IN THE NAME OF GOD وزارت بهداشت درمان و آموزش پزشكي اداره كل آزمايشگاه مرجع سالمت سید علی ناظری کارشناس ارشد سم شناس.
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Slide 1
IN THE NAME OF GOD
Slide 2
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
Slide 3
كارگاه
آزمایشهای تشخیص مواد مخدر و روان گردان
31اردیبهشت 94
دانشگاه علوم پزشکی شهید صدوقی یزد
Slide 4
عناوین مورد بحث
رویکرد جدید آزمایشهای تشخیص مواد مخدر و روانگردان
بررس ي انواع نمونه های بیولوژیکی ( ادرار،عرق،بززاق ،وزون و )...مزورد اسزاداده بزرای آزمزایا مزواد مخزدرو روان
گردان
مدت زمان تشخيص مواد مخدرو روان گردان درنمونه ادرار
بررس ی ( Cross-reactivityواکنشهای ماقاطع ،تداوالت دارویی)
اصول نمونه گیری و شناسایی انواع مواد مداوله گر در آزمایشهای تشخیص مصرف مواد مخدر و روان گردان
روشهای تشخیص مواد مخدر و روان گردان
-روش غربالی
-کروماتو گرافی الیه نازک )(TLC
TLC Troubleshooting -
تدسیر ناايج آزمايشهاي تشخيص مواد مخدر و روان گردان ( ناایج مثبت آمداامین و مت آمداامین )
کنترل کیدی و اعابار بخش ی روش آزمایا
Slide 5
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
Slide 6
مقدمه
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.
Slide 7
دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس
ردرسدسردتنسگردتن
دسززاوراللمل آزمززایا مززواد مخززدر و روان گززردان بززر اسززاس بنززد 5مصززوبات جلسززه 126
سززااد مبززارزه بززا مززواد مخززدر ریاسززت جمهززوری کززه بززه توشززی ریاسززت محتززرم جمهززوری در
تززاری 90/2/6بززا امءززا وزیززر محتززرم کشززور و دبیززر کززل محتززرم سززااد مبززارزه بززا مززواد مخززدر
به کلیه دساگاههای ذیربط ازجمله وزارت بهداشت ابالغ شده است:
ب ززر اس ززاس ای ززن دس ززاوراللمل ابالش ززی ،انف ززام آزم ززایا تش ززخیص مص ززرف م ززواد مخ ززدر و
روانگردان توسزط آزمایشزگاههای مربوطزه بزه صزورت یز تکلیزق نزانونی در آمزده اسزت و
دس ز ززاگاههای اجرای ز ززی موظدن ز ززد نس ز ززبت ب ز ززه جم ز ززع آوری و ارس ز ززال والص ز ززه آم ز ززاری نا ز ززایج
آزمایشززها ،هززر 3مززاه یز بززار بززه دبیززر وانززه سززااد مبززارزه بززا مززواد مخززدر ریاسززت جمهززوری
اندام نمایند.
Slide 8
دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس
ردرسدسردتنسگردتن
در اين دساور اللمل:
آزمایشگاه :به عنوان آزمایشگاه تشخیص مواد مخدر و روانگردان شناواه می شود.
س ززو مص ززرف م ززواد :ش ززامل مص ززرف کلی ززه م ززواد مخ ززدر و روانگردان ززی وواه ززد ب ززود ک ززه در
نانون ممنوع می باشد.
نمون ززه مناس ززا ب ززرای آزم ززایا :درآزمایشز ززگاههای تش ززخیص م ززواد مخ ززدر و روانگ ززردان بز ززه
منظ ززور ازدواا ،اس ززاخدام ،پوش ززه وري و ...فق ززط نمون ززه ادرار ) )Urineوواه ززد ب ززود و از
اوذ سایر نمونه های بیولوژی مثل وون ،عرق ،مو و ...وود داری شود.
Slide 9
نحوه انفام آزمایشهای تشخیص مواد مخدر و روانگردان
مطززابا ایززن دسززاوراللمل کززه نجیفززه مصززوبات کمیاززه فمززی سززااد مبززارزه بززا مززواد مخززدر و
نیز مطابا با اساانداردهای بین املللزی مزی باشزد روش حزبی بزرای تشزخیص مزواد مخزدر
و روان گززردان بززه ایززن ترت ززا اسززت کززه اباززدا بززا رعایززت کلیززه نکززات مربززو بززه هیزه نمونززه
حززبی
(جلززوگیری از تقلبهززای رایززج) ،اباززدا ایززن آزمایشززها بززا روش هززای غربززالگری س زریع
(روشززهای مطلززوق و نابززل نبززول از نظززر کنتززرل کیدززی کززه بززه تاییززد وزارت بهداشززت رسززیده
باشززد) انفززام شززده سز
درهمان آزمایشگاه).
ناززایج مثبززت بززا روش کرومززاتو گرافززی تاییززد شززوند( .ترجیحززا
Slide 10
گرچز ز ززه روش مرجز ز ززع بز ز ززرای تشز ز ززخیص مز ز ززواد مخز ز ززدر روش GC/MSمز ز ززی باشز ز ززد ولز ز ززی
محز ز ززدودی های ایز ز ززن روش اسز ز ززاداده از آن را محز ز ززدود مز ز ززی کنز ز ززد و در ز ز ززال ا ز ز ززر از
روشز ززهای کرومز ززاتوگرافی سز ززاده تز ززر مثز ززل کرومز ززاتو گرافز ززی الیز ززه نز ززازک ) (TLCبز ززرای
آزمایشززهای روزانززه اسززاداده م ززی شززود( .الباززه ب ززا رعایززت کلیززه نک ززات فمززی مرب زو ب ززه
مرا ل انفام آزمایا و نکات مربو به تدسیر ناایج).
Slide 11
برخی از محدودی های روشهایی نظیر GC/MS,GCبه شرح زیر است:
این س سامها ویلی گران هسجند .
اساداده از مواد مشاا ساز و مزواد شزیمیایی بزا درجزه ولزو بزاال هزینزه آزمایشزها را بزاال
می برد.
-3 نیاز به افراد ماخصص داشاه تا توانایی کار با این دساگاهها و تدسیر طیدهای بدست
آمده را داشاه باشد.
نیازمند ودمات پ ازفروش نوی می باشد.
عدم دسترس ی و امکان پذیر نبودن اساداده آنها در نقا دور دست.
Slide 12
نگهداري سوابق و مستندات انجام آزمايشها
سوابا انفام آزمایشها بصورت حبی بایگانی و نگه 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
Slide 13
Specimen (matrix)
Slide 14
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.
Slide 15
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).
Slide 16
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.
Slide 17
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.
Slide 18
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
Slide 19
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
Slide 20
DRUG DETECTION TIMES IN
URINE
Slide 21
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
Slide 22
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.
Slide 23
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
Slide 24
Amph, Meth, MDMA
Slide 25
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)
Slide 26
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)
Slide 27
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
Slide 28
Cross-reactivity
Slide 29
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.
Slide 30
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.
Slide 31
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.
Slide 32
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.
Slide 33
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...
Slide 34
Cross-reactivity
Amphetamin Test:
Methylenedioxyamphetamine (MDMA)
Phentermine
Benzphetamin
Ephedrine
Pseudoephedrine
Selegiline
Deprenyl
Methamphetamine
L-Methamphethamine (Vick’s inhaler)
Slide 35
Phenylpropanolamine
Phenylephrine
Ranitidine
Tyramine , ….
Metamphetamine Test:
Methylenedioxymethamphetamine (MDMA)
Amphetamine
Ephedrine
Phenyletylamine
Chloroquine
…
Slide 36
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.
Slide 37
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.
Slide 38
Cross-reactivity
Opiate,Opioids tests:
Morphine
Codeine
Poppy seed
Dextromethorphn
Diphenhidramine
Hydrocodone
Hydromorphon
Nalorphine
Procaine
Tebaine
Naloxane
Slide 39
Regulatory Process
Slide 40
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
Slide 41
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.
Slide 42
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.
Slide 43
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
Slide 44
Methods
Immunochromatography
Chromatography
Slide 45
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 …,
Slide 46
Principles of on-site immunoassay devices
Direct competition technology****
Displacement technology
Trapping technology
Slide 47
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.
Slide 48
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.
Slide 49
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 )
Slide 50
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.
Slide 51
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.
Slide 52
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%
Slide 53
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
Slide 54
Validation protocol
Sensitivity ≥ 90%
Specificity ≥ 90%
Efficiency ≥ 95%
Accuracy (Control-Comparison method- Recovery- St.add.)
Precision
Intera assay ) ( درون سنجی
Inter assay ))برون سنجی
Cross - reactivity
Cut - off
Stability
(Accel, Real, in use)
Slide 55
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
Slide 56
Accuracy
Accuracy is a measure of the closeness of test results obtained
by a method to the true value.
Re-Co-Comp...
Slide 57
(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
•
Slide 58
•
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
Slide 59
Chromatography Methods
:رتحلسدستت تء
)Exteraction(س اد س دردسآمالی سس،تن ررت
)Mobile Phas) فا س حرکس
(Stationary Phase) فا سثاب سس
(Detecore) ظایرسکممد
Slide 60
Liquid liquid extraction (LLE)
Slide 61
Solid phase Extraction (SPE)
Slide 62
Slide 63
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. ....
Slide 64
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.
Slide 65
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.
Slide 66
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.
Slide 67
Thin Layer Chromatography
Slide 68
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.
Slide 69
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.
Slide 70
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
Slide 71
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).
Slide 72
Sample Application (spotting)
Dissolve solid sample in MeOH. Use TLC
capillary to transfer and spot dissolved
sample.
Slide 73
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.
Slide 74
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).
Slide 75
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 .
Slide 76
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.
Slide 77
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
Slide 78
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.
Slide 79
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.
Slide 80
Using Silica plates impregnated with methanolic
KOH (0.1 mol/l).
Detection time ( 1-1.5 h)
Dark
Temperature
Moisture
Experienced & Expert person
Slide 81
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,
Slide 82
Thin Layer Chromatography (TLC)
Slide 83
Thin Layer Chromatography (TLC)
Slide 84
Slide 85
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.
Slide 86
Troubleshooting TLC
The sample runs as a downward crescent.
The adsorbent was disturbed during the spotting,
causing the crescent shape.
Slide 87
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.
Slide 88
Interpretation of Results
Slide 89
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.
Slide 90
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.
Slide 91
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.
Slide 92
Slide 93
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).
Slide 94
Interpretation of Amphetamine Positive
Results
Some drugs can metabolise to metamphetamine and
amphetamine ( benzphetamine and selegeline).
Slide 95
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.
Slide 96
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
Slide 97
Thin Layer Chromatography
Slide 98
Thin Layer Chromatography
Slide 99
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 100
Thin Layer Chromatography
Slide 101
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 102
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 103
TLC, Multi Drug
Slide 104
Thin Layer Chromatography
Slide 105
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 106
TLC, Amphetamine
Slide 107
TLC, Amphetamine
Slide 108
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 109
Thin Layer Chromatography
Slide 110
Thin Layer Chromatography
Slide 111
Thin Layer Chromatography
Slide 112
Paper chromatography
Slide 113
Paper chromatography
Slide 114
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
Slide 115
HPTLC analysis
Slide 116
Slide 117
Thanks for your attention
Slide 118
پذیرش و نمونه گیری
Slide 119
نکاتی در مورد پذیرش و نمونه گیری
•
نمونه گیری بایستی ءوری و یا با اساداده از دور بین مدار بساه انفام
شود.
•
مکان نمونه گیری بایستی از هویه و نور کافی برووردار باشد.
•
در محل نمونه گیری صابون و مواد سدید کننده نابل دسترس ی برای
مراجله کنندگان موجود نباشد.
•
ارائه كارت شناسايي يا ملرفي نامه عك
نمونه با نمونه فرد ديگر.
جلوگیري از جايگزيمي
•
موارد ا افي (كت ،كيق و ) ....وارا از محل نمونه گیري تحويل گرفاه
شود .تي بازرس ي بدني در صورت لزوم.
دار
Slide 120
مکا
سدر دردسپذیر
دسم دماسگیری
•
شستن دس ها نبزل از نمونزه گیزري :زيزر نزاون ،چسزا زوزم ویزا آلزوده كزردن دسزت
به موادي كه موجا ایفاد اواالل در آزمايا مي شوند.
