1. INTRODUCTION:
1.1 Cyproheptadine hydrochloride:
Cyproheptadine hydrochloride(CYP) chemically known as
4-(5H dibenzo[a,d]-cyclohepten-5-ylidene)-1-methyl-piperidine HCl. Cyproheptadine
is an antihistamine used to relieve allergy symptoms such as watery eyes, runny
nose,itching eyes/nose, sneezing , hives, and itching. It works by blocking a
certain natural substance(histamine) that our body makes during an allergic
reaction. This medication also blocks another natural substance in your
body(serotonin).
This medication should not be used in newborn or
premature infants. Cyproheptadine hydrochloride is a serotonin antagonist and
histamine H1 blocker used as antipruitic, appetite stimulant, antiallergic and
for the post-gastrectomy dumping syndrome etc. Cyproheptadine HCl is the
hydrochloride salt of synthetic methyl–piperidine derivative with
antihistaminic and the antiserotoninergic properties.
Cyproheptadine exhibits anticholinergic andsedative properties
and has been shown to stimulate appetite and weight gain.Cyproheptadine
hydrochloride (anhydrous) is the hydrochloride salt of cyproheptadine.[3] There
were many analytical method performed of cyproheptadine hydrochloride such as
HPLC [10],extraction and determination of CPHin human urine by DLLME-HPLC
method [7],quantification of CPH in human plasma by HPLC coupled to
electrospray tandem mass spectrometry in bioequivalence study [17], stability
indicating HPLC method [16], first derivative synchronous spectrofluorimetric
method [15],ion association titration[9], spectroscopic method[2], Isocratic
HPLC method for the Quantitation of cyproheptadine in human milk and plasma
using solvent and solid phase extraction techniques [18],etc., In “Development
and validation of UV-spectrophotometric method for estimation of cyproheptadine
hydrochloride in bulk and tablet formulation by co-solvency approach” water and
methanol used[1] but we developed another UV method in which only distilled
water used as solvent which more economical and safe to use.
1.2 Drug profile-
Fig1: Structure of cyproheptadine hydrochloride
2. MATERIAL AND METHODS:
2.1 Apparatus and Equipments:
A Shimadzu UV-Visible spectrophotometer (UV-1800 Shimadzu
Corporation, Kyoto, Japan) was used for all absorbance measurements with 1 cm
paired quartz cell.
2.2 Reagents and Chemicals:
Pharmaceutical grade cyproheptadine hydrochloride was
supplied as a gift sample from Raptakos, Brettand Co. Ltd, Thane, India.
Distilled water used as solvent.
2.3 Preparation of standard stock solution:(100ug/ml)
An accurately weighed quantity of 10mg cyproheptadine
HCl was transferred to 100 ml volumetric flask dissolved with 50 ml of
distilled water and sonicated for 10 min ,volume was then made up to the mark
with same distilled water.
2.4 Selection of wavelength for analysis:
Prepared appropriate dilution of standard stock
solution and solution containing 10ug/ml of cyproheptadine hydrochloride, these
dilution solutions were scanned in range 200-400 nm.Drug showed λmax at
286nm in (Fig 2).
Fig 2: UV spectra of cyproheptadine hydrochloride(CPH)
2.5 METHOD VALIDATION:
The UV Spectrophotometric method was validated as per
ICH guidelines for method validation. The performance parameters like
linearity, precision, and accuracy were evaluated.[5]
1. Limit of detection (LOD):
LOD is the lowest level of concentration of analyte in
a sample that can be detected, though not necessarily quantitated. It was
calculated by using the formula,[4,5]
LOD=
Where,
=standard deviation of the response
S=slope of calibration curve.
2. Limit of Quantitation(LOQ):
LOQ is the lowest concentration of analyte in a sample
that may be determined with acceptable accuracy and precision when the required
procedure is applied. It was calculated by using the formula,[5]
LOQ=
Where,
standard deviation of the respose
S=slope of calibration curve.
3. Linearity:
Linearity was studied by diluting standard stock
solution of cyproheptadine HCl 2-10 ug/ml concentrations (n=3).Calibration
curve with concentrations verses absorbance were plotted at their respective
wavelengths and the obtained data was subjected to regression analysis using
the least square method. The standard curve of drug showed in (fig 2).[4]
Table1: calibration study data of cyproheptadine
HCl(CPH)
|
Sr.No.
|
Concentration(ug/ml)
|
Absorbance
at 286nm
|
|
1
|
2
|
0.053
|
|
2
|
4
|
0.115
|
|
3
|
6
|
0.184
|
|
4
|
8
|
0.240
|
|
5
|
10
|
0.305
|
Fig 3: Calibration curve of cyproheptadine
hydrochloride (2-10ug/ml)
4. Specificity:
Specificity is ability to measure unequivocally the
desired analyte in the presence of components such as excipient and impurities.
For specificity determination, lactose monohydrate was used as excipient and
from which is prepared stock solution (B) (100ug/ml).Spiking Lactose
monohydrate in 3 different levels 80,100,120 % respectively from the stock
solution (B) and the stock solution (A) which is spike 100% and mix with 3 different
level of lactose monohydrate solution to determine the amount of % recovery at
286nm.[4,5]
5. Accuracy:
To check the accuracy of the developed methods and to
study interference of formulation additives, analytical recovery experiments
were carried out by using standard addition method. Reference standard solution
of each drug was added to tablet samples at three different concentrations
level (80,100,120%). At each level, samples were prepared in triplicate and the
mean percentage recoveries and percentage RSD value were calculated.[4,5]
6. Precision:
Precision is the degree of agreement among individual
test results when the procedure is applied repeatedly to multiple samplings. It
was determined by studying repeatability, intra-day and inter-day precision of
method. The average recovery of the analyte of 80,100,120 % solution of
standard stock solution. For intraday, the analysis was carried out at
different intervals on the same day and for inter day, the analysis was carried
on different days.[4,5]
Table 4: Precision study data for CPH
|
Sample
No.
|
Intra-day
(%Assay)
|
Inter
-day (%Assay)
|
|
1
|
102.9611
|
102.9873
|
|
2
|
103.9735
|
101.4572
|
|
3
|
102.0273
|
100.335
|
|
Mean
|
102.9873
|
101.5932
|
|
SD
|
1.36721
|
1.383102
|
|
%RSD
|
1.335226
|
1.366404
|
Robustness:
Robustness of the proposed method is determined by
analysis of aliquots from homogenous slots by different analysts using similar
operational and environmental conditions. [4,5]
Table 5: Robustness study data for CPH
|
Sample
no.
|
|
Wavelength
(nm)
|
|
|
|
285nm
|
282nm
|
290nm
|
|
1.
|
101.2208
|
98.56688
|
98.03609
|
|
2.
|
102.8132
|
100.1592
|
99.62845
|
|
3.
|
103.8747
|
101.7516
|
101.2208
|
|
Avg.
% Recovery
|
102.6362
|
100.1592
|
99.62845
|
|
SD
|
1.335781
|
1.300154
|
1.300154
|
|
%
RSD
|
1.301471
|
1.298087
|
1.305003
|
3. RESULT AND DISCUSSION:
Development and optimization of UV-spectrophotometric
method:
The UV-spectrophotometric method was developed for
quantitative determination of cyproheptadine hydrochloride. For that selection
of proper wavelength depends upon the nature of the sample and its solubility
parameter. For the proper result different trials with different diluents
concentration were performed and best result obtained which used for
quantitative estimation of drug.
Selection of wavelength by scanning standard stock
solution in UV -spectrophotometric between 200 nm to 400nm on spectrum mode,
using distilled water as a blank. Cyproheptadine hydrochloride shows λmax
at 286nm.
Method validation:
According to ICH guideline validation parameter was
performed. The following result were obtained.
Table 6: Summary of various validation parameters
|
Sr.No.
|
Parameter
|
Result
|
|
1.
|
λmax
|
286nm
|
|
2.
|
Slope
|
0.0314
|
|
3.
|
Intercept
|
0.0093
|
|
4.
|
Linearity and range (ug/ml)
|
2-10 ug/ml
|
|
5.
|
Accuracy (% recovery)
|
91.27513
|
|
6.
|
Precision (% RSD)
Interday precision (n=3)
Intraday precision(n=3)
|
RSD<2%
1.335226
1.366404
|
|
7.
|
Limit Of Detection
Limit Of Quantitation
|
0.013471μg/ml
0.040822μg/ml
|
4. CONCLUSION:
The developed UV methods were found to be more
accurate, precise and reproducible. This validation parameter of these methods
shows good results. The recovery studies revealed excellent accuracy and high
precision of the method. The methods were found to be simple, safe and
economical. These methods could be applied for routine analysis in quality
control laboratories and also it will useful in stability study of drug by
using UV spectrophotometric method.
5. ACKNOWLEDGMENT:
The authors are thankful to the management and
trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for
providing necessary chemicals and analytical facilities and to Raptakos, Brett
and Co. Ltd, Thane, India. gift sample.
6. REFERENCE:
1. Madhu M, Chanti Naik M, Gireesh Kumar E,
Chand Basha S, Gopinath.C Development and validation of spectroscopic method
for the estimation of cyproheptadine HCl in tablet dosage form Journal of
Global Trends in Pharmaceutical Sciences,2016,2982-2986.
2. Wagh Yogesh B., Bhushan Bhairav A., and
Saudagar Ravindra B.”Development and validation of UV-spectrophotometric method
for estimation of cyproheptadine hydrochloride in bulk and tablet formulation
by co-solvency approach”, World Journal of Pharmaceutical Research, Volume 5,
1924-1930.
3. Cyproheptadine hydrochloride, available on
website of WebMD.
4. ICH Harmonized Triplicate Guidelines,
“Validation of analytical procedures: text and methodology, Q2 (R1),” in International
Conference on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use, 2005.
5. International Conference of Harmonization
(ICH) of Technical Requirements for the Registration of Pharmaceuticals for
Human Use, Validation of Analytical Procedures: Methodology, Adopted in Geneva
1996.
6. Indian Pharmacopoeia 2014, volume 2,
Published by the Indian Pharmacopoeia commission Ghaziabad 1st
January 2014, 1489-1491.
7. Mehdi Mahama, Vahid Kiarostamib, Syed
Waqif-Husaina, et al., ”Extraction and determination of cyproheptadine in
human urine by DLLME-HPLC Method.”Iranian Journal of pharmaceutical Research
2013,12:2,311-318.
8. Alaa El-Gindy, Fawzy El-Yazby, Ahmed
Mostafa, Moustafa M. Maher, "HPLC and chemometric methods for the
simultaneous determination of cyproheptadine hydrochloride, multivitamins, and
sorbic acid",Journal of Pharmaceutical and Biomedical Analysis 2004,
35,703–713.
9. Madihalli S. Raghu and Kanakapura Basavaiah,
Application of Ion-Association Titration for the Assay of Cyproheptadine
Hydrochloride in Pharmaceuticals”. International Scholarly Research Network,
(ISRN Analytical Chemistry) Volume 2012, 1-7.
10. Burrows GW, Alliger CL, High-performance
liquid chromatographic determination of cyproheptadine hydrochloride in tablet
formulations", J Pham Sci.1983;72(10):1212-3.
11. Madihalli S. Raghu and Kanakapura Basavaiah,
Sensitive and selective methods for the determination of cyproheptadine in
tablets using N-Bromosucinimide and two dyes", Association of the chemical
Engineering of serbia (AchE), (Chemical industry and chemical Engineering
Quarterly 2012, 18 (3), 449-458.
12. Sayana, T. Veeraiah and Ch. Venkata Ramana
Reddy, Determination of cyproheptadine hydrochloride in pure and pharmaceutical
forms:A Spectrophotometric study. Oriental journal of chemistry, 31,2231-5039.
13. Xesus Feasab, Lei Yeb Development and
validation of LC–MS/MS method for the determination of cyproheptadine in
several pharmaceutical syrup formulations."Journal of Pharmaceutical and
Biomedical Analysis, 2009, 50:5, 1044-1049.
14. Kanakapura Basavaiah and, Vaidyanathan Shakunthala
Charan, Ion-Pair Complexometric determination of cyproheptadine hydrochloride
using Bromophenol Blue. Science Asia 2004, 30,163-170.
15. Fathy M. Salama, Khalid, A.M. Attia, Ragad
A.M. Said, Ahmed EI-Olemy, et al. First derivative synchronous
spectrofluorimetric determination of cyproheptadine hydrochloride in presence
of its oxidative degradation product at critical micelle concentration.” Journal
of Advanced pharmacy Research, 2018, 2:2,104-112.
16. M.K. Sharaf El-Din, F. Belal, M.M. Tolbaand
H. Elmansi, Stability indicating HPLC Method Coupled with Fluorescence
Detection for the Determination of Cyproheptadine Hydro-chloride in Its Tablets
Studies on Degradation Kinetics. Analytical Chemistry Letters, 2018, 8:4,
565-577.
17. Gustavo Duarte Mendesa, Andre Arrudaa, Lu
Shi Chenb, Jose Cassio de Almeida Magalhaesa, Khalid M Alkharfyd and Gilberto
De Nuccia, Quantification of cyproheptadine in human plasma by
high‐performance liquid chromatography coupled to electro spray tandem
mass spectrometry in abioequivalence study.Biomed. Chromatography. 2012; 26: 129–136.
18. John E. Koundourellis, Kenneth O. Ebeteand Eleftheria
T. Malliou, Isocratic high performance liquid chromatographic method for the
quantitation of cyproheptadine in human milk and plasma using solvent and solid
phase extraction techniques. Journal of Liquid Chromatography and Related
Technologies, 22:4, 603-614.