiency ≥ 95%

Other considerations






Temperature / Humidity (Stability)
Shelf life (length of storage)
Range of drugs /classes
Time requirements to perform a test
Documentation / storage of results
Costs & Time
Validation protocol
Sensitivity ≥ 90%
Specificity ≥ 90%
Efficiency ≥ 95%
Accuracy (Control-Comparison method- Recovery- St.add.)
Precision
Intera assay )‫( دردنسنمت س‬
Inter assay ) ‫)بردنسنمت‬
Cross - reactivity
Cut - off
Stability
(Accel, Real, in use)

Precision
Precision is a measure of the reproducibility of the whole
analytical method (including sampling, sample preparation
and analysis) under normal operating circumstances.
Precision is determined by using the method to assay a sample
for a sufficient number of times to obtain statistically valid
results .The precision is then expressed as the relative standard
deviation.
%RSD = std dev x 100% / mean

Accuracy
Accuracy is a measure of the closeness of test results obtained
by a method to the true value.
Re-Co-Comp...
(Chromatography Methods) Confirmatoryt Tests -B
، ‫ تفكيك مرفين و كدئين – حساسيت كم‬:
Paper Chromatography
•
‫زمان طوالني‬
‫تفكيك مناسبتر‬
: Thin layer Chromatography (TLC)
‫ زمان كوتاهتر‬، ‫ حساسيت بيشتر‬، ‫ كدئين‬،‫مرفين‬
•
‫•‬
‫‪Chromatography (GC) ,GC/MS‬‬
‫‪ : Gas‬تفكيك بسيار خوب‬
‫مرفين وكدئين ‪ ،‬و ساير آناليتها ‪،‬حساسيت بسيار باال‪ ،‬گران قيمت ‪ ،‬نيازمند تخصص ويژه است‪.‬‬
‫• ‪MS/MS, LC/MS, HPLC‬‬
Chromatography Methods
:‫رتحلسدستت تء‬
)Exteraction(‫س اد س دردسآمالی سس‬،‫تن ررت‬
)Mobile Phas) ‫فا س حرکس‬
(Stationary Phase) ‫فا سثاب سس‬
(Detecore) ‫ظایرسکممد‬




Liquid liquid extraction (LLE)
Solid phase Extraction (SPE)
Introduction




Thin layer chromatography (TLC) has become one of
the most commonly used techniques for the separation
and identification of illicitly manufactured drugs .
TLC is a widely-used chromatography technique used
to separate chemical compounds.
It is a technique used to separate and identify individual
components in a mixture.
It can be used to determine the pigments a plant
contains, to detect drugs , pesticides or insecticides in
food , in forensics to analyze or to identify compounds
present in a given substance. ....
Introduction



TLC is rapid, sensitive (sub-milligram quantities of analyte
required), enormously flexible in both the stationary and the
mobile phase, and thus amenable to a wide variety of
substances, in base and salt form, ranging from the most polar
to the most non-polar materials.
It is also amenable to a variety of visualization techniques.
TLC is one of the easiest of the many chromatographic
techniques, and it is inexpensive.
TLC Theory
It involves :
 A stationary phase consisting of a thin layer of adsorbent
material, usually silicagel, alumina, cellulose immobilised
onto a flat, inert carrier sheet.
 A mobile Phase (solvent )(eluent) travels up the matrix by
capillarity, moving the components of the samples at various
rates because of their different degrees of interaction with the
matrix (stationary phase) and solubility in the developing
solvent.
TLC Theory

Non-polar solvents will force non-polar compounds to the top
of the plate, because the compounds dissolve well and do not
interact with the polar stationary phase.
Thin Layer Chromatography
Technique (Operation involved)

Choice of adsorbent (stationary phase,plate)
 Preparation of plate
 Preparation & application of sample (spotting)
 Choice of solvent (mobile phase)
 Development of chromatogram
 Drying of chromatogram
 Location of spot (visualization)
 Quantitative estimation.
Choice of adsorbent


Two general properties decides the selection are
Particle size and homogeniscity.
Factor affecting selection:
There should not be any reaction with substance to be
separated. It should be insoluble with mobile phase and
solvent used for elution. It should not catalyses or
decompose off substance. It should be colour less.
Classification of adsorbents used
Classification according to bonding strength:
 Weak adsorbent; eg. Sucrose, starch, talc ,cellulose.
Intermediate adsorbent eg. Silica gel, calcium
carbonate, calcium phosphate, magnesia
 Strong adsorbent: alumina ,charcoals
Stationary Phase (adsorbents)
 A special finely, ground matrix (silicagel,alumina,or
similar material) is coated on a glass plate,a metal or a
plastic film as a thin layer (~0.25 mm).

In addition a binder like gypsum is mixed into the
stationary phase to make it stick better to the slide.
 In many cases, a fluorescent powder is mixed into the
stationary phase to simplify the visualization later on (e.g.
bright green when you expose it to 254 nm UV light).
Sample Application (spotting)

Dissolve solid sample in MeOH. Use TLC
capillary to transfer and spot dissolved
sample.
Plate preparation (spotting)



The plate is dried and activated by heating in an oven for
thirty minutes at 80-110 C. (90)(120)
Spotting must be done carefully, without damaging the
plate’s surface.
The starting point of the run, i.e., the “spotting line, ”
should be 1-2 cm from the bottom of the plate. The
solvent level has to be below the starting line of the TLC,
otherwise the spots will dissolve away.
Plate preparation (spotting)


The spacing between applications of sample (spotting
points) should be at least 1 cm, and spots should not be
placed closer than 1.5 cm to the side edge of the plate.
To avoid diffuse spots during development, the size of the
sample spot should be as small as possible ( 2 mm).
Spotting and developing



Remove plate from the development tank as soon as the
solvent reaches the development line marked beforehand; until
~1 cm from the top. otherwise, diffuse spots will occur.
Do not allow the solvent to run over the edge of the plate.
Next, let the solvent evaporate completely .
Choice of solvent


Selection of M.P. depends on:
nature of substance to be separated ,Viscosity & Polarity of
S.P. The solvent used may be a single or double phase
system.
N-hexane, cyclohexane, carbon tetra chloride, benzene,
toluene, trichloro ethylene, chloroform, diethyl ether, ethyl
acetate, n-butanol, acetone, ethanol, methanol and water.
Choice of solvent
Solvent systems (mobile phases)

System A:
Methanol 100 : Concentrated ammonia 1.5
 System B:
Ethyl acetate 85: Methanol 10 : Concentrated ammonia 5

System C:
Cyclohexane 75: Toluene 15: Diethylamine 10
Visualization/detection

Plates must be dried prior to visualization: The solvent can be
allowed to evaporate at room temperature, or removed with a
hot air blower.
 If hot air is used,care must be taken because of the volatility of
the ATS free bases.
 It is important for proper colour development that all traces of
ammonia be removed from the plate.
Visualization of TLC Results
There are various techniques to visualize the compounds.
Iodine , Sulforic ,…., RF, UV, long wavelength (background
green, spots dark), short wavelength (plate dark, compounds
glow)
Be sure not to use the UV lamp outside of the cabinet and
wear the safety glasses at all times while viewing.
Using Silica plates impregnated with methanolic
KOH (0.1 mol/l).
 Detection time ( 1-1.5 h)
 Dark
 Temperature
 Moisture
 Experienced & Expert person

Interpretation
After visualization, mark spots (e.g., by pencil), and calculate
retardation factor (Rf) values:
Rf =
Migration distance: from origin to centre of analyte zone (spot)/
Development distance: from origin to solvent front
It is very common to express retention factors as Rf x 100,
Thin Layer Chromatography (TLC)
Thin Layer Chromatography (TLC)
Troubleshooting TLC

About the first time you run a TLC, and see spots
everywhere and blurred , streaked spots?
Run the TLC again after diluting your sample.
Or, your sample might just contain many components,
creating many spots which run together and appear as a
streak.
Troubleshooting TLC

The sample runs as a downward crescent.
The adsorbent was disturbed during the spotting,
causing the crescent shape.
Troubleshooting TLC

