Eurasian Journal of Analytical Chemistry, 2018, 13(5), em44 ISSN:1306-3057 OPEN ACCESS Research Paper https://doi.org/10.29333/ejac/94233

© 2018 by the authors; licensee Modestum Ltd., UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/).

yahyaalbayti@yahoo.com (*Correspondence)

Preparation of Selective Sensors for Cyproheptadine Hydrochloride based on Molecularly Imprinted Polymer used N, N-

Diethylaminoethyl Methacrylate as Functional Monomer Yehya Kamal Al-Bayati 1*

1 University of Baghdad, College of Science, Chemistry Department, Al-Jaderia, Baghdad, IRAQ

Received 24 June 2018 ▪ Revised 21 July 2018 ▪ Accepted 21 July 2018

ABSTRACT The research involved the preparation of four selective sensors for cyproheptadine hydrochloride using molecularly imprinted polymers (MIPs) method. The process of polymerization was used for preparation of MIP using cyproheptadine hydrochloride as the template with N, N-(diethylaminoethyl methacrylate) (NDMAT) as monomer, N, O-bismethacryloyl ethanol amide (NBMEA) as cross linker, and 2, 2-azobisisobutyronitrile (AIBN) as initiator. Preparation of membrane as sensors for electrode construction based on different plasticizers, di-isodecyl adipate (DIA), di-isobutylmaleate (DIBM), 2-nitrophenyldodecylether (NPDE) and 3-trimethyltrimellitata (TMTM). The selective sensors were tested and their susceptibility to the estimation of the cyproheptadine hydrochloride, the selectivity coefficients of inorganic ions, sugars, amino acids, and drugs were also studied. The experimental results showed that the best electrode was based on DIA and TMTM as plasticizers, displaying a linear range from1×10-1-3×10-4M and 1×10-1-2×10-4M with a Nernstian slope of 58.4 mV/decade and 55.8 mV/decade, correlation coefficient of 0.9998 and 0.9993. The detection limit was 3.3×10-5 M and 2.4×10-5, the lifetime was around 30 and 48 days respectively. The proposed electrodes were applied successfully to the determination of cyproheptagine in a pharmaceutical preparation.

Keywords: cyproheptadine hydrochloride determination, molecularly imprinted polymers, N, N-diethylaminoethyl methacrylate monomer, electrodes specifications

INTRODUCTION Molecular imprinting technique has been used successfully and widely to prepare polymers offering high affinity binding sites for a variety of molecules; including organic, inorganic, and even biological molecule or Ions [1]. These materials are useful for various applications; such as biomaterials, sensor technologies, molecular and ionic separation, and catalysis. MIPs are recognition materials that have been used in sensors which possess attributes of both of the above class [2]. MIPs are synthetic polymers that can be prepared inexpensively and readily often from commercially available starting materials. The recognition properties of MIPs can also be tailored using a molecular templation process as shown in Scheme 1.

Cyproheptadine hydrochloride (CYPH) has been using a treatment of cases as sedative a histamine antagonist with anticholinergic. CYPH was also used as an appetite stimulant to assist weight gain. Chemically known as 4 (5H-Dibenzo [a, d] cyclohepten-5-ylidene) -1-methylpiperidine hydrochloride Figure 1 [18,22] and United States Pharmacopeia [23]. Crystal violet as an indicator for indicating the endpoint [15]. Different methods were used for the application of estimation of CYPH in pharmaceutical formulations. (LC-MS) [20,25] and (HPLC) [9,11,13,16,17,19,24,26]. Recently, applicate visible spectrophotometric methods [3,7,8,10,12,14,21] and its derivatives for the determinate CPH in a two-component system was used for cyproheptadine determination.

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Several papers were published for drug determination by MIP such as ibuprofen [4,5,6]. In this work MIP of cyproheptadine was prepared based on N, N-(diethylaminoethyl methacrylate) monomer and different types of plasticizers. The best electrodes were used for determination of cyproheptadine in pharmaceutical samples.

EXPERIMENTAL

Chemicals and Apparatus Two models of pH meters were used (WTW model, Germany) and (WTW model pH 720, Germany). Reference

electrode saturated calomel electrode (Gallenkamp, USA) and silver/silver chloride as working electrode, with highest purity for using all chemical reagents: poly (vinyl) chloride (PVC) high molecular weight, di-isodecyl adipate (DIA) (98%), di-isobutylmaleate (DIBM) (99%), 2- nitrophenyldodecylether (NPDE) (99%),and 3-trimethyltrimellitata(TMTM)(98%),N,N-(diethylaminoethylmethacrylate) (NDMAT) (99%), as monomer, used N,O-bismethacryloyl ethanol amide (NBMEA) (99%), 2,2-azobisisobutyronitrile (AIBN) (98%), methanol(MeOH) and tetrahydrofurane (THF) were purchased from Fluka and Sigma Aldrich. The performance of synthesis electrodes based on measuring their potential in the concentration range 10-6- 10-1 M of cyproheptadine hydrochloride. Stock solution 10-2 M cyproheptadine hydrochloride (CYPH) was prepared by dissolving 0.1619 g in 50 mL methanol and a series of solutions were prepared gradual dilution of the stock solution.

Scheme 1. Schematic representation of the principle of molecular imprinting technology

Figure 1. Structure of cyproheptadine hydrochloride

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Synthesis of Imprinted Polymer (CYPH-MIP) The polymerization process was done in 50 mL thick-walled glass tube with screw cap by dissolving 3 mmol

(g) CYPH with methanol (18 mL). A functional monomer N, N-(diethylaminoethyl methacrylate) (NDMAT) 9 mmol (g), cross-linker N, O-bismethacryloyl ethanolamide (NBMