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897
Research Article
Shinde. EUROPEAN JOURNAL OF PHARMACEUTICAL
European Journal of Pharmaceutical and Medical Research
AND MEDICAL RESEARCH ISSN 2394-3211

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DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS

ESTIMATION OF MECLIZINE HYDROCHLORIDE AND CAFFEINE IN BULK AND
TABLET DOSAGE FORM

Shinde Ganesh Shashikant1*

1
Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Rahata, Ahmednagar
423109.

*Corresponding Author: Shinde Ganesh Shashikant

Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Rahata, Ahmednagar 423109.

Article Received on 27/03/2019 Article Revised on 17/04/2019 Article Accepted on 07/05/2019

ABSTRACT
A simple and more economic RP-HPLC method was developed and subsequently validated for the simultaneous
determination of Meclizine hydrochloride and Caffeine in bulk and pharmaceutical dosage form. The
chromatographic conditions were standardized using a Grace C18, (250×4.6mm,5µ). The analyte detection was
carried out by using a UV detector set at a wavelength of 228 nm. The mobile phase consisted of Methanol: Water
(80:20) at pH 3 adjusted with ortho phosphoric acid (OPA) with the flow rate of 1.0 ml/min and retention time of
Meclizine hydrochloride and Caffeine was found to be 4.128 min and 5.107 min respectively. The calibration
curves of two drugs were linear with correlation coefficients of 0.9991 and 0.9992 over a concentration range of 5-
25μg/ml for Meclizine hydrochloride and 4-20μg/ml for Caffeine. This method has been validated and shown to be
accurate, precise, specific, sensitive, linear, robust and fast. Meclizine hydrochloride and Caffeine were subjected
to different degradation stress conditions. The degradation products were well resolved from that of pure standard
drugs (Meclizine hydrochloride and Caffeine) with significant different retention time values. The current method
has been statistically validated according to the ICH guidelines and this method has been subsequently developed
and applied successfully to determine the levels of Meclizine hydrochloride and Caffeine in a combined
formulation and in the routine quality control analysis with good accuracy and sensitivity.

KEYWORDS: Meclizine hydrochloride, Caffeine, RP-HPLC.

INTRODUCTION formula C27H27CIN2•2HCI•H2O. The chemical name is

Meclizine is approved by the U.S. Food and Drug 1-(p-chloro-alpha-phenylbenzyl)-4-(m-methyl-benzyl) –
Administration (FDA) to treat symptoms of motion piperazine dihydrochloride monohydrate. [4]
sickness. Safety and efficacy in children younger than
twelve years of age has not been established; therefore,
use in this population is not recommended. Meclizine
should be taken with caution in the elderly due to
increased risk of confusion and amnesia.[1]

Meclizine is an antagonist at H1 receptors. It

possesses anticholinergic, central nervous system
depressant, and local anesthetic effects.[2] Its antiemetic
and antivertigo effects are not fully understood, but its
central anticholinergic properties are partially
responsible. The drug depresses labyrinth excitability
and vestibular stimulation, and it may affect the
medullary chemoreceptor trigger zone. Meclizine also is
Figure No. 1: Chemical structure of Meclizine
a dopamine antagonist at D1-like and D2-like
hydrochloride.
receptors but does not cause catalepsy in mice, perhaps
because of its anticholinergic activity.[3] Meclizine
Caffeine is a central nervous system (CNS) stimulant of
hydrochloride, an oral antiemetic, is a white, slightly
the methylxanthine class.[5] It is the world's most widely
yellowish, crystalline powder which has a slight odor and
consumed psychoactive drug. Unlike many other
is tasteless. It has the following structural (fig.no.1) and
psychoactive substances, it is legal and unregulated in

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nearly all parts of the world. There are several

known mechanisms of action to explain the effects of
caffeine. The most prominent is that it reversibly blocks
the action of adenosine on its receptor and consequently
prevents the onset of drowsiness induced by adenosine.
Caffeine also stimulates certain portions of
the autonomic nervous system. Pure anhydrous caffeine
is a bitter-tasting, white, odorless powder with a melting
point of 235–238 °C.[6] Caffeine is moderately soluble in
water at room temperature (2 g/100 mL), but very
soluble in boiling water (66 g/100 mL).[7] It is also
moderately soluble in ethanol (1.5 g/100 mL).[8] It is
weakly basic (pKa of conjugate acid = ~0.6) requiring Figure No. 2: Chemical structure of Caffeine.
strong acid to protonate it. It has the following structural
(fig.no.2) and formula C8H10N4O2. The chemical name is
1,3,7-trimethylpurine-2,6-dione.

MATERIAL AND METHOD

List of Instruments
Table 1: List of apparatus/ instruments used.
Sr. No. Name Model Manufacturer/Supplier
1. weighing balance PGB 100 Wensar
2. Digital pH meter pH cal Analab Instrument
3. Ultra-Sonicator WUC-4L Wensar
4. UV- Spectrophotometer and Software UV2450 UV probe v 2.3.3 Shimadzu
HPLC 3000 series
P- 3000-M reciprocating
Analytical
5. HPLC (binary pump)
Technologies ltd.
UV-3000-M (UV-Visible
Detector)

List of Chemicals
Table 2: List of chemical used.
Sr. No. Reagents and Chemicals Make Details
1. Water MI HPLC grade
2. Methanol Finar HPLC grade
3. Ortho-phosphoric acid (85% pure) Merck specialties private limited HPLC grade

Table 3: List of API used.

Sr. No. Name Specification Manufacturer/Supplier
1. Meclizine hydrochloride Working standard Swapnroop drugs and pharmaceuticals
2. Caffeine Working stand Ard Swapnroop drugs and pharmaceuticals

Table No. 4: Formulation Used.

Sr. No. Name Contents Product Developed By
Meclizine
1. Pregnidoxin tab. hydrochloride 25mg UCB India Pvt. Ltd.
Caffeine 20mg

Preparation of mobile phase Standard Stock Solution

Mixed a HPLC grade Methanol: Water with pH 3.0
(80:20) in volumetric flask. Filter through 0.45μ filter
under vaccum filtration.
Diluent preparation: Use mobile phase as diluent.
Procedure
Accurately weighed quantity of Meclizine hydrochloride
and Caffeine 10 mg individually dissolved in 10 ml
volumetric flask using mobile phase and solution was
sonicated for 20 minutes and volume is make up to the
mark to get 1000 μg/ml and filtered through 0.45μm
membrane filter. 1ml from each solution taken and
dissolved in 10ml volumetric flask seperately using
mobile phase to get 100 μg/ml.

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Preparations of working standard solution
Procedure
From the standard stock solution 1.5 ml Meclizine
hydrochloride and 1.2 ml Caffeine solution taken and
added in 10 ml volumetric flask and diluted up to the
mark with mobile phase.
Preparation of Sample solution
Procedure
20 tablets were weighed and powdered, tablets powder
equivalent to 10 mg of Meclizine hydrochloride and
caffeine was transferred 100 ml volumetric flask,
sufficient amount of mobile phase was added and
dissolved by 20minutes ultrasonication. then made the
volume up to the mark with the mobile phase and filtered
with 0.45µ filter paper. Pipette out 1.5 ml from above
solution and diluted to 10 ml mobile phase and use for
sample injection.
Selection of analytical wavelength
Accurately weighed quantity of Meclizine hydrochloride
and Caffeine 10 mg dissolved in 100 ml volumetric flask
using methanol and volume is make up to the mark to get
100 μg/ml. From this solution 2.5 ml was taken and
added in 10 ml volumetric flask and diluted up to the
mark using methanol. Solution was scanned using UV-
Visible Spectrophotometer in the spectrum mode
between the wavelength ranges of 400 nm to 200 nm.
The wavelength selected was 228 nm.

Fig. 3: Wavelength of Meclizine hydrochloride and Caffeine.

Optimized chromatographic condition
In the present study the separation of Meclizine
hydrochloride and Caffeine was achieved by using
column Grace C18, (250×4.6mm,5µ) with mobile phase
consisting of mixture of methanol and Water (pH 3.0) in
the ratio of 80:20 at a flow rate 1.0 ml/min with uv
detection wavelength of 228nm at ambient temperature.
The retention time for Meclizine hydrochloride and
Caffeine were found to be 4.128min and 5.107min
respectively.

Figure No. 4: Chromatogram for standard solution.

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Shinde. European Journal of Pharmaceutical and Medical Research

Theoretical Asymmetry
Name RT(min) Area Resolution
plate factor
Meclizine hydrochloride 4.128 735204 3.50 5806 1.36
Caffeine 5.107 563628 0.00 6857 1.26

Figure No. 5: Chromatogram for sample solution.

Time Area Resolution Th. Plate Asymmetry

4.193 219397 3.70 6525 1.36
5.593 172323 0.00 6963 1.24

RESULT AND DISCUSSION
System Suitability
HPLC system was optimized as per the chromatographic
conditions. 20 µl of standard solutions of drugs were
injected in triplicate into the chromatographic system.
The chromatogram were recorded and measure the
response for the major peak. system suitability parameter
such as retention time, theoretical plate and asymmetry
factor. then the %RSD of all parameter were calculated.

Table 5: System suitability parameters for Meclizine hydrochloride and Caffeine.

System suitability parameters Meclizine hydrochloride Caffeine
Retention time 4.193 min 5.593 min
Theoretical plate no. 6525 6963
Tailing factor 1.36 1.24
Resolution 3.70 0.0

Assay of the Developed RP-HPLC Method

Table No. 6: Assay Result of Meclizine hydrochloride.
Sr. No. Concentration(µg/ml) Area of Standard Area of Sample % Assay(w/v)
1 15 734056 733728 99.95%

Table No. 7: Assay Result of Caffeine.

Sr. No. Concentration(µg/ml) Area of Standard Area of Sample % Assay(w/v)
1 12 565152 562338 99.50%

Validation of the Developed Method
A. Linearity
Linearity of an analytical method is its ability to elicit
test results that are directly proportional to the
concentration of analyte in samples within a given range.
analysis of samples with analyte concentrations across
the claimed range. Area is plotted graphically as a
function of analyte concentration. Percentage curve
fittings are calculated.

The linearity of the analytical method is determined by

mathematical treatment of test results obtained by

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Table No. 8: Data of Linearity for Meclizine hydrochloride and Caffeine.

Conc. Of Meclizine
Area Conc. Of Caffeine (μg/ml) Area
HCL (μg/ml)
5 219397 4 172323
10 472788 8 381423
15 734056 12 565152
20 988106 16 741246
25 1285615 20 942472

From the linearity study of Meclizine hydrochloride and coefficient value(R2) were found to be 0.9991 and
Caffeine was found to be linear in the concentration 0.9992 for Meclizine hydrochloride and Caffeine
ranges from 05-25µg/ml and 4-20µg/ml. Correlation respectively.

Figure No. 6: Chromatogram for linearity of Meclizine hydrochloride.

Figure No. 7: Chromatogram for linearity of Caffeine.

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Shinde. European Journal of Pharmaceutical and Medical Research

B. Accuracy (%recovery) The accuracy of an analytical method is determined by

The accuracy of an analytical method is the closeness of applying the method to analyzed samples, to which
test results obtained by that method to the true value. known amounts of analyte have been added. The
Accuracy may often the expressed as percent recovery accuracy is calculated from the test results as the
by the assay of known added amounts of analyte. percentage of analyte recovered by the assay.

Table No. 9: %Recovery of Meclizine hydrochloride.

Amount Amount added Amount % Mean
% composition Area
taken (µg/ml) (µg/ml) recovered (µg/ml) Recovery % recovery
10 5 731163 14.83 98.86
50% 10 5 734056 14.88 99.20 99.24
10 5 737545 14.95 99.66
10 10 984217 19.61 98.05
100% 10 10 989876 19.71 98.59 98.41
10 10 989942 19.72 98.60
10 15 1284624 25.28 101.12
150% 10 15 1263586 24.88 99.55 100.45
10 15 1278976 25.17 100.68

Table No. 10: %Recovery of Caffeine.

% Amount Amount added Amount recovered % Mean
Area
composition taken (µg/ml) (µg/ml) (µg/ml) Recovery % recovery
8 4 560448 11.99 99.98
50% 8 4 561552 12.02 100.16 100.15
8 4 562418 12.03 100.33
8 8 742277 15.82 98.87
100% 8 8 745145 15.88 99.29 99.07
8 8 743789 15.85 99.06
8 12 940787 20.00 100.02
150% 8 12 938960 19.96 99.83 99.92
8 12 939732 19.98 99.91

From the results shown in the accuracy table it was
found that recovery value of pure drugs were between
98.0% to 102%. Mean recovery was found 99.36% for
Meclizine hydrochloride and 99.71% for Caffeine.
C. Precision
The precision of an analytical procedure expresses the
closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple sampling
of the same homogeneous sample under the prescribed
conditions.
Precision of the method was determined by Repeatability
(intraday) and Intermediate Precision (inter-day) studies.
Precision study was carried out by injecting a sample
into HPLC without changing the assay procedure and the
result are shown the %RSD is less than 2% for Meclizine
hydrochloride and Caffeine. The low RSD value indicate
that the method was precise.
i. Repeatability (intraday)
This study was performed with a minimum of three
replicate measurement of sample solution at morning and
evening in a same day.

Table No. 11: Repeatability (intra-day) result of Meclizine hydrochloride.

Conc. Of Meclizine
Morning Evening Mean %RSD
HCL (μg/ml)
734056 734384
15 735204 732975 734350.83 0.27%
737645 731841

Table No. 12: Repeatability (intra-day) result of Caffeine.

Conc. Of Caffeine
Morning Evening Mean %RSD
(μg/ml)
565152 568362
12 563628 564481 565994 0.33%
567433 566909

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The relative standard deviation values for repeatability ii. Intermediate precision (inter-day)
precision was found less than 2%. %RSD of repeatability Intermediate precision was performed by injecting the
was 0.27% for Meclizine hydrochloride and 0.33% for sample solution in to HPLC at two different day.
Caffeine.

Table No. 13: Intermediate (inter-day) result of Meclizine hydrochloride.

Conc. Of Meclizine HCL (μg/ml) Day 1 Day2 Mean %RSD
734056 735252
15 735204 735654 738795 0.24%
737645 738795

Table No. 14: Intermediate (inter-day) result of Caffeine.

Conc. Of Caffeine (μg/ml) Day 1 Day2 Mean %RSD
565152 562415
12 563628 564202 564493 0.30%
567433 564493

The relative standard deviation values for intermediate
precision was found less than 2%. %RSD of intermediate
precision was 0.24% for Meclizine hydrochloride and
0.30% for Caffeine.
D. Robustness
The robustness of an analytical method is determined by
analysis of aliquots from homogenous lots by differing
physical parameters that may differ but are still within
the specified parameters of the assay.
The sample along with standard was injected under
different chromatographic conditions as shown below.
1. Effect of variation of wavelength
A study was conducted to determine the effect of
variation in wavelength at different wavelength like
228nm, 230nm, 226nm. The system suitability
parameters were evaluated and found to be within the
limits.

Table No. 15: Result of change in wavelength for Meclizine hydrochloride.

Conc.
Wavelength (nm) Area Mean SD %RSD
(μg/ml)
5 228 219397
5 230 220720 219760 839.5767 0.38 %
5 226 219163

Table No. 16: Result of change in wavelength for Caffeine.

Conc.
Wavelength (nm) Area Mean SD %RSD
(μg/ml)
4 228 172323
4 230 173204 172911 509.224 0.29 %
4 226 173206

2. Effect of Variation of PH hydrochloride and Caffeine which is pH 3, pH 3.4 and

A study was conducted to determine the effect of pH 2.6. The system suitability parameters were evaluated
variation in PH of Standard solution of Meclizine and found to be within the limits.

Table No. 17: Result of change in pH for Meclizine hydrochloride.

Conc.(μg/ml) PH Area Mean SD %RSD
5 3 219397
5 3.4 217360 218746 1200.74 0.54%
5 2.6 219480

Table No. 18: Result of change in pH for Caffeine.

Conc.(μg/ml) PH Area Mean SD %RSD
4 3 172323
4 3.4 171784 172541 885.79 0.51%
4 2.6 173515

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Shinde. European Journal of Pharmaceutical and Medical Research
The result of robustness were found to be satisfactory
within range. %RSD of change in wavelength was found
0.38% for Meclizine hydrochloride and 0.29% for
Caffeine. %RSD of change in pH was found 0.54% for
Meclizine hydrochloride and 0.51% for Caffeine.
E. Limit of Detection (LOD) and limit of quantitation
(LOQ)
Limit of detection
The lowest conc. of the analyte in the sample that the
method can detect but not necessarily quantify under the
stated experimental conditions simply indicates that the
sample is below or above certain level. Limit test
prescribed as percentage or as parts per million. The
limit of detection will not only depend on the procedure
of analysis but also on type of instrument.
S/N= 2/1 or 3/1
Where,
S= Signal
N=Noise
It may be calculated based on standard deviation (SD) of
the response and slope of the curve(S).
LOD=
Where, SD= Standard deviation
S= Slope
LOD was found to be 0.08 μg/ml for Meclizine
hydrochloride and 0.24μg/ml for Caffeine.
Limit of quantitation
From the linearity data calculate the limit of quantitation,
using the following formula
LOQ =
Where,
SD = standard deviation
S = slope
LOQ was found to be 0.06 μg/ml for Meclizine
hydrochloride and 0.18μg/ml for Caffeine.
CONCLUSION
The developed RP-HPLC method offers several
advantages such as rapidity, usage of simple mobile
phase and easy sample preparation steps. From the
present study, it can be concluded that the proposed
method is simple, specific, sensitive, precise, accurate
and reproducible. Results of validation parameters
demonstrated that the analytical procedure is suitable or
appropriate for its intended purpose. Further, improved
sensitivity makes it and reliable specific for its intended
use. Hence, this method can be applied for the analysis
of pharmaceutical dosage forms and pure drug.
ACKNOWLEDGEMENT
The authors express their gratitude to the Pravara Rural
College of Pharmacy, Loni for providing all the facilities
and Swapnroop drugs and pharmaceuticals, for providing
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me the gift samples of Meclizine hydrochloride and
Caffeine.
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