Transcript lab 1 and2

PHG 461
King Saud University
Pharmacy Collage
Pharmacognosy Depaartment
LAB#1
Chemical & chromatographic
detection of A.B in milk
Part I
N.B:Chemical reaction depend on color
(reaction with A.B + specific reagent)
Why antibiotic is add to our food
product?
* A.B is found in milk in small amount since it is used
as animal feed additives to promote growth (weight
gain) &prevent infection.
*Chlortetracycline, for example is added by commercial fisherman to the ice in which ocean fish are
packed in order to reduce decomposition.
Milk is NOT allowed to be marketed if the milk gives
+ve penicillin test, Why?
• Sensitization of consumers to penicillin
• The emergence of strains of pathogenic
microorganisms resistant to these drugs
• Allergic reactions in people sensitive to
penicillin might result from its ingestion
Chemical tests for detection
of antibiotics in milk
1- a.Detection of penicillin in milk
Few mgs + 2-3 drp sat. aq. phosphomolybdic acid
close T.T
With cotton
Intense blue color
Boiling
water
Principle of reaction: color reaction involve 2 steps:-
a. Liberation of parent acid from its salt by phosphomolybdic
acid
b. Hydrolysis of the organic acid to give penicillamine which is a
mercaptane (b, b-dimethyl cysteine) and is immediately oxidized to the
corresponding disulfide by the phosphomoybdic acid with
simultaneous formation of molybdenum blue.
Na
Oxid.
Di sulfide + molybdenum metal
( blue)
-Test is given by all pen.
antibiotics
Penicillamine
(mercaptan)
1)b. detection of Penicillin G in milk:Principle:this test depend on forming ferric hydroxamate whish is
pinkish in color, it is formed when hydroxylamine reacts
with the B-lactam group in the presence of fecl3
Principle of reaction:
cont.
NH2OH
Na
Fe+3
ferric
hydroxamate
Test is given by all b-lactam -containig antibiotics
(pen.,cephalosporine) & can be used for their
detection.
Procedure:
*test:- place apiece of filter paper on top of 250ml beaker
1-add 2 drops
1%KOH/ANHYDROUS MEOH
In center of the paper
follow immediatly
2-add 10 drops unhydrous
fecl3 /MEOH saturated with
NH2OH.HCL
Sprinkle milk powder
over wet area of the
paper
1)b. Procedure for detection
Penicillin G in milk:-
Observe the paper
against light
Remove exss.
Pinkish spots
cont.
Detection of some A.B in milk:cont.
2-Test for detection of doxycycline & oxytetracycline :-
*Sakagushi test:-place drops of reagent in petri dish
Intense red
(oxytetracycline)
Intense yellow
( doxycycline)
Sprinkle milk powder
on the surface of
reagent
-Sakagushi test differentiate between the different types
of tetracyclines.
- it is give –ve result with penicillin &chloramphinicol.
Detection of some A.B in milk:cont.
3- Detection of chloramphenicol :*Principle:Chloramphenicol is only known A.B which has
character of an aromatic nitro compound. The NO2
group can readily be reduced to NO (nitroso) group
by warming an aqeous sol. Of chloramphenicol with
calcium chloride and zinc. The nitroso compound thus
produced condenses, in acetic acid solution, with
alfa-naphthylamine to yield aviolet azo dye.
Principle of reaction:
CaCl2
Zn
a-naphthyl
amine/acetic
acid
Boiling
water
 The NO2 group can be reduced to the NO (nitroso) group by warming
with CaCl2 and Zn
 The nitroso compound produced condenses to yield a violet azo dye
3- Procedure for detection
cont.
of chloramphenicol :Milk(solid) contain
chloramphenicol
WB.
2min
+
2 drops 10%cacl2
2drops 5%sol.alfanaphthylamine/acetic
acid sol.
+ several
mg zn dust
WB.
Intense violet color
Chromatographic detection
of pen.G in milk:-
1-Thin layer chromatography (TLC):
Use: precoated silica plate
Solvent system: aceton-CHCL3-glacial acetic acid (50:45:5)
RF =
Distance traveled by the substance
Distance traveled by the solvent
Rate flow
**Dry plate then spray it with potassium ferriccyanide followed by
exposing the plate to iodine vapor
Solvent front
Dist.travell
by sub.
Drop of sample(Milk
sample+2drop of
solvent )
1cm
Dist. Travell by
solv.
RF value of penicillin G spot is consider as reference for
comparison with the result obtained from our sample .
RF pen. G =0.6-0.7
Part II
• Preparation of Procaine Penicillin G
•
•
•
•
Detection of procaine penicillin G
Calculation of % yield
Determination of physical constants
Calculation of I.U of Penicillin G Na.
Preparation of procaine penicillin G



Procaine penicillin, is a combination of
benzylpenicillin with the local anesthetic agent
procaine.
Following deep intramuscular injection, it is slowly
absorbed into the circulation and hydrolysed to
benzylpenicillin .
It is used where prolonged low concentrations of
benzylpenicillin are required.
Preparation of procaine penicillin G
- It is not effective orally.
- Different prepration to give different dose
- Procaine penicillin G is prepared by
mixing equimolar of pen. G salt (Na or K )
and procaine HCL.
Preparation of Procaine penicillin
Procaine
HCl
Na
Penicillin G
sodium
Penc.G + Procain HCl
Procaine penicillin
G
ProcainePen. G + NaCl
Preparation of procaine penicillin G
- Test for detection of procaine penicillin G :
*Phosphomolybdic acid test:- give +ve result with all penicillin
Procaine pen. G +2-3 drops saturated aqueous
phosphomolybdic acid sol.
Cotton
pecie
WB
Intense blue
color
**Color reaction involve two steps:First:- libration of parent acid from its salt by phosphomolybdic
acid.
Second:- hydrolysis of acid to give penicillamine which meroptane=
B,B dimethyl cysteine
oxidation of penicillamine
to give the disulfide by phosphomolybdic acid to give disulfid with
MO metal (blue in same time )
Physical constants of Procaine
Penicillin G
1)
2)
3)
Solubility
PH
Melting point
Physical constants of Procaine Penicillin G
1)
Solubility

Pulverize the dry procaine penicillin you prepared

Weigh out 100mg and determine how much water is required
to dissolve this quantity.

Add 10ml of water to the salt in 50ml conical flask and shake
vigorously for several minutes.

If the salt has not dissolved, add 5ml more of water and
repeat

Continue the process, and record your results
Physical constants of Procaine Penicillin G
2) PH
Measure the PH of the previously prepared solution of procaine
penicillin using a universal PH paper or a PH meter.
3) Melting point
Using the melting point instrument
International unit of penicillin salt :-
The strength and dosage of penicillin are
measured in terms of international units.
The international unit of penicillin is the specific
penicillin activity contained in 0.6 mg of the crystalline
sod. Salt of Penicillin G
1mg pencillin G Na =1667 I.U.
Exampels 1
- How many I.U in 1mg of procaine pen.G?
(procaine pen.G MWT=588.7 , PEN.G Na MWT =356.4 )
1mmolProcaine pen. G
588.7
1mmol Pen.G Na
356.4
1mg
X
X= 1 x 356.4/588.7 =0.605 mg
1mg pen.G
1667 I.U.
0.605 mg
X
x= 1008.535 I.U.
Exampels2
- How many I.U in 1mg of benzathine penicillin?
(benzathine pen. MWT=909.1 , PEN.G Na MWT =356.4 )
(N.B:- 2mmol pen.G Na =1mmol benzathine penicillin)
King Saud University
Pharmacy Collage
Pharmacognosy Depaartment
LAB#2
Glucose urine analysis
& interaction with A.B.
*Urinary glucose detection is of great importance for
monitoring the condition of diabetic patients. The detection is
done by the following reagents:
1- Benedict,s reagent:
- The reagent has clear deep blue color
- It contains acomplex of cupric citrate, when mixed with an
equal volume of urine then warmed gently glucose will
reduct to formed ppt.of cuprus oxide (CUO2)which is
bright red
Red.
Cupric citrate
If glucose more or equal 0.15%
Cuprous oxide (CuO2)
+ve result
(bright red color)
2- Clinitest reagent:
-The reagent comes as tablets, so we should prepare
fresh solution for urine test.
-It contain copper acetat, so the reaction with the urine
glucose with same principle (between cupric ion &
glucose in urine )
bright red
Advantage of Clini test over Benedict,s reagent:-
Advantage:
1-Clinitest provide fresh prepared solution of cupric
acetat that superior sensitivity to Benedict,s solution
which tend to give in accurat result when stored for
extended period of time.
Interaction of A.B:
*Some anti-infective agent give false-positive test
for glucose when use any reagent for testing
e.g,(tetracyclines, chloramphenicol,streptomycin,
P.amino salicylic acid, ascorbic acid in high dose
(>1.5G/day) )
Comment:-
1- tetracyclines, chloramphenicol,streptomycin give
false-positive result with glucose in urine due to
their reducing property.
2-all cephalosporins interfer by different mechanism
a-react with CU ion produce insoluble brownish
black ppt. of cupric sulfat(CUS), these ppt. will
mask +ve color of test.
Comment:-
cont.
b-Moxalactam is acephalosporin A.B that lacks
sulfer in its ring structure, so it does not cause this
interference
How the test is do it ?
-1ml of benedic,s sol or
-1mg of clini powder
+1pelet of NaOH
WB
+ 1ml of sample
Observe color
Sample
1) Sugar free urine
2) Sugar containing urine
3) Solution of a tetracycline
4) Solution of Cephalexin
5) Solution of Streptomycin
Benedict,s
Cilin test
orange
Urine+ceph.
Red or
orange
brown
Urine+strep.
blue
blue
brown
green
Sugar+urine
Urine+TCN
brown
To overcome this a disadvantage of
previous reagent, lilly company has
special tape which is selective only for
the dextrose :it is test-tape
Glucose enzymatic test strip
=test-tape method:-
This reagent is in form of paper strips
impregnated with :
*2 oxidizing enzyme: (glucose oxidase,peroxidase)+
oxidizable substrate(o-tolidine)+
yellow dye.
How test-tape is work?
when paper impregnated into urine contain
O2 from
air
glucose
Glucose
Glucose
oxidase
H2O2 +o-tolidine
gluconic acid +H2O2
peroxidase
blue color
With the addition of a yellow dye ,The color range from
deep blue
light green
Yellow
** compare color result with the chart and
determine the % of glucose in urine.
** yellow color mean NO glucose in urine
* Non of the anti-infective agents interfere with the results of tes-tape
method
* Tes-tape method should be the method of choice for urinary glucose
detection if the patient is receiving any of these agents