PHG 461

King Saud UniversityPharmacy Collage Pharmacognosy Depaartment

LAB#1

Chemical & chromatographic detection of A.B in milk

N.B-:

Chemical reaction depend on color (reaction with A.B + specific reagent)

Part I

* 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.

Why antibiotic is add to our food

product?

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 milkFew mgs + 2-3 drp sat. aq. phosphomolybdic acid

Intense blue color

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.

Boiling water

close T.T With cotton

Penicillamine (mercaptan)

Di sulfide + molybdenum metal ( blue)

Na

Oxid.

-Test is given by all pen. antibiotics

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

1)b. detection of Penicillin G in milk-:

Principle of reaction:

NH2OH

Fe+3

ferric hydroxamate

Test is given by all -lactam -containig antibiotics (pen.,cephalosporine) & can be used for their detection.

NH

ONH

OFe+3

cont.

Na

*test:- place apiece of filter paper on top of 250ml beaker

1-add 2 drops

1%KOH/ANHYDROUS MEOHIn 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

Procedure:

1)b. Procedure for detection Penicillin G in milk-: cont.

Remove exss .

Observe the paper against

light

Pinkish spots

2-Test for detection of doxycycline & oxytetracycline-:

*Sakagushi test-:-place drops of reagent in petri dish

Sprinkle milk powder on the surface of

reagent

Intense red (oxytetracycline)

Intense yellow (doxycycline)

Detection of some A.B in milk-:cont.

-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:

CaCl2Zn

ON }

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

N ]AzodyeNC10H17

naphthyl amine/acetic acid

Boiling water

Milk(solid) contain chloramphenicol + 2 drops

10%cacl2

+several mg zn

dust

WB.

2min

2drops 5%sol.alfa-naphthylamine/ace

tic acid sol.

WB.

Intense violet color

3 -Procedure for detection

of chloramphenicol-: cont.

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

1cm

Drop of sample(Milk

sample+2drop of solvent )

Solvent front

Dist.travell by sub.

Dist. Travell by solv.

**Dry plate then spray it with potassium ferriccyanide followed by exposing the plate to iodine vapor

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

• 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.

Part II

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

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.

Na

Penicillin G sodium

Procaine penicillin G

Procaine HCl

Preparation of Procaine penicillin

Penc.G + Procain HCl 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 )

1) Solubility2) PH3) Melting point

Physical constants of Procaine Penicillin G

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) PHMeasure the PH of the previously prepared solution of

procaine penicillin using a universal PH paper or a PH meter.

3) Melting pointUsing the melting point

instrument

International unit of penicillin salt-:

1mg pencillin G Na =1667 I.U.

The international unit of penicillin is the specific penicillin activity contained in 0.6 mg of the crystalline sod. Salt of Penicillin G

The strength and dosage of penicillin are measured in terms of international units.

- 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 1mmol Pen.G Na

588.7 356.4

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.

Exampels 1

-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)

Exampels2

King Saud UniversityPharmacy 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

If glucose more or equal 0.15% +ve result

( bright red color )

Cupric citrate Cuprous oxide (CuO2)

Red.

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-:

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.

Advantage:

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-:

b-Moxalactam is acephalosporin A.B that lacks sulfer in its ring structure, so it does not cause this interference

cont.

-1ml of benedic,s sol or-1mg of clini powder+1pelet of NaOH

+1ml of sample

WBObserve color

How the test is do it?

1( Sugar free urine

2( Sugar containing urine

3( Solution of a tetracycline

4( Solution of Cephalexin

5( Solution of Streptomycin

1( Sugar free urine

2( Sugar containing urine

3( Solution of a tetracycline

4( Solution of Cephalexin

5( Solution of Streptomycin

Sample

Benedict,sCilin test

Sugar+urineRed or orange

orange

Urine+ceph.brownbrown

Urine+strep.blueblue

Urine+TCNbrowngreen

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 glucose

Glucose gluconic acid +H2O2

H2O2 +o-tolidine blue color

With the addition of a yellow dye ,The color range fromYellowlight greendeep blue

O2 from air

Glucose oxidase

peroxidase

**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