The plate solvent front runs crookedly.
Either the adsorbent has flaked off the sides of the plate
Or ; the sides of the plate are touching the sides of the container
as the plate develops.
(Or ; the paper used to saturate the container 30-45 min)
Crookedly run plates make it harder
to measure Rf values accurately.
Interpretation of Results
Interpretation of Opiate Positive Results
Confirmation of Codein and Morphine by GC/MS.
Use of 300ng/ml criterion resulted in a large number of
positive results that drived from ether poppy- seed
ingestion or therapeutic doses for codeine.
Where there was a prescription for codeine or
morphine….
However,in the absence of a prescription for codeine or
morphine , “ clinical signs of opiate use “ befor reporting
verified opiatepositive result back to the employer.
Interpretation of Amphetamine &
Metamphetamine Positive Results

A laboratory report Metamphetamine as positive only if :
its concentration is 500 ng\ml or greater, and if that of
amphetamine is 200 ng\ml or greater by GC\MS.
This reporting criterion was introduced hn 1991 after
several specimens were reported as positive for
methamphetamine when the specimen contained larg amounts
of Pseudoephedrine or Ephedrine .
It was discovered (Hornbeck et al. 1993) that
hydroxylated sympathomimetics (Pseudoephedrine or
Ephedrine) could convert to metamphetamine in either the
extraction or chromatographic stage of analysis. None of
these specimens contained amphetamine when tested. and
therfor; false–positive.
It was discovered (Hornbeck et al. 1993) that hydroxylated
sympathomimetics (Pseudoephedrine or Ephedrine) could
convert to metamphetamine in either the extraction or
chromatographic stage of analysis. None of these specimens
contained amphetamine when tested. and therfor; false–
positive.
Pre – oxidation step with preiodate to prevent the
possibility of this happening.
Interpretation of Amphetamine Positive
Results

If both amines are present in concentration greater than
500 ng\ml by GC\MS, both are reported as positive and
threre is no confusion.
Dificalty araises when the amphetamine between 200
and 500ng\ml , (Less than the amphethamine
confirmation cut-off).
Interpretation of Amphetamine Positive
Results

Some drugs can metabolise to metamphetamine and
amphetamine ( benzphetamine and selegeline).
Interpretation of Amphetamine Positive
Results

The Confusion in differentiation of ,
D - and L-Metamphetamine:
Vicks Inhaler (decongestant product); It’s use can result
(false positive) in the detection of amphetamine and
metamphetamine in urine. (low concentration and crossreactivity to the L- isomer of
metamphetamine and
amphetamine; a unlikely positive (False Positive) , following
normal use.
In such case can request a GC/MS separation and
isomers. These are usualy analysis and reported as X% disomer and %Y L-isomer. Where the L-Isomer is greater than
80% , the result as negative.
L-methamphetamine isn’t really anything like the
D-methamphetamine isomer that is found in street
drugs. D-methamphetamine is psychoactive,
L-methamphetamine isn’t very
while
psychoactive at all
Thin Layer Chromatography
Thin Layer Chromatography
Thin Layer
Layer Chromatography
Chromatography
Thin
Thin Layer Chromatography
Thin Layer
Layer Chromatography
Chromatography
Thin
Thin Layer
Layer Chromatography
Chromatography
Thin
TLC, Multi Drug

