A Review of Analytical Methods for Determination of

Chlorpheniramine in Pharmaceutical and Biological

Samples
Taral N. Patel1, Dr. Hasumati A. Raj2
Abstract: Chlorpheniramine is an antihistaminic drug which is available in the
different pharmaceutical dosage forms. It is used in cough and nasal congestion assosiated
with upper respiratory tract conditions. . This article reviews the current analytical
methods for identification and quantitative determination of Chlorpheniramine in
pharmaceutical and biological samples. The clinical and pharmaceutical analysis of this drug
requires effective analytical procedures for quality control and pharmacodynamic and
pharmacokinetic studies as well as stability study. an extensive survey of the literature
published in various analytical and pharmaceutical chemistry related journals has been
conducted and the instrumental analytical methods which were developed and used for
determination of Chlorpheniramine as single or combination with other drugs in bulk
drugs and formulations have been reviewed. this review covers the time period from 1994 to
2012 during many analytical methods including spectrophotometric methods like uv and
derivative; and chromatographic method including HPLC, HPTLC, GC were reported. The
application of these methods for the determination of Chlorpheniramine in pharmaceutical
formulations and biological samples has also been discussed.
INTRODUCTION
Chlorphenamine (INN) or chlorpheniramine (USAN, former BAN), commonly
marketed in the form of chlorpheniramine maleate, is a first-generation alkylamine
antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis
and urticaria. Its sedative effects are relatively weak compared to other first-generation
antihistamines.
It is named according to IUPAC rules as 3-(4-chlorophenyl)-N,N-dimethyl3-pyridin-2-yl-propan-1-amine.[1] Chlorpheniramine occurs as an odorless, white crystalline
powder.[1][2] It is Freely soluble in water, soluble in alcohol &chloroform, slightly soluble in
ether & benzene.[2] Chlorpheniramine is commonly available as the Chlorpheniramine
Maleate. pKa value of Chlorpheniramine is 9.13 and it mealts at 196-200C.[1]

Figure 1: chemical structures of Chlorpheniramine

Chlorpheniramine competes with histamine for H1 receptor sites, there by

antagonizing many histamine effects to reduce allergy signs and symptoms[3]. It is rapidly
absorbed from the gastrointestinal tract. The first-pass through the hepatic portal vein results
in some of the drug's being metabolized Primarily by Cytochrome P450(CYP450) enzyme[3]
Execretion occur by kidney.
The use of the Chlorpheniramine as a drug essential in pharmaceutical formulations
highlights the requirement for its determination and quantification with appropriate analytical
methods. This paper gives an overview of the analytical techniques that are available and
nowadays have been used for determination of Chlorpheniramine in pharmaceutical samples.

METHODS FOR THE DETERMINATION OF CHLORPHENIRAMINE

Official Methods
Chlorpheniramine is official in Indian pharmacopoeia, British Pharmacopoeia and

United state pharmacopoeia.

Table 1: Summary of Compendial Methods
Pharmacopoeia
Method
[1]
IP
Potentiometry:
Weigh accurately 0.2 g and dissolve in 20 mI anhydrous glacial acetic acid.
Titrate with 0.1 M perchloric acid, determining the. end-point
potentiometrically. Carry out a blank titration.
I mI of 0.1 M perchloric acid is equivalent to 0.01954 g of C20H23ClN2O4.
BP
Potentiometry:[2]
Dissolve 0.15 g in 25 ml anhydrous acetic acid. Titrate with 0.1 M perchloric
acid, determining the end-point potentiometrically.
1 ml of 0.1 M perchloric acid is equivalent to 19.54 mg of C20H23ClN2O4.
USP
Titrimetry:[4]
Dissolve 500 mg in 20 ml anhydrous acetic acid. Add 2 drops of crystal violet.
Titrate with 0.1 M perchloric acid. Perform a blank determination.
1 ml of 0.1 M perchloric acid is equivalent to 19.54 mg of C20H23ClN2O4.

Reported Methods
Analytical Methods for Single Drug
Table 2: Summary of Analytical Methods for Single Drug
SR NO
1.

METHOD
HPLC[5]

2.

TLCDensitometry
[6]

3.

RIA[7]

TITLE & ABSTRACT

Rapid Quantitative Analysis of Chlorpheniramine in Plasma,
Saliva and Urine by High-Performance Liquid Chromatography.
A method was developed for the rapid quantitative analysis of
Chlorpheniramine in plasma, saliva and urine using HPLC. A diethyl
ether or hexane extract of the alkalinized biological samples was
extracted with dilute acid which was chromatographed on a reversedphase column. Ultraviolet absorption at 254 nm was monitored for the
detection and brompheniramine was employed as the internal standard
for the quantitation. The effects of buffer, pH, and acetonitrile
concentration in the mobile phase on the chromatographic separation
were investigated. A mobile phase 20% acetonitrile in 0.0075 M
phosphate buffer at a flow-rate of 2 ml/min was used for the assays of
plasma and saliva and urine samples.
Rapid Identification and Quantification of Chlorpheniramine
maleate or Pheniramine maleate in Pharmaceutical Preparations
by Thin-Layer Chromatography-Densitometry.
Thin-layer chromatography (TLC)-densitometry was used to
separate, identify, and quantitate Chlorpheniramine maleate (CPM) or
Pheniramine maleate (PM) when present in combination with other
drugs in pharmaceutical preparations of tablets, syrups, eye and ear
drops, etc. CPM or PM was extracted (tablets, capsules, etc.) or diluted
(liquid preparations, if needed) with 80% ethanol and isolated from
other ingredients by TLC on silica gel G using cyclohexane chloroform
methanoldiethylamine (4.5 + 4.0 + 0.5 + 1.0, v/v) as the mobile phase.
Separated CPM and PM were detected under shortwave ultraviolet light
and quantitated by scanning densitometry at 260 nm.
Subnanogram Quantitation of Chlorpheniramine in Plasma by
a New Radioimmunoassay and Comparison with a Liquid
Chromatographic Method.
A new Radio Immuno Assay allows the determination of
Chlorpheniramine levels up to 96 h after oral administration of a single
4-mg tablet to healthy volunteers. This procedure was sensitive to a
156-pg/mL plasma concentration when a 100-L plasma sample was
used. The mean coefficient of variation over the linear range from 0.156
to 20 ng/ml was 3.79%. The specificity of the assay was investigated,
and the antisera showed 7% cross-reactivity with the N,N-didemethyl
analogue and 17% cross-reactivity with the N-demethyl analogue. This
high degree of specificity was also evident from the findings that the
plasma concentrations determined by this newly described RIA
procedure gave a strong correlation (r2 = 0.88) with values obtained by
an HPLC-UV procedure. The antiserum cross-reacted 100% with
brompheniramine and, thus, can be used for its analysis in plasma

Analytical Methods For Combined Dosage Forms

SR
NO
1.

Table 3: Summary of Analytical Methods for Combined Dosage Forms

METHOD
TITLE & ABSTRACT
Spectrophotometry[8]

2.

Spectrophotometry[9]

3.

Spectrophotometry[10]

4.

HPLC[11]

Simultaneous Estimation & Validation of Paracetamol,

Phenylephrine Hydrochloride and Chlorpheniramine
Maleate in Tablets by Spectrophotometric Method.
This method utilize 0.1N NaOH as solvent. Simultaneous
equation develops using 256.8 nm, 236.8 nm and 222.4 nm as the
max of Paracetamol, Phenylephrine HCl and Chlorpheniramine
maleate respectively. Calibration curves were linear over the
concentration ranges of 0-35 g/ml for all drugs. The results
demonstrated that the procedure is accurate, precise and
reproducible (relative standard deviation < 1 %), while being
simple, cheap and less time consuming, and hence can be suitably
applied for simultaneous determination of three drugs in
laboratory prepared mixtures and in commercial tablet
preparation.
Spectrophotometric
Determination
of
Chlorpheniramine Maleate and Phenylpropanolamine
Hydrochloride in Dosage Forms.
A rapid and simple method for simultaneous determination
of Chlorpheniramine Maleate (CPM) and Phenylpropanolamine
HCl (PPM) by first derivative UV spectrophotometry has been
developed in combined pharmaceutical dosage forms. The
proposed method was successfully applied for the determination
of drugs in physical mixture and commercial formulations and
results showed good linearity, precision and reproducibility.
A Validated UV Spectrophotometric Method for the
Simultaneous Estimation of Dextromethorphan
Hydrobromide and Chlorpheniramine Maleate in Syrup
Formulation.
A novel spectrophotometric method for the simultaneous
estimation of Dextromethorphan HBr and Chlorpheniramine
Maleate in combined liquid dosage form,(i.e., syrup). This is a
derivative spectroscopic method. Dextromethorphan HBr has
absorbance maxima at 289.2nm and Chlorpheniramine maleate
has absorbance maxima at 262.6nm in methanol. The proposed
method was validated in terms of Linearity, Accuracy, Specificity,
Precision, Ruggedness, Beers Law was obeyed in the
concentration range of 10-70 g/ml for both the drugs. r2 =
0.999.
Simple HPLC Method for Simultaneous Determination
of Acetaminophen, Caffeine and Chlorpheniramine maleate
in Tablet Formulations.

5.

HPLC[12]

6.

RP-HPLC [13]

7.

RP-HPLC[14]

A simple, rapid and accurate, routine-HPLC method is

described for simultaneous determination of Acetaminophen,
Caffeine and Chlorpheniramine maleate in a new tablet
formulation Chromatographic separation of the three
pharmaceuticals was achieved on a Hypersil CN column (150
5.0 mm, 5 m) using a mobile phase comprising a mixture of
acetonitrile, an ion-pair solution and tetrahydrofuran (13:14:87,
v/v,pH4.5). The flow-rate was changed from 1.0 ml/min(in 07.5
min) to 1.8 ml/min (after 3.5 min). was complete in <10 min.
Simultaneous
High-Performance
Liquid
Chromatographic
Determination
of
Paracetamol,
Phenylephrine HCl, and Chlorpheniramine maleate in
Pharmaceutical Dosage Forms.
A rapid, precise, and specific HPLC method is described for
the simultaneous determination of Paracetamol, Phenylephrine
HCl, and Chlorpheniramine maleate in combined pharmaceutical
dosage forms. The method involves the use of a Bondapak CN
RP analytical column (125 , 10 m, 3.9 150 mm) at 22C as the
stationary phase with the mixture of acetonitrile and phosphate
buffer (pH 6.22, 78:22) as the mobile phase. Derivatization of the
drugs is not required.
A Validated RP-HPLC Method for the Simultaneous
Estimation of Dextromethorphan Hydrobromide and
Chlorpheniramine Maleate in Syrup Formulation.
The proposed method is a smethod for the simultaneous
estimation
of
Dextromethorphan
HBr
(DXM)
and
Chlorpheniramine maleate (CPM) in bulk and syrup formulation.
Stationary phase consist of Eclipse-XDB C18 column (150
4.6mm, 5m) and mobile phase with gradient mode consisting of
phosphate buffer (pH 3.0): acetonitrile (80 : 20 v/v) was used.
The flow rate was set at 1.0 ml/min and UV detection was carried
out at 272 nm. The retention time of DXM and CPM were 9.05
min and 7.53 min respectively. The % recovery of DXM and CPM
was found to be 99.58 1.33 and 98.24 1.97 respectively. DXM
and CPM drugs were found to be linear over the concentration
range of 2-50 g/ml and 0.8 - 20 g/ml respectively.
Development and Validation of a RP-HPLC Method for
the Determination of Chlorpheniramine maleate and
Phenylephrine in Pharmaceutical Dosage Form.
Developed for the simultaneous determination of
Chlorpheniramine maleate and Phenylephrine in tablet dosage
forms. A RP C18 column (250 mm 8 mm, 10 m) column with

8.

RP-HPLC[15]

9.

RP-HPLC[16]

10.

RP-HPLC[17]

mobile phase consisting of acetonitrile and phosphate buffer

55:45 (v/v) (pH 5.6 0.02, adjusted with triethylamine) was
used. The flow rate was 1.0 ml/ min and effluents were
monitored at 255 nm. The retention times of chlorpheniramine
maleate and phenylephrine were found to be 3.13 min and 4.58
min, respectively.
Validated Stability Indicating RP-HPLC Method for
Simultaneous Estimation of Codeine phosphate and
Chlorpheniramine maleate from Their Combined Liquid
Dosage Form.
Development of quick stability indicating RP-HPLC method
for the simultaneous estimation of Codeine phosphate and
Chlorpheniramine maleate in the presence of its degradation
products. Both drugs & their combination drug product were
exposed to acid, base, oxidation, dry heat, and photolytic stress
conditions, and the stressed samples were analysed by proposed
method. The proposed HPLC method utilizes the Phenomenex C18
column (5) using a mixture of 1% o-phosphoric acid in
water:acetonitrile:methanol (78:10:12) mobile phase with pH
adjusted to 3.0 in an isocratic elution mode at a flow rate of
1ml/min, at 23C with a load of 20l. The detection was carried
out at 254nm.
Simultaneous Determination of Chlorpheniramine
maleate, Paracetamol and Pseudoephedrine Hydrochloride
in Pharmaceutical Preparations by RP-HPLC.
A simple, precise and specific RP-HPLC method
wasdeveloped for identification and simultaneous determination
of Chlorpheniramine maleate, Paracetamol and Pseudoephedrine
HCl in capsules and liquids. The separation of three components
was performed on
C18, 150 x 4.6 mm, 5 HPLC column using
gradient mobile phase methanol ,sodium perchlorate (0.043M, 2
ml triethylamine, pH 5.0) at a flow rate of 1.0 ml/min , detection
was at 204nm for Chlopheniramine maleate, Pseudoephedrine
HCl and 300 nm for Paracetamol.
Simultaneous Determination of Chlorpheniramine
maleate, Dextromethorphan HBr and Phenylephrin HCl in
Syrup Using
RP-HPLC.
A simple, precise and specific HPLC method was developed
for simultaneous determination of Chlorpheniramine maleate,
Dextromethorphan HBr and Phenylephrin HCl in syrup. The
separation of three components was performed on
Zorbar C18,
(4.6 x 250 mm ,5) HPLC column using acetonitrile :phosphate

buffer pH 3.5 (15:85 v/v) at a flow rate of 0.9 ml/min , detection

was at 280nm .
11.

RP-HPLC[18]

12.

RP-HPLC[19]

13.

GC[20]

CONCLUSION

Simultaneous Determination of Chlorpheniramine

maleate, Phenylephrine Hydrochloride, Paracetamol and
Caffeine in Pharmaceutical Preparation by RP-HPLC.
A RP-HPLC method was successfully developed for the
simultaneous determination of quaternary mixture of
Chlorpheniramine maleate (CPM), Phenylephrine HCl (PE),
Paracetamol (PCM) and Caffeine in pharmaceutical preparation.
The separation of the drugs was carried out using an Inertsil ODS
C18 column using 0.05M dibasic phosphate buffer: acetonitrile
(93: 7; v/v) as the mobile phase. The flow rate was adjusted to
1.5 ml/min and the column oven temperature was kept at 30 C.
All the drugs were resolved successfully with retention times
2.74 (CPM), 3.48 (PE), 9.5 (PCM) and 26.32 (caffeine) min when
detection was carried out at 215 nm. The r2 was found to be
0.999, 0.998, 0.999 and 0.999, respectively for CPM, PE, PCM and
caffeine.
Analysis of Cough and Analgesic Range of Pharmaceutical
Active Ingredients using RP-HPLC Method.
A novel and single RP-HPLC method was developed for the
determination of ten active ingredients (Codeine phosphate,
Paracetamol,
Chlorpheniramine
Maleate,
Theophylline,
Pseudoepidrine HCl, Ambroxol, Salbutamol, Guaifenesin,
Dextromethorphan and Diphenhydramine HCl) in all pharmaceutical
dosage forms, along with preservative (Sodium benzoate). The
separation was achieved on a X-Terra C18, 150 x 4.6mm, 3.5 in the
simple gradient mode using buffer and Acetonitrile with 0.8 ml/min
flow rate. Column oven temperature maintained at 40C and
performed the analysis with 220 nm.
Simultaneous Determination of Some Active Ingredients in
Cough and Cold Preparations by Gas Chromatography, and
Method Validation.
A simple and rapid gas chromatographic (GC) method has been
developed for the simultaneous determination of combinations of
Acetaminophen,
Phenylpropanolamine
HCl,
Guaifenesin,
Pseudoephedrine HCl, Caffeine, Chlorpheniramine maleate, and
Dextromethorphan HBr in cough and cold tablets and syrups. After
extraction of the analyte with alkaline ethyl acetate, 2 l extract was
injected (splitting ratio 50:1) into a CBP1-M25-025 fused silica
capillary column (25 m x 0.22 mm; film thickness, 0.25 m). The
column temperature 150C for 5 min, increased to 175 C at 3
C/min, and increased to 270 C at 10 C/min. The temperatures of
the flame ionization detector and injector were maintained at 300C.

Presented systematic review covers the current analytical methods for the
determination of Chlorpheniramine in pharmaceutical and biological samples. The
limitation of the reported methods requires developing new optimized method which would
be suitable for intended analytical purpose for analyzing the content of Chlorpheniramine in
pharmaceutical and biological samples. The new trends and advances for quantification of
Chlorpheniramine are based on using high-pressure liquid chromatography which is widely
available and flexible method . The HPLC method could be automated; there are different
column fillings; different solvents with different polarity as mobile phases and different
detection modes. The faster time, high sensitivity, specificity and better separation
efficiency enable HPLC to be used frequently for the simultaneous qualitative and
quantitative determination of Chlorpheniramine in the comparison with the other methods.
The development of a new established method should reduce existing analytical problems
as well as improving the resolution which can give accurate results for the concentration
of all Chlorpheniramine samples and could be used for routine analysis.

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