PHG 414 Practical course

Quali tati ve & Quanti tati ve

Analysi s
of

and other adulterants
















1
PHG 414 Practical course

I NTRODUCTI ON


Heroin ( diacetylmorphine ) is a semisynthetic drug first made from morphine
at St, May's hospital (London) in 1874 . it was introduce in 1898 as remedy for
cough and for morphine addiction . Some years passed , however before it was
appreciated that it cured morphine addiction by substituting itself as the addicting
agent . It is converted to morphine in the body .
It is commonly stated that heroin is the most potent of all dependence-
producing drugs .
The great advantage of heroin, from the point of view of illicit traffickers, is
that it seems capable of almost unlimited dilution with other substances, while still
the addict obtains from it some gratification for his desire. Nearly every small
peddler who resells heroin first mixes his supply with at least an approximately
equal amount of lactose or powdered sugar. As his heroin is usually highly
adulterated and diluted even before he gets it, it frequently reaches the final user
containing no more than 5% of the actual alkaloid, or even sometimes as little
as1% . This illicit "heroin" is actually a salt of the base, namely the hydrochloride.
The impure heroine, containing small amounts of acetylcodeine and
papaverine derived from the original opium, and of poorly acetylated heroine,
containing more or less monoacetylmorphine and residual morphine.

CLASSIFICATION OF OTHER SUBSTANCES
The various substances that may be found in a heroin sample, besides
diacetylmorphine, the substances may be classified as follows:
A. DILUENTS:
Generally lactose or powdered sugar, but sometimes mannitol or some other
substance may be used.
B. ADULTERANTS:
While the diluents may also be called adulterants in the usual meaning of the
term, it is convenient to distinguish the substances which are merely diluents
from the drugs of more or less activity which are added either to conceal the
comparative lack of real heroine, or to enhance its effect. Quinine is the most
common adulterant and probably occurs in more than half the heroin samples in
the United States and Canada. It is probably added, for one reason, to enhance
the bitter taste of the mixture, in case the addict tries to assay the strength by
tasting. It is most often added as the hydrochloride; sometimes as the sulfate.
Some years ago, procaine was very commonly used. The amount of quinine
2
PHG 414 Practical course
usually equals or exceeds that of heroine, but the procaine was only about one-
sixth to one-third the amount of the heroine, and possibly it was used chiefly for
its effect as a local anaesthetic. Other adulterants that have been found are
barbiturates, caffeine, acetphenetidin, methadone, and amphetamine.
C. IMPURITIES OF MANUFACTUR:
These include monoacetylmorphine and morphine, remaining from imperfect
acetylation. The absence of such impurities may be significant of diversion from
legal manufacture.
D. IMPURITIES OF ORIGIN:
These include codeine (present as acetylcodeine after the acetylation),
papaverine, meconic acid, and brown coloring matter. The absence of these
substances may not mean much, but their presence proves that a rather crude
process was used, not only in the manufacture and purification of the heroine, but
before that, in the extraction of the morphine.



LAB WORK


Lab work includes the qualitative and of heroin by color tests, Microscopically tests
and TLC and quantitative analysis by HPLC.
Col our Test s :
1. Marquis' reagent (Formaldehyde in concentrated or (6 +1) H
2
SO
4
) -purple red
changing to purple.
2. Frohde's reagent (Molybdate in concentrated H
2
SO
4
) strikes violet , quickly
changing to strong purplish red, fading out to weaker brown or brownish, then
developing green.
3. Mecke's (or Lafon's) reagent (Selenious acid in concentrated H
2
SO
4
)
green,quickly greenish blue, changing to blue, slowly to bluish green with
yellow-brown edge, then olivaceous green.
Comment on tests 1 to 3: These sulfuric acid reagents provide highly
characteristic colour tests for the spot-plate, and most adulterants and diluents
do not seriously interfere. Rarely, a sample may contain so little heroine that
charring of the diluent sugar with the concentrated acid will obscure the
colours. Of course in that case, a separation is necessary. Imperfect acetylation
does not matter as morphine and monoacetylmorphine give the same colours.
The initial colour with the hydrochloride is slightly different from that with
the free alkaloid or sulfate, bluer with Frohde's reagent and more yellow-
brown with Mecke's; and this effect is increased if additional chloride is
present.
3
PHG 414 Practical course
4. Nitric acid-light yellow solution, gradually bright green. Concentrated HNO
3

is usually used, but a (4 +1) acid is somewhat better (4 parts concentrated
HNO
3
mixed with 1 part H
2
O).
Comment: This highly characteristic test is not very sensitive and often
enough it may not be obtained on adulterated, diluted samples. Morphine
gives an orange red color fading to yellow; sometimes with a little morphine
present a red-orange is obtained at first and later the green of
diacetylmorphine develops. Monoacetylmorphine is similar to morphine.
5. Copper test. To a little of the powder on the spot plate add several drops of
water, 2 or 3 drops of 3% H
2
O
2
, a drop or two of NH 4OH and stir with a
piece of copper. A pink to red color is produced. This test is given by various
phenols and their acetyl derivatives including morphine and diacetylmorphine.


Mi c r osc opi c Test s :
1. Platinum chloride, H
2
PtCl
6
. A little of the powder being examined is
dissolved in a drop of water, or better in diluted acetic acid, on the microscpoe
slide, and a drop of the 5% reagent solution is added, then crystals are looked
for under the microscope. They form gradually and are needles in rosettes.The
crystals grow larger and form blades in rosettes in the presence of acetic acid,
which also diminishes the interface of quinine, and may be used up to (1+1)
strength. (Lews, New York International Revenue Laboratory.)
2. Mercuric iodide in HCl. (The solvent solution contains about 27% by volume
of concentrated HCl, or 10% by weight of HCl, and is saturated with Hgl
2
)
This reagent is applied to a little of the dry substance on the microscope slide.
The crystals are branching threads and splinter-plates.
Comment: The test is extremely sensitive and usually succeeds even with
highly adulterated samples. The reagent can also be applied to the aqueous
solution.








4
PHG 414 Practical course
Thi n Layer Chr omat ogr aphy (TLC) :

Sample preparation :

- If the sample provided ,suspected to contain heroin, is in powdered from : It
is boiled with dichloromethane (CH
2
Cl
3
) , Filtered then the residue (after
evaporation of solvent) is dissolved in 1 ml CHCl
3
:MeOH .
- If the sample is provided in solution , it should be alkalinizing (using
NH
4
OH) then extracted with CH
2
Cl
2
as mentioned before.

Condition :
o Stationary phase : Silica gel 60 F
254
(5X10)
o Mobile phase :MeOH (10ml) :NH
4
OH ( 5 drops) OR 5%
MeOH/CHCl
3
(using 4-5 drops)
o Detection : by UV at 254 nm then spray with Dragendorff's
reagent
o References : Heroin 0.05% in CHCl
3
/MeOH .


N.B. Heroin, distributed in the market is usually mixed with adulterants and
diluents. The average purity of wholesale samples (45%) was only slightly
higher than the purity of retail samples (30%). It may contain: Paracetamol,
phenobarbitone, caffeine, which a can be detected easily on TLC plate by
using references of these compounds.




Tests for adulterants :

1. Quinine or other fluorescent compounds. A little of the powder is dissolved in
dilute sulfuric acid in a test tube or on the microscope slide and observed
under ultraviolet light. Nupercaine also fluoresces strongly.
2. Sanchez test for procaine and other primary aromatic amines. A solution of
furfural in acetic acid, applied to the dry powder on the spot plate, yields a
bright red colour with any speck of procaine or other primary aromatic amine.
3. Chromium sulfate-chloride reagent for methadone. This reagent can be
applied either to a drop of solution or directly to a little of the dry powder on
the microscope slide, and will readily yield the methadone crystals even in the
presence of much more heroine or quinine or both.