•
دماي نمونه تزا 4دنيقزه بلزد از نمونزه گیزري انزدازه گیزري شزود.
: )33موثرترين راهها براي تشخيص رنيا سازي
•
نمونه گیر بايد فردي مطمئن ،هوشيار و آگاه باشد.
(-37 C
Slide 121
بعدست سم دماسگیروس
.1
دماي نمونه تا 4دنيقه بلد از نمونه گیري 33-37 Cاست.
.2
رنگ ،شدافيت ، PH،وزن مخصو نمونه بررس ي شود .اگر نمونه مشكوك
به تقلا است دوباره نمونه گیري انفام مي شود.
.3
كراتي تین كمتر از 20mg/dl
مشكوك
وزن مخصو بايد 1.002-1.04باشد پايین تر نابل نبول نوست.
PHنمونه 4.5-8.5نابل نبول است.
نمونه مهر و موم شده (به روش مناسا نگهداري شود و دسترس ي به نمونه تا زمان
آزمايا براي افراد مادرنه امكان پذير نباشد).
Slide 122
روشهاي مداوله (تقلا ) در نمونه گیري
تلويض نمونه )(Substituation
•
جابفايي نمونه با نمونه ديگر ويا اساداده از موادي كه شبيه ادرار باشند مثل چاي،آق سوا
و...
• رنيا نمودن نمونه )(Diluation
• -رنيا سازي توسط آق سبا مي شود كه غلظت دارو به دي كاها يابد كه نابل
تشخيص نباشد.
• -نوشيدن آق و مايلات ديگر سبا مي شود كه غلظت دارو كمتر از غلظت د
مرزي ) (Cut Offبرسد.
• افزايا سرعت دفع :سركه ،وياامین ،Cآق ليمو ،داروهاي ديورتيك
•
تزريا آق و مايلا ت ديگر به مثانه (عدونت و آسوا بافتي)
• اساداده از ادرار ليوفيلیزه كه براي اين منظور به صورت تفارتي ساواه موشود.
Slide 123
•افزودن مواد وارجي )(Adulteration
آزمايشگاه
بطور كلي افزودن مواد وارجي به نمونه با هدف به اشجباه انداوتن
ودريافت پاسخ مندي كاذق ِ Adulterationگداه موشود.اين مواد بطور كلي دو گروه
هسجند :
• گروه اول مواد وانگي هسجند كه به را تي در دسترس مي باشند.
صابون ،پاك كننده هاي وانگي )(Detergent سديد كننده ها )(Bleaching agent جوهر ليمو كلريد سديم سركه وياامین Cو...• گروه دوم موادي هسجند كه به را تي در دسترس نبوده و اساداده از آنها نياز به اطالعات
وتخصص دارد .تلدادي از اين مواد به شرح زير هسجند:
Slide 124
دتدیسکاسبطدرس ع دلسدرسدن رسسمین مد
- كلر()Chlorine
- آق اكسیژنه
- نمكها 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 تفارتي Clean choce
- گلوتاريك آلدهيد )(Glutaric aldehyde
- مثل موادي با نامهاي تفارتي Instant clean
- مصرف موادي تحت عنوان Golden seal,Guick caps ,Test cleanو...
Slide 125
• سه مورد اویر تحت عنوان كلي Urine Lockنیز نا ميده مي شود.
• : Mixed reagentممكن است تركيا چند مورد از مواد فوق با هم توسط فرد
مورد آزمايا مورد اساداده نرار گیرد كه در اين الت به آنها Lock Labگداه مي شود
.
Bleaching agent , Detergent
** تاثیر اين مواد روي بلض ي مواد مورد آزمايا بوشتر و در مواردي كمتر است همچنین تاثیر
اين مواد در روشهاي مخالق باهم فرق ميكند.
** ووشبخاانه افزودن اين مواد در مر له نمونه گیري صورت مي گیرد كه با دنت در انفام
نمونه گیري ءوري مي توان از اين دساه تقلبها جلوگیري كرد.
Slide 126
مکان سم عمل موادی که برای تقلا اساداده 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.
Slide 127
Thanks for your attention
IN THE NAME OF GOD
Slide 2
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
Slide 3
كارگاه
آزمایشهای تشخیص مواد مخدر و روان گردان
31اردیبهشت 94
دانشگاه علوم پزشکی شهید صدوقی یزد
Slide 4
عناوین مورد بحث
رویکرد جدید آزمایشهای تشخیص مواد مخدر و روانگردان
بررس ي انواع نمونه های بیولوژیکی ( ادرار،عرق،بززاق ،وزون و )...مزورد اسزاداده بزرای آزمزایا مزواد مخزدرو روان
گردان
مدت زمان تشخيص مواد مخدرو روان گردان درنمونه ادرار
بررس ی ( Cross-reactivityواکنشهای ماقاطع ،تداوالت دارویی)
اصول نمونه گیری و شناسایی انواع مواد مداوله گر در آزمایشهای تشخیص مصرف مواد مخدر و روان گردان
روشهای تشخیص مواد مخدر و روان گردان
-روش غربالی
-کروماتو گرافی الیه نازک )(TLC
TLC Troubleshooting -
تدسیر ناايج آزمايشهاي تشخيص مواد مخدر و روان گردان ( ناایج مثبت آمداامین و مت آمداامین )
کنترل کیدی و اعابار بخش ی روش آزمایا
Slide 5
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
Slide 6
مقدمه
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.
Slide 7
دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس
ردرسدسردتنسگردتن
دسززاوراللمل آزمززایا مززواد مخززدر و روان گززردان بززر اسززاس بنززد 5مصززوبات جلسززه 126
سززااد مبززارزه بززا مززواد مخززدر ریاسززت جمهززوری کززه بززه توشززی ریاسززت محتززرم جمهززوری در
تززاری 90/2/6بززا امءززا وزیززر محتززرم کشززور و دبیززر کززل محتززرم سززااد مبززارزه بززا مززواد مخززدر
به کلیه دساگاههای ذیربط ازجمله وزارت بهداشت ابالغ شده است:
ب ززر اس ززاس ای ززن دس ززاوراللمل ابالش ززی ،انف ززام آزم ززایا تش ززخیص مص ززرف م ززواد مخ ززدر و
روانگردان توسزط آزمایشزگاههای مربوطزه بزه صزورت یز تکلیزق نزانونی در آمزده اسزت و
دس ز ززاگاههای اجرای ز ززی موظدن ز ززد نس ز ززبت ب ز ززه جم ز ززع آوری و ارس ز ززال والص ز ززه آم ز ززاری نا ز ززایج
آزمایشززها ،هززر 3مززاه یز بززار بززه دبیززر وانززه سززااد مبززارزه بززا مززواد مخززدر ریاسززت جمهززوری
اندام نمایند.
Slide 8
دن درستلع ل فرتیمدیای آ اییاا س یریصس دتدس
ردرسدسردتنسگردتن
در اين دساور اللمل:
آزمایشگاه :به عنوان آزمایشگاه تشخیص مواد مخدر و روانگردان شناواه می شود.
س ززو مص ززرف م ززواد :ش ززامل مص ززرف کلی ززه م ززواد مخ ززدر و روانگردان ززی وواه ززد ب ززود ک ززه در
نانون ممنوع می باشد.
نمون ززه مناس ززا ب ززرای آزم ززایا :درآزمایشز ززگاههای تش ززخیص م ززواد مخ ززدر و روانگ ززردان بز ززه
منظ ززور ازدواا ،اس ززاخدام ،پوش ززه وري و ...فق ززط نمون ززه ادرار ) )Urineوواه ززد ب ززود و از
اوذ سایر نمونه های بیولوژی مثل وون ،عرق ،مو و ...وود داری شود.