Thin Layer Chromatography
Thin Layer
Layer Chromatography
Chromatography
Thin
TLC, Amphetamine
TLC, Amphetamine
Thin Layer
Layer Chromatography
Chromatography
Thin
Thin Layer Chromatography
Thin Layer Chromatography
Thin Layer Chromatography
Paper chromatography
Paper chromatography
HPTLC analysis
HPTLC analysis is particularly appreciated in the
following fields :
- chemical / medical: detection of illicit substances,
quality controls and pharmaceutical and cosmetic
purity
- Human and animal nutrition: additives; monitoring
composition and stability; quality control
- Environment: pesticides, water and soil pollution,
plant extracts, etc
HPTLC analysis
Thanks for your attention
‫پ یرش و ناونل گیری‬
‫نکاتی درمورد پ یرش و ناونل گیری‬
‫•‬
‫ناونل گیری بایستی زءوری و یا با اسبراده از دور بین مدار بسبل ان ام‬
‫شود‪.‬‬
‫•‬
‫مکان ناونل گیری بایستی از هویل و نور کافی بر وردار باشد‪.‬‬
‫•‬
‫در محل ناونل گیری صابون و مواد سرید کننده ابل دسترس ی برای‬
‫مراجعل کنندگان موجود نباشد‪.‬‬
‫•‬
‫ارائل كارت شناسايي يا معرفي نامل عكس دار‬
‫ناونل با ناونل فرد ديگر‪.‬‬
‫جلوگیري از جايگزيمن‬
‫•‬
‫موارد ا افي (كت‪ ،‬كين و ‪ ) ....‬ارج از محل ناونل گیري تحويل گرفبل‬
‫شود‪ .‬زتن بازرس ن بدني در صورت زوم‪.‬‬
‫مکا‬
‫سدر دردسپذیر‬
‫دسم دماسگیری‬
‫•‬
‫شسييتن دسي ها بييل ازناونييل گیييري ‪ :‬زيييرنييا ن ‪ ،‬چسييب ز ييم ویييا آ ييوده كييردن دسيت بييل‬
‫موادي كل موجب ای اد ا بالل درآزماي مي شوند‪.‬‬
‫•‬
‫دماي ناونيل تيا ‪ 4‬د يقيل بعيد از ناونيل گیيري انيدازه گیيري شيود‪.‬‬
‫‪ :‬موثرترين راژها براي تشخيص ر يا سازي‬
‫•‬
‫ناونل گیر بايد فردي مطائن ‪ ،‬ژوشيارو آگاه باشد‪.‬‬
‫(‪)33-37 C‬‬
‫بعدست سم دماسگیروس‬
‫‪.1‬‬
‫دماي ناونل تا ‪ 4‬د يقل بعد ازناونل گیري ‪ 33-37 C‬است‪.‬‬
‫‪.2‬‬
‫رنگ ‪ ،‬شرافيت ‪ ، PH،‬وزن مخصوز ناونل بررس ن شود‪ .‬اگر ناونل مشكوك بل‬
‫تقلب است دوباره ناونل گیري ان ام مي شود‪.‬‬
‫‪‬‬
‫‪‬‬
‫‪‬‬
‫‪.3‬‬
‫كراتي تین كاتراز ‪20mg/dl‬‬
‫مشكوك‬
‫وزن مخصوز بايد ‪ 1.002-1.04‬باشد پايین تر ابل بول ن ست‪.‬‬
‫‪ PH‬ناونل ‪ 4.5-8.5‬ابل بول است‪.‬‬
‫ناونل مهر و موم شده (بل روش مناسب نگهداري شود و دسترس ن بل ناونل تا زمان‬
‫آزماي براي افراد مبرر ل امكان پ يرنباشد)‪.‬‬
‫روشهاي مدا لل (تقلب ) درناونل گیري‬
‫تعويض ناونل )‪(Substituation‬‬
‫•‬
‫جاب ايي ناونل با ناونل ديگرويا اسبراده ازموادي كل شبيل ادرارباشند مثل چاي‪،‬آو س ب و‪...‬‬
‫• ر يا ناودن ناونل )‪(Diluation‬‬
‫• ‪ -‬ر يا سازي توسط آو سبب مي شود كل غلظت دارو بل زدي كاژ يابد كل ابل‬
‫تشخيص نباشد‪.‬‬
‫• ‪ -‬نوشيدن آو و مايعات ديگرسبب مي شود كل غلظت دارو كاترازغلظت زد‬
