KIMIA FARMASI KUALITATIF II

ANTIBIOTICS
Dr. Friardi, Apt
 Antibiotic/Antimicrobial
• Antibiotic: Chemical produced
by a microorganism that kills or
inhibits the growth of another
microorganism
• Antimicrobial agent: Chemical
that kills or inhibits the growth of
microorganisms
Microbial
Sources
of
Antibiotics
Antibiotic Spectrum of Activity

• No antibiotic is effective against all

microbes
 Mechanisms of
Antimicrobial Action
• Bacteria have their own enzymes
for
– Cell wall formation
– Protein synthesis
– DNA replication
– RNA synthesis
– Synthesis of essential
metabolites
The History of Chemotherapy
• Paul Ehrlich and Sahachiro Hata
developed Salvarsan
(Arsphenamine) against
syphilis in 1910: The concept of
chemotherapy to treat microbial
diseases was born.
• Sulfa drugs (sulfanilamide)
discovered in 1932  against
Gram+ bacteria
 The History of Chemotherapy
1928: Fleming discovered penicillin
1940: Howard Florey and Ernst
Chain performed first clinical trials
of penicillin. Fig 20.1
β-Lactams
H
R N
C S
Me

O Me
N
O

H
COOH
R N
S
C

O
N OAc

CO2H
 History
1928- Alexander Fleming discovers a mold which inhibits
the growth of staphylococcus bacteria
1940- penicillin is isolated and tested on mice by
researchers at Oxford
1941- penicillin mass produced by fermentation for use
by US soldiers in WWII
1950’s- 6-APA is discovered and semi-synthetic
penicillins are developed.
1960’s to today- novel β-lactams/ β-lactamase inhibitors
are discovered and modified from the natural products
of bacteria
 Cephalosporins

Fungi of genus
Cephalosporium
 4 Generations of
cephalosporins

1. First-generation: Narrow spectrum, gram-positive

2. Second-generation: Extended spectrum includes
gram-negative
3. Third-generation: Includes pseudomonads; mostly
injected, some oral.
4. Fourth-generation: Most extended spectrum
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
 Cephalosporins cont.
Structure and mode of action resembles penicilins

1. More stable to
bacterial -
lactamases than
penicilins

2. Broader
spectrum  used
against penicillin-
resistant strains

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings

Penicillinase (b Lactamase)

Figure 20.8
 -: 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

Makroskopis
• Bau amis
• Serbuk kekuningan
 Detection of penicillin
1. Phosphomolybdic acid
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

(β, β-dimethyl cysteine) and is immediately oxidized to the corresponding
disulfide by the phosphomoybdic acid with simultaneous formation of
molybdenum blue.
 N
a

Oxid.
Di sulfide + molybdenum metal
( blue)

-Test is given by all pen. antibiotics Penicillamine

(mercaptan)
 FeCl3 .2

-: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
NH
Fe+3 O
NH
OFe+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*

add 2 drops-1 In center of the paper

1%KOH/ANHYDROUS MEOH
follow immediatly

add 10 drops unhydrous-2

FeCl3 /MEOH saturated with
Sprinkle sample (milk
NH2OH.HCL
powder) over wet area
of the paper
 -:Procedure for detection Penicillin G in milk
.cont

Observe the paper

.Remove exss against light

Pinkish spots
3. Sulfuric acid
This reagent is prepared by dissolving 1 g of
potassium nitrite in 10 ml of sulphuric acid
Benzathine penicillin, benzoic acid,
benzylpenicillin yellow
 Chromatographic detection
-:of pen.G

:Thin layer chromatography (TLC) .4

Use: precoated silica plate

Solvent system: aceton-CHCL3-glacial acetic acid (50:45:5)

Distance traveled by the substance

= RF
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 Dist. Travell by

.by sub .solv

Drop of sample
sample+2drop of(
) solvent
1cm
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
 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 Procaine penicillin G

