1.
1
Principles and procedures involved in usage of reagents in
pharmaceutical analysis
PRESENTED BY:
T. LAKSHMI BHAVANI
(2015MPH40023)
Pharmaceutical Analysis -1sem
Under the guidance of:
Dr. G.RAJITHA MADAM
M.Pharm.,Ph.D
SPMVV - TIRUPATHI

2.
CONTENTS
INTRODUCTION
REAGENTS
 PDAC
 NINHYDRIN REAGENT
 1,2 NAPHTHOQUINONE - 4- SULFONATE
REAGENT PROFILE
REACTION AND MECHANISM
PRINCIPLE
PROCEDURE
APPLICATIONS
EXAMPLES
REFERENCE

3.
INTRODUCTION
 The reagents are mainly used for quantitative and
qualitative analysis of drugs.
 The reagents have a greater application in the
pharmacy field.
 Reagents are also applicable to recent analytical
techniques.

4.
PARA DIMETHYLAMINO
CINNAMALDEHYDE(PDAC) REAGENT
• Para dimethyl amino cinnamaldehyde(PDAC) is an
aromatic compound and chromogenic in nature. which is
chemically 4-(dimethyl amino) cinnamaldehyde
containing amine and aldehyde moities.
• PDAC reagent is used for determination of hydrazine.

5.
Reagent profile:
 Synonym : Ehrlich’s reagent
 IUPAC name: 4-(dimethyl amino) cinnamaldehyde
 Structure:
 Molecular formula : C11 H13 NO
 Molecular weight: 175.23 g/mol
H
CHN
CH3
CH3
CH C O

6.
 Color : dark yellow powder
 Solubility:
 soluble in warm dioxane(500mg/ml- clear ,deep
greenish yellow solution)
 soluble in mixture of chloroform: ethanol (1:1).

7.
CH
N
CH3
CH3
CH
CH
O
+ R NH2
N
CH3
CH3
R
N
+ HOH
PDAC Amines
Dehydration
Schiff base
General reaction

8.
 Chromophore formed with PDAC is more intense than
with PDAB because of additional double bond.
EXAMPLE:
CH
N
CH3
CH3
CH
CH
O
+
N
CH3
CH3
N
+ HOH
PDAC
Dehydration
Schiff base
NH2
Aniline

9.
PRINCIPLE
 The reaction between primary amine and carbonyl
compound form the condensation product are referred as
schiff’s base.
 The primary amine group which is present in the structure
of drug react with the carbonyl group in PDAC reagent
and forms schiff’s base (colored compound) which can be
measured by using colorimetry.

10.
MECHANISM OF PDAC REAGENT:
CH
N
CH3
CH3
CH
CH
O
+ R NH2
N
CH3
CH3
R
N
+ HOH
PDAC 10
Amines
H
OH
N
CH3
CH3
R
NH
Schiff’s base(colored
compound)

11.
PREPARATION OF REAGENT
 Preparation of 2% PDAC reagent:
weigh 200mg of PDAC reagent and
transfer in to a 10ml volumetric flask add 0.4ml
H2SO4 mix well and make up with distilled water.

12.
APPLICATIONS OF PDAC REAGENT
For detection of indole derivatives.
Determination of aromatic amines.
Chromogenic reagent for the detection of benzodiazepine
and flavanoids.
Used for spectrophotometric determination of ranitidine,
ganciclovir, sumatriptane, succinate, mesalamine,
lenalidomide etc.

13.
EXAMPLES
1.ASSAY OF RANITIDINE
PRINCIPLE:
The primary amine group of ranitidine reacts with
carbonyl group of PDAC reagent and forms schiff’s base
which can be measured colorimetrically.

14.
REACTION:
SCHIFF’S BASE(COLORED COMPOUND)
+ CH N
CH3
CH3CHCH
O
Ranitidine PDAC
OH2-
N
CH3
CH3
N
CH3
CH3
O
S
NH N
+
CH3
N
+
O
-
O

15.
PROCEDURE:
PREPARATION OF STOCK SOLUTION:
A standard stock solution containing 1000µg/ml of ranitidine
drug in distilled water and diluting to volume of 10ml in
volumetric flask.
PREPARATION OF CALIBRATION STANDARD:
From the above stock solution 0.2, 0.4, 0.6, 0.8 & 1ml of
solutions was pipetted out in to 10ml volumetric flask.

16.
After colour development cool the solution later diluted to
volume with distilled water.
The resulting solutions were measured for the absorbance at
632nm.
The calibration curve was plotted using concentration on x-
axis & absorbance on y-axis.
From the above stock solution , to each flask add 0.5 ml of
PDAC reagent, mix well and heat on hot plate for 15min at
1000c.

17.
ASSAY OF RANITIDINE HCl TABLETS
3 tablets were weighed and powdered.
A powder equivalent to 100mg of ranitidine HCl was
weighed and transferred into 10ml volumetric flask.
From this 10ml was pipette out.
Now add 0.5ml of PDAC reagent and heated at 800c for
15mins.
After development of color, cool the solution and later
diluted with distilled water.
The resulting solution was measured at 632nm.

18.
2.Estimation of Lenalidomide in pharmaceutical
formulation by PDAC reagent
PRINCIPLE:
 Lenalidomide(LLD) is an immuno modulatory agent with anti
angiogenic and anti-neoplastic properties.
 Lenalidomide is used to treat multiple myeloma in people who
have received at least on other therapy.
 The A primary amine group of Lenalidomide reacts with
carbonyl group of PDAC reagent and forms schiff’s base which
can be measured colorimetrically.

19.
 LLD in combination with dextramethasone is indicated for
the treatment of multiple myeloma patients.
 The chemical name of LLD is 3-[4-amino-1-oxo 1,3-
dihydro-2H-isoindol-2-yl] piperidine 2,6-dione.

20.
+ N
CH3
CH3
OHN
H
NH2
N
O O
O
H2SO4 - H2O
N
H
N
N
O
O
O
N
CH3
CH3
Lenalidomide PDAC
Schiff's base(red colored
compound)

21.
ASSAY OF LENALIDOMIDE CAPSULES
 Aliquots of LLD solution (100μg/ml) ranging from 0.1 to
0.5 ml were transferred in to a series of 10ml volumetric
flask.
 To each flask 1.0ml of PDAC(0.2%w/v) solution and
0.1ml of H2SO4 were added.
 After 5min. The volume was brought up to 10ml with
methanol and the absorbance of red colored species was
measured at 530nm against reagent blank.

22.
NINHYDRIN REAGENT
 It is a chromogenic reagent which is chemically 2,2-
dihydroxy indane-1,3-dione.
 It is used in colorimetric estimation of drugs containing
free amine group.
 It gives deep blue or purple color which is popularly
known as Ruhemann’s purple.

23.
REAGENT PROFILE
Synonym: Indanetrione
IUPAC name: 2,2-dihydroxy indane-1,3-dione.
Structure:
Molecular formula:C9 H6 O4
Appearance/form : white crystalline powder
Solubility : soluble in water and alcohol.
slightly soluble in ether and chloroform.
O
O
OH
OH

24.
PRINCIPLE
 Ninhydrin tautomerizes to 1,2,3-indane trione which
forms a schiff base when react with amine group
containing amino acids.
 The ketamine formed undergoes decarboxylation yielding
the aldehyde and was isolated by ruhemann.
 Condensation of this intermediate amine with another
molecule of ninhydrin follows to form the expected
chromophore, Ruhemann’s purple.

25.
Reaction with Amino acids:
O
O
OH
OH
R
NH2O
OH
+
O
O
N
O
O
+ C
O
H R
+ co2 H2O+
Ninhydrine
Amino acid
Purple coloured complex

26.
O
O
OH
OH
+
O
O
N
O
O
+ + co2 H2O+
Ninhydrine Phenyl alanine
Purple coloured complex
H
NH2
O
OH
CH3
O
REACTION

27.
REACTION WITH IMINO ACIDS
O
O
OH
OH
+
Ninhydrine L- Proline
Red coloured complex
O
NH
OH
O
OO
O
OH
N

28.
REACTION WITH PRIMARY AMINE
O
O
OH
OH
+
Ninhydrin
Primary amine
R NH2
O
O
N
R
Colored compound

29.
O
O
OH
OH
O
O
O
NH2
R
O
O
-
H
NH2
R
N R
O
O
-H
O
O
N R
H
O
O
CO2
O H
H
MECHANISM OF NINHYDRIN REAGENT
Ninhydrine

30.
N R
H
O
O
-
O H
H
N R
H
O
O
H
R-CHO
NH2
O
O
H
N
O
O
O
O
-
H2O,H+
H+
Ruhemann’s purple

31.
APPLICATIONS
 Used for both qualitative and quantitative analysis of
amino acids and drugs containing amine groups.
 Used as a stain to amino acids.
 It is mostly used to detect fingerprints, as the terminal
amines of lysine residues reacts with ninhydrin.
PREPARATION OF 0.1%W/V NINHYDRIN
REAGENT: 0.1g of ninhydrin reagent was
Dissolved in 100ml of distilled water.