Slide 9
نحوه انفام آزمایشهای تشخیص مواد مخدر و روانگردان
مطززابا ایززن دسززاوراللمل کززه نجیفززه مصززوبات کمیاززه فمززی سززااد مبززارزه بززا مززواد مخززدر و
نیز مطابا با اساانداردهای بین املللزی مزی باشزد روش حزبی بزرای تشزخیص مزواد مخزدر
و روان گززردان بززه ایززن ترت ززا اسززت کززه اباززدا بززا رعایززت کلیززه نکززات مربززو بززه هیزه نمونززه
حززبی
(جلززوگیری از تقلبهززای رایززج) ،اباززدا ایززن آزمایشززها بززا روش هززای غربززالگری س زریع
(روشززهای مطلززوق و نابززل نبززول از نظززر کنتززرل کیدززی کززه بززه تاییززد وزارت بهداشززت رسززیده
باشززد) انفززام شززده سز
درهمان آزمایشگاه).
ناززایج مثبززت بززا روش کرومززاتو گرافززی تاییززد شززوند( .ترجیحززا
Slide 10
گرچز ز ززه روش مرجز ز ززع بز ز ززرای تشز ز ززخیص مز ز ززواد مخز ز ززدر روش GC/MSمز ز ززی باشز ز ززد ولز ز ززی
محز ز ززدودی های ایز ز ززن روش اسز ز ززاداده از آن را محز ز ززدود مز ز ززی کنز ز ززد و در ز ز ززال ا ز ز ززر از
روشز ززهای کرومز ززاتوگرافی سز ززاده تز ززر مثز ززل کرومز ززاتو گرافز ززی الیز ززه نز ززازک ) (TLCبز ززرای
آزمایشززهای روزانززه اسززاداده م ززی شززود( .الباززه ب ززا رعایززت کلیززه نک ززات فمززی مرب زو ب ززه
مرا ل انفام آزمایا و نکات مربو به تدسیر ناایج).
Slide 11
برخی از محدودی های روشهایی نظیر GC/MS,GCبه شرح زیر است:
این س سامها ویلی گران هسجند .
اساداده از مواد مشاا ساز و مزواد شزیمیایی بزا درجزه ولزو بزاال هزینزه آزمایشزها را بزاال
می برد.
-3 نیاز به افراد ماخصص داشاه تا توانایی کار با این دساگاهها و تدسیر طیدهای بدست
آمده را داشاه باشد.
نیازمند ودمات پ ازفروش نوی می باشد.
عدم دسترس ی و امکان پذیر نبودن اساداده آنها در نقا دور دست.
Slide 12
نگهداري سوابق و مستندات انجام آزمايشها
سوابا انفام آزمایشها بصورت حبی بایگانی و نگه 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
Slide 13
Specimen (matrix)
Slide 14
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.
Slide 15
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).
Slide 16
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.
Slide 17
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.
Slide 18
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
Slide 19
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
Slide 20
DRUG DETECTION TIMES IN
URINE
Slide 21
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
Slide 22
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.
Slide 23
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
Slide 24
Amph, Meth, MDMA
Slide 25
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)
Slide 26
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)
Slide 27
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
Slide 28
Cross-reactivity
Slide 29
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.
Slide 30
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.
Slide 31
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.
Slide 32
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.
Slide 33
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...
Slide 34
Cross-reactivity
Amphetamin Test:
Methylenedioxyamphetamine (MDMA)
Phentermine
Benzphetamin
Ephedrine
Pseudoephedrine
Selegiline
Deprenyl
Methamphetamine
L-Methamphethamine (Vick’s inhaler)
Slide 35
Phenylpropanolamine
Phenylephrine
Ranitidine
Tyramine , ….
Metamphetamine Test:
Methylenedioxymethamphetamine (MDMA)
Amphetamine
Ephedrine
Phenyletylamine
Chloroquine
…
Slide 36
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.
Slide 37
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.
Slide 38
Cross-reactivity
Opiate,Opioids tests:
Morphine
Codeine
Poppy seed
Dextromethorphn
Diphenhidramine
Hydrocodone
Hydromorphon
Nalorphine
Procaine
Tebaine
Naloxane
Slide 39
Regulatory Process
Slide 40
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
Slide 41
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.
Slide 42
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.
Slide 43
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
Slide 44
Methods
Immunochromatography
Chromatography
Slide 45
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 …,
Slide 46
Principles of on-site immunoassay devices
Direct competition technology****
Displacement technology
Trapping technology
Slide 47
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.
Slide 48
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.
Slide 49
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 )
Slide 50
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.
Slide 51
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.
Slide 52
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%
Slide 53
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
Slide 54
Validation protocol
Sensitivity ≥ 90%
Specificity ≥ 90%
Efficiency ≥ 95%
Accuracy (Control-Comparison method- Recovery- St.add.)
Precision
Intera assay ) ( درون سنجی
Inter assay ))برون سنجی
Cross - reactivity
Cut - off
Stability
(Accel, Real, in use)
Slide 55
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
Slide 56
Accuracy
Accuracy is a measure of the closeness of test results obtained
by a method to the true value.
Re-Co-Comp...
Slide 57
(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
•
Slide 58
•
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
Slide 59
Chromatography Methods
:رتحلسدستت تء
)Exteraction(س اد س دردسآمالی سس،تن ررت
)Mobile Phas) فا س حرکس
(Stationary Phase) فا سثاب سس
(Detecore) ظایرسکممد
Slide 60
Liquid liquid extraction (LLE)
Slide 61
Solid phase Extraction (SPE)
Slide 62
Slide 63
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. ....
Slide 64
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.
Slide 65
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.
Slide 66
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.
Slide 67
Thin Layer Chromatography
Slide 68
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.
Slide 69
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.
Slide 70
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
Slide 71
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).
Slide 72
Sample Application (spotting)
Dissolve solid sample in MeOH. Use TLC
capillary to transfer and spot dissolved
sample.
Slide 73
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.
Slide 74
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).
Slide 75
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 .
Slide 76
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.
Slide 77
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
Slide 78
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.
Slide 79
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.
Slide 80
Using Silica plates impregnated with methanolic
KOH (0.1 mol/l).
Detection time ( 1-1.5 h)
Dark
Temperature
Moisture
Experienced & Expert person
Slide 81
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,
Slide 82
Thin Layer Chromatography (TLC)
Slide 83
Thin Layer Chromatography (TLC)
Slide 84
Slide 85
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.
Slide 86
Troubleshooting TLC
The sample runs as a downward crescent.
The adsorbent was disturbed during the spotting,
causing the crescent shape.
Slide 87
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.
Slide 88
Interpretation of Results
Slide 89
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.
Slide 90
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.
Slide 91
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.
Slide 92
Slide 93
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).
Slide 94
Interpretation of Amphetamine Positive
Results
Some drugs can metabolise to metamphetamine and
amphetamine ( benzphetamine and selegeline).
Slide 95
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.
Slide 96
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
Slide 97
Thin Layer Chromatography
Slide 98
Thin Layer Chromatography
Slide 99
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 100
Thin Layer Chromatography
Slide 101
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 102
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 103
TLC, Multi Drug
Slide 104
Thin Layer Chromatography
Slide 105
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 106
TLC, Amphetamine
Slide 107
TLC, Amphetamine
Slide 108
Thin Layer
Layer Chromatography
Chromatography
Thin
Slide 109
Thin Layer Chromatography
Slide 110
Thin Layer Chromatography
Slide 111
Thin Layer Chromatography
Slide 112
Paper chromatography
Slide 113
Paper chromatography
Slide 114
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
Slide 115
HPTLC analysis
Slide 116
Slide 117
Thanks for your attention
Slide 118
پذیرش و نمونه گیری
Slide 119
نکاتی در مورد پذیرش و نمونه گیری
•
نمونه گیری بایستی ءوری و یا با اساداده از دور بین مدار بساه انفام
شود.
•
مکان نمونه گیری بایستی از هویه و نور کافی برووردار باشد.
•
در محل نمونه گیری صابون و مواد سدید کننده نابل دسترس ی برای
مراجله کنندگان موجود نباشد.
•
ارائه كارت شناسايي يا ملرفي نامه عك
نمونه با نمونه فرد ديگر.
جلوگیري از جايگزيمي
•
موارد ا افي (كت ،كيق و ) ....وارا از محل نمونه گیري تحويل گرفاه
شود .تي بازرس ي بدني در صورت لزوم.
دار
Slide 120
مکا
سدر دردسپذیر
دسم دماسگیری
•
شستن دس ها نبزل از نمونزه گیزري :زيزر نزاون ،چسزا زوزم ویزا آلزوده كزردن دسزت
به موادي كه موجا ایفاد اواالل در آزمايا مي شوند.