5
PHG 414 Practical course


Hi gh P erformance Li qui d Chromatogarphy
(HP LC)

Condi t i ons:
Column : -porasil [Normal phase] ( 150 X 3.9 mm)
Mobile phase : 3% MeOH in CHCl
3
.
Detector : UV at 254 nm
Flow rate : 2 ml/ min
CS : 1 cm / min
Volume injection : 20 l

Pr oc edur e:
1. For standard curve:
Heroin is prepared in concentration ( 0.05%) in CH
2
Cl
2
( 0.5 mg/1 ml). Then
dilute different volumes : 4, 3, 2, and 1 ml to each to 5 ml (in a volumetric
flask) with CH
2
Cl
2
, representing: 0.4, 0.3, 0.2, and 0.1 mg/ml.
Inject 20 ml, in duplicate, into the HPLC system. The standard curve is
plotted using different concentrations of heroin (mg/1mg) versus area under
the peak.
A linear relationship confirm the possible use of this curve for
quantization.

2. For the Sample:
For determination of heroin in the sample provided : prepare 0.1 0.2
% in CH
2
Cl
2
and inject 20l, in duplicate, into the HPLC system. Obtain from
the read-out sheat the area under the peak corresponding to the injected
samples refer to the standard curve to calculate the percentage of heroin in the
sample.












6
PHG 414 Practical course
Opiates






Morphine, Heroin, Codeine,
Hydromorphone,
Hydrocodone


Trade Names
Narcotic analgesic Classification
Morphine-high; Heroin-high;
Codeine-moderate;
hydromorphone-
high
Physical/
Psychological
Dependence :
Swallowed or injected
Methods of
Administration:
White, brown, or black
powder,
liquids (injectable), tablets,
capsules
of various sizes and colors
Physical
Appearance :
3 days
Approximate
Detection Time
in Urine :
Euphoria, analgesia,
drowsiness,
respiratory depression
Clinical Effects/
Symptoms:
The Basi c s
Opiates are drugs such as heroin, codeine and morphine,
which can produce a very high physical and/or
psychological dependence by their users. Samples may
be tested for codeine and morphine (a metabolite of both
codeine and heroin), and follow-up testing for 6
acetylmorphine, also a metabolite of heroin, is allowed
on morphine-positive samples.
Testing for the opiates, hydrocodone (Vicodin, Lortabs),
hydromorphone (Dilaudid), is performed on
nonregulated samples. Feelings of euphoria, analgesia,
drowsiness, and respiratory depression are reported by
users.
Sampl e
Random urine specimen in a properly sealed and labeled
urine container.
Met hod and I nst r ument
Preliminary screening for drugs of abuse in urine is
performed by immunoassay, Bayer ADVIA 2400.
Confirmation by Gas Chromatography/Mass
Spectroscopy.
St abi l i t y
After proper collection, concentration of codeine,
morphine or other opiates in urine will not change
significantly for several days at room temperature, for
several weeks at refrigerated temperature or indefinitely
when frozen.
Pur pose:
Urine positive indicates recent usage.
Nor mal Resul t s
Negative.
Abnor mal Resul t s
The original immunoassay has been confirmed by
GC/MS. The screening cutoff for opiates in regulated
samples is 2000 ng/mL. The confirmation cutoff for
either codeine or morphine in these samples is 2000
ng/mL. This concentration is consistent with ingestion of
the analyte or the analyte-producing medication
sometime within the 72-hour period preceding the urine
collection.
I nt er f er enc es
Though no compounds have been tested that cannot be
separated from codeine and morphine by GC/MS
analysis, there are numerous prescription medications
which contain codeine. In addition, poppy seeds contain
morphine and codeine in varying amounts, so careful
evaluation of confirmed opiate-positive samples by an
experienced professional can be essential in avoiding a
false accusation of drug abuse.










































7