32.
1. Estimation of ampicillin in pharmaceutical formulation by
Ninhydrin reagent
PRINCIPLE:
 Ampicillin is an antibacteral drug which is chemically
(6R)-6(α-phenyl)-O-glycyl amino pencillanic acid.
 It contains primary amine group so it can be estimated by
using ninhydrin.
EXAMPLES

33.
 Tautomerised form of ninhydrin react with ampicillin to
form schiff’s base.
 The ketamine formed under goes decarboxylation and this
on condensation, with another molecule of ninhydrin
gives a colored complex which can be estimated at
absorbance of λ max.

34.
PROCEDURE
PREPARATION OF 0.1N HCl:
0.85 ml of HCl was made up to 10ml with distilled
water.
PREPARATION OF STANDARD STOCK SOLUTION:
10mg of ampicillin was dissolved in 10ml of 0.1N
HCl to give 1000µg/ml stock solution.

35.
CONSTRUCTION OF CALIBRATION
CURVE:
 From the standard stock solution 1,1.5,2,2.5 were pipetted
out in to 10ml volumetric flask.
 To each flask add 2ml of 0.2% ninhydrin reagent,1.5 ml of
0.1N HCl ,2ml of methanol, heat on the water bath until
color develops.
 After the color develops make up the volume to 10ml with
0.1N HCl, Measure the absorbance at 490nm.

36.
REACTION
O
O
OH
OH
O
O
O +
H
NH2
O
NH
N
S
O
OH
CH3
O
CH3
N
O
NH
N
S
O
OH
CH3
O
CH3
O
O
H
O
O
NH2
H
O
OO
O
N
Ninhydrin 1,2,3-indanetrione
Ampicillin
Schiff's base
Intermediate amineColored complex

37.
ANALYSIS OF AMPICILLIN CAPSULES
 Two capsules were weighed and powder equivalent to
10mg was dissolved in 10ml of 0.1N HCl.
 From the above solution pipette out 2ml and transfer to
10ml volumetric flask and then add 2ml of 0.1%w/v
Ninhydrin reagent,1.5ml of 0.1N HCl, 2ml of CH3OH was
added and heated on a water bath until color develops.
 Make up the volumetric flask to 10ml with 0.1N HCl and
measure absorbance at 490nm.

38.
2.Estimation of levodopa in pharmaceutical
formulation by ninhydrin reagent
PRINCIPLE
 Levodopa {(-)-3-[3,4-dihydroxyl phenyl-1-alanine] a
precursor of dopamine, is an important neurotransmitter
which is used for medication of neural disorder such as
Parkinson's disease.
 After administration levodopa is converted into dopamine
through enzymatic reaction catalyzed by
dopadecarboxylase.
 Different methods to determine levodopa have been
employed such as high performance liquid
chromatography, fluorescence spectroscopy,
electrochemical method.

39.
REACTION
OH
OH
NH2
O
OH
+
O
O
OH
OH
OH
OH
O
OH
OH
+
O
O
OH
OH
O
O
O
O
N
O
O
Levodopa Ninhydrin
Hydrindatin
Ruhemann's purple

40.
ANALYSIS OF CAPSULES
 Weigh accurately 100mg of levodopa , add 60ml of DMF
and stirred.
 It was filtered with whatmann filter paper size≠1 and
washed with DMF.
 The filtrate was diluted to 100ml with DMF.
 To 5ml of this solution ,2ml of 0.2% ninhydrin solution
was added.
 The mixture was heated on a water bath at 80 to 850C for
5mins.

41.
 The mixture was cooled to room temperature and then
transferred to a 100ml measuring flask.
 Final volume was made upto 100ml with DMF and
scanned in the UV- visible range at 400 to 800nm,for the
determination λmax of levodopa by using blank solution.

42.
1,2 NAPHTHOQUINONE – 4 – SULFONATE
REAGENT (NQS)
 NQS is the most widely used chromogenic reagent for the
estimation of drugs containing aromatic amine.
 It is a derivative of O-quinone

43.
REAGENT PROFILE
 SYNONYM: Sullivan reagent
Follin’s reagent
 STRUCTURE:

44.
PRINCIPLE
 When the NQS reacted with any amine containing
compound that will release hydroxyl group and the
sodium sulphonate group is replaced with the aromatic
amine group.