•
دماي نمونه تزا 4دنيقزه بلزد از نمونزه گیزري انزدازه گیزري شزود.
: )33موثرترين راهها براي تشخيص رنيا سازي
•
نمونه گیر بايد فردي مطمئن ،هوشيار و آگاه باشد.
(-37 C
Slide 121
بعدست سم دماسگیروس
.1
دماي نمونه تا 4دنيقه بلد از نمونه گیري 33-37 Cاست.
.2
رنگ ،شدافيت ، PH،وزن مخصو نمونه بررس ي شود .اگر نمونه مشكوك
به تقلا است دوباره نمونه گیري انفام مي شود.
.3
كراتي تین كمتر از 20mg/dl
مشكوك
وزن مخصو بايد 1.002-1.04باشد پايین تر نابل نبول نوست.
PHنمونه 4.5-8.5نابل نبول است.
نمونه مهر و موم شده (به روش مناسا نگهداري شود و دسترس ي به نمونه تا زمان
آزمايا براي افراد مادرنه امكان پذير نباشد).
Slide 122
روشهاي مداوله (تقلا ) در نمونه گیري
تلويض نمونه )(Substituation
•
جابفايي نمونه با نمونه ديگر ويا اساداده از موادي كه شبيه ادرار باشند مثل چاي،آق سوا
و...
• رنيا نمودن نمونه )(Diluation
• -رنيا سازي توسط آق سبا مي شود كه غلظت دارو به دي كاها يابد كه نابل
تشخيص نباشد.
• -نوشيدن آق و مايلات ديگر سبا مي شود كه غلظت دارو كمتر از غلظت د
مرزي ) (Cut Offبرسد.
• افزايا سرعت دفع :سركه ،وياامین ،Cآق ليمو ،داروهاي ديورتيك
•
تزريا آق و مايلا ت ديگر به مثانه (عدونت و آسوا بافتي)
• اساداده از ادرار ليوفيلیزه كه براي اين منظور به صورت تفارتي ساواه موشود.
Slide 123
•افزودن مواد وارجي )(Adulteration
آزمايشگاه
بطور كلي افزودن مواد وارجي به نمونه با هدف به اشجباه انداوتن
ودريافت پاسخ مندي كاذق ِ Adulterationگداه موشود.اين مواد بطور كلي دو گروه
هسجند :
• گروه اول مواد وانگي هسجند كه به را تي در دسترس مي باشند.
صابون ،پاك كننده هاي وانگي )(Detergent سديد كننده ها )(Bleaching agent جوهر ليمو كلريد سديم سركه وياامین Cو...• گروه دوم موادي هسجند كه به را تي در دسترس نبوده و اساداده از آنها نياز به اطالعات
وتخصص دارد .تلدادي از اين مواد به شرح زير هسجند:
Slide 124
دتدیسکاسبطدرس ع دلسدرسدن رسسمین مد
- كلر()Chlorine
- آق اكسیژنه
- نمكها 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 تفارتي Clean choce
- گلوتاريك آلدهيد )(Glutaric aldehyde
- مثل موادي با نامهاي تفارتي Instant clean
- مصرف موادي تحت عنوان Golden seal,Guick caps ,Test cleanو...
Slide 125
• سه مورد اویر تحت عنوان كلي Urine Lockنیز نا ميده مي شود.
• : Mixed reagentممكن است تركيا چند مورد از مواد فوق با هم توسط فرد
مورد آزمايا مورد اساداده نرار گیرد كه در اين الت به آنها Lock Labگداه مي شود
.
Bleaching agent , Detergent
** تاثیر اين مواد روي بلض ي مواد مورد آزمايا بوشتر و در مواردي كمتر است همچنین تاثیر
اين مواد در روشهاي مخالق باهم فرق ميكند.
** ووشبخاانه افزودن اين مواد در مر له نمونه گیري صورت مي گیرد كه با دنت در انفام
نمونه گیري ءوري مي توان از اين دساه تقلبها جلوگیري كرد.
Slide 126
مکان سم عمل موادی که برای تقلا اساداده 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.
Slide 127
Thanks for your attention