‫مرزي )‪ (Cut Off‬برسد‪.‬‬
‫• افزاي سرعت دفع ‪ :‬سركل‪ ،‬ويبامین ‪ ،C‬آو ياو‪ ،‬داروژاي ديورتيك‬
‫•‬
‫تزريا آو و مايعا ت ديگربل مثانل (عرونت و آس ب بافتن)‬
‫• اسبراده ازادرار يوفيلی ه كل براي اين منظور بل صورت ت ارتي سا بل م شود‪.‬‬
‫•افزودن مواد ارجي )‪(Adulteration‬‬
‫آزمايشگاه ودريافت‬
‫بطور كلي افزودن مواد ارجي بل ناونل با ژدف بل اشجباه اندا تن‬
‫پاسخ منري كاذو ِ‪ Adulteration‬گربل م شود‪.‬اين مواد بطور كلي دو گروه ژسجند ‪:‬‬
‫• گروه اول مواد انگي ژسجند كل بل رازتن دردسترس مي باشند‪.‬‬
‫ صابون‪ ،‬پاك كننده ژاي انگي )‪(Detergent‬‬‫ سريد كننده ژا )‪(Bleaching agent‬‬‫ جوژر ياو‬‫ كلريد سديم‬‫ سركل ويبامین ‪ C‬و‪...‬‬‫• گروه دوم موادي ژسجند كل بل رازتن دردسترس نبوده و اسبراده ازآنها نيازبل اطالعات‬
‫وتخصص دارد‪ .‬تعدادي ازاين مواد بل شرح زيرژسجند‪:‬‬
‫دتدیسکاسبطدرس ع دلسدرسدن رسسمین مد‬
‫‪ - ‬كلر(‪)Chlorine‬‬
‫‪ - ‬آو اكسیژنل‬
‫‪ - ‬ناكها مثل‪ :‬سديم برومايد ‪ ،‬سد يم نيتريت و ‪...‬‬
‫‪ - ‬نيتريت )‪ (Nitrite‬مثل ‪Whizziez , Klear‬‬
‫‪ - ‬كرومات و پیريدينيوم كرومات )‪(Chromat , Pyridinium chromat‬‬
‫‪ - ‬پراكسيد و پراكسيداز )‪(Peroxid , Peroxidas‬‬
‫مثل موادي با نامهاي ت ارتي ‪Clean choce‬‬
‫‪ - ‬گلوتاريك آ دژيد )‪(Glutaric aldehyde‬‬
‫‪ - ‬مثل موادي با نامهاي ت ارتي ‪Instant clean‬‬
‫‪ - ‬مصرف موادي تحت عنوان ‪ Golden seal,Guick caps ,Test clean‬و‪...‬‬
‫• سل مورد ا یرتحت عنوان كلي ‪ Urine Lock‬نی نا ميده مي شود‪.‬‬
‫• ‪ : Mixed reagent‬ماكن است تركيب چند مورد ازمواد فوق با ژم توسط فرد مورد‬
‫آزماي مورد اسبراده رارگیرد كل دراين زا ت بل آنها ‪ Lock Lab‬گربل مي شود ‪.‬‬
‫‪Bleaching agent , Detergent‬‬
‫** تاثیراين مواد روي بعض ن مواد مورد آزماي ب شترو درمواردي كاتراست ژاچنین تاثیراين‬
‫مواد درروشهاي مخبلن باژم فرق ميكند‪.‬‬
‫** وشبخبانل افزودن اين مواد درمرزلل ناونل گیري صورت مي گیرد كل با د ت دران ام‬
‫ناونل گیري زءوري مي توان ازاين دسبل تقلبها جلوگیري كرد‪.‬‬
‫مکان سم عال موادی کل برای تقلب اسبراده می شود‬
‫‪ ‬پرستکنیدسدسپرستکنیدت‬
‫ بعمدتنستکنیدتم سد لس سکممدس‪.‬‬‫دت‪،‬م سآم سبادوسدسآم سژن‬
‫رریبسد لسآم ی سدتر اللسدرسس‬
‫‪ ‬کرد ا سدسپیریدسمید سکرد ا‬
‫‪ -‬قابلس قایناسباس دردسباال‬
‫‪ ‬گلد اریکسآلدسیید ) ‪)c-o-c‬‬
‫‪-‬‬
‫ر لسکردنسد لسآم ی سسبرصدصسرد سس‪Emit‬‬
‫‪ ‬نریدسکممد سیاس‬
‫ت سیكسن سباسدتردسبامدس سیدمدس( ینل) دتر اللسدرس‬
‫دت‪،‬م سآم سبادوسدسآم سژن‬
‫‪‬‬
‫‪-‬‬
‫تنیدسدسقلیا‬
‫دتدیس ثلستنیدسیاسدسقلیایاسبهاس یییهرس‪ pH‬باده س‬
‫یییرسنار انسپرد مینسدتر اللسدرسبامهدسیهدنسآم ه س‬
‫بادیسدسآم سژنس سیدمدسدردیهایستی دمدتن سرتس حه س‬
‫اثیرسقرترس یدیمد‪.‬‬
Thanks for your attention