45.
GENERAL RECTIONNa
O
O
SO O
O
+ R NH2
- OH
O
O
NH
R
- OH
O
O
NH
NQS
Aliphatic amine
colored chromogen
NH2
+
O
O
SO O
O
Na

46.
APPLICATIONS
 Sulpha drugs
 Succinyl sulphathiazole
 Chloramphenicol
 Nimesulide
 Nifedipine
 Nitrendipine

47.
EXAMPLES
1.Estimation of cefadroxil in pharmaceutical
formulation by NQS reagent
 Cefadroxil is an antibacterial drug. As it contains amine
group, it reacts with NQS reagent.
 The principle involved is nucleophilic substitution in
alkaline condition.
 In the presence of 0.1NaOH ,NQS reagent reacts with
amino group of cefadroxil to form a colored complex
which is measured at λmax of 453nm.

48.
REACTION
Cefadroxil
. H2O +
OH
NH2
O
O
O
N
N
S
O CH3
OOHOrange colored product
NQS

49.
ANALYSIS OF CEFADROXIL TABLETS
 5 Tablets were weighed and powdered.
 The powder equivalent to 50mg of cefadroxil was taken
in to a 10 ml volumetric flask and make up with distilled
water .
 From Above stock solution 0.1ml was pipetted out and
again dilute to 10ml with distilled water , then add 1ml of
0.1N NaOH(0.4g of NaOH in 100ml d.w.)and add 1ml of
0.5%w/v NQS reagent. Measure the absorbance at 453nm.

50.
2. Spectrophotometric Method for Determination of
Primaquine in Pharmaceutical Formulations via
Derivatization with 1,2-Naphthoquinone-4-Sulfonate
PRINCIPLE:
 Primaquine is anti malarial drug.
 The nucleophilic substitution reaction proceeds
quantitatively at PH 10 buffer solution with absorption
maximum at 485 nm.
 The method is based on the formation of a brown color
product from the reaction between PQ and NQS.

51.
N
O
CH3
NH
CH3
NH2
+
O
O
S OO
ONa
N
O
CH3
N
CH3
NH
O
O
O
O
2
+ 2 NaHSO4
Primaquine
1, 2 - Naphthoquinone
- 4 sulfonate
Brown coloured compound
Sodium hydrogen sulphate
REACTION

52.
PROCEDURE:
PREPARATION OF STANDARD AND SAMPLE
SOLUTION
 Stock standard solution of Primaquine
diphosphate (PQ -200 μg/ mL)
An accurately 0.02 g of PQ standard was
dissolved in distilled water and transferred in 100 mL
volumetric flask diluted to mark. The solution was further
diluted to obtain working solution.

53.
 Sample Solution
Four tablets (PQ 15 mg ∕ tablet) were weighed and
finely powdered. A portion of the powder equivalent to 0.09 g
of the drug was dissolved in distilled water and transferred into
50 mL volumetric flask the solution was completed to mark,
shaken well filtered and then analyzed by spectrophotometric
method.
 1,2-napthoquinone-4-sulfonate Reagent{ NQS (0.4%, w/v) }
It was prepared by dissolving 0.4 g in 100 mL
volumetric flask and complete the volume with distilled water.
The solution was freshly prepared.

54.
Analysis of primaquine tablets
 A 2.0 mL of 200 μg ∕ mL primaquine diphosphate standard
was transferred in 10 mL volumetric flask.
 2.0 mL of NQS was added and followed by 1.5 mL of
buffer solution PH 10.
 The flask was completed to volume with distilled water
and the absorbance was measured at 485 nm against
blank.

55.
REFERENCES
 Text book of pharmaceutical analysis by P.D.chaithanya
sudha
 Austin journal of analytical and pharmaceutical
chemistry by Aida Makram Nouralla Altigani, Abdalla
Ahmed Elbashir* ,University of Khartoum, Faculty of
Science, Chemistry Department, Sudan .

56.
 El-Kommos ME, Emara KM, Alexandria
Spectrophotometric determination of certain
pharmaceutical secondary aromatic amines using p-
dimethylamino-cinnamaldehyde J. Pharm. Sci. 1988; 2:
171-176.
 Aida Makram Nouralla Altigani, Abdalla Ahmed
Elbashir* University of Khartoum, Faculty of Science,
Chemistry Department, Sudan