Penc.G + Procain HCl ProcainePen. G + NaCl

 : Test for detection of procaine penicillin G -

-:Phosphomolybdic acid test*

give +ve result with all penicillin -

Procaine pen. G +2-3 drops saturated aqueous WB

.phosphomolybdic acid sol

Cotton Intense blue

pecie 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 )
Tetracyclines
Origin
Tetracyclin, oxytetracyclin isolated from
Streptomyces aureofaciens
Minocycline, doxycyclin are synthetic
Macroscopic
• Tetracyclines are a yellow or a light-yellow
crystalline powder, odorless, bitter on test.
identifikasi
• The reaction the a concentrated sulfuric acid – forming
the anhydrotetracycline’s derivatives, which have a
specific colour
tetracycline – violet-red,
Oxytetracycline – red
the colour transfers in a yellow at the adding of water:
• ТLC method comparing with a standard
pharmacopoeial sample.
• Hydrochloride’s of tetracyclines give the
reaction of chlorides.
• Determine an specific optical rotation.
• UV-spectrometry. Determine the maximum and
minimum absorption and calculate a specific
absorption rate.
• Formation of the coloured complex salts in an
alcohol medium with FeCl3- brown and red-
brown (present of the phenolic hydroxyl in the
10-th position).
• Formation of the coloured complexes with the
salts of copper (II), zinc.
• Formation in certain conditions, the fluorescent
products.
• In an express analysis of tetracycline drugs
there is used color reactions with sodium
nitroprusside, n-dimethylaminobenzaldehide,
Nessler reagent, diazoreagent.
-: Test for detection of doxycycline & oxytetracycline
:Sakagushi test* α-naphthol in alkali
place drops of reagent in petri dish-
Sprinkle sample (milk
powder) on the surface
of reagent

Intense red
)oxytetracycline(

Intense yellow
)doxycycline (

Sakagushi test differentiate between the different types-

.of tetracyclines
.it is give –ve result with penicillin &chloramphinicol -
 Chloramphenicol
Intro

 An antibiotic produced by Streptomyces

venezuelae, an organism first isolated in
1947 from a soil sample collected in
Venezuela.
Macroscopics
• Appearance:
White or white with a slight greenish
yellowish sheen crystalline powder,
odorless, bitter taste.
• Solubility:
Slightly soluble in water, easily soluble in
95% alcohol, soluble in ethyl acetate R,
hardly soluble in chloroform R.
chloramphenicol molecule has 2 asymmetric carbon atoms, and
therefore it is possible the existence of four isomers:
D- and L-threo, D- and L-erythro-,
Which differ by the spatial arrangement of functional groups:
Chloramphenicol is a leftrotatory threo-isomer
of the D row.
Mixture of D(-) and L(+) threoisomers of
laevomycetin is called racemic mixture,
optically inactive compound known as
synthomycin (has 50% of the Chloramphenicol
physiological activity).

Erythro-forms is not used in medicine because

it is a toxic compounds.
identifikasi
A. To 0,1 g of preparation add 5 ml of the
diluted sodium hydroxide solution R and
heated, the yellow coloration appears that
with further heating transforms into a red-
orange.
At the boiling the color of solution
increases, allocated brick-red precipitate
and appears the smell of ammonia:
В. After filtration of the mixture obtained in Test A, 2 ml of filtrate
acidified by nitrate diluted acid R, add 0,4 ml of silver nitrate R1,
mix and defend; formed white amorphous precipitate.
The liquid above the sediment is decantated. Precipitate is
washed
with three portions of water R 1 ml of each. This operation is
conducted quickly in the protected from bright light place, thus
allowed that liquid over the sediment was not completely
transparent.
Precipitateis suspendered in 2 ml of water R and add 1,5 ml of
ammonia R; residue quickly dissolved; allowed the presence of a
few crystals, which dissolve slowly:

NaCl + AgNO3 → AgCl↓ + NaNO3;

AgCl↓ + 2NH4OH → [Ag(NH3)2]Cl + 2H2O.
c. CaCl2-Zn

-: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 a-naphthyl
ON } amine/acetic acid
Zn
Boiling
water C10H17 N N ]Azodye

 The NO2 group can be reduced to the NO (nitroso) group by warming with CaCl 2 and
Zn
 The nitroso compound produced condenses to yield a violet azo dye
 Milk(solid) contain
several +
chloramphenicol + drops 10%cacl2 2
mg zn dust

.WB 2drops 5%sol.alfa- .WB

naphthylamine/acetic
2min .acid sol

Intense violet color

d. Reaksi NaOH
• Hydrolysis in acidic or basic mediums with the
following identification of the formed
products. Thus, at the heating of laevomycetin
with sodium hydroxide at first yellow color
appears,
• that transfers to the red-orange (as a result of
formation of acynitroform),
• after the following heating brick-red
precipitate formed and smell of ammonia
appeared:
e. HCl
• Laevomycetin stearate at the heating with concentrated
chloride acid hydrolyzed – stearic acid is formed, which floats
to the surface in the form of oily drops which harden when
cooled:
 f. CuSO4
• In the express-analysis use reaction of laevomycetin with
copper (II) sulfate in alkali medium in the presence of n-
butanol – alcoholic layer painted in blue-violet color
• due to the formation of complex salt, which supposedly has
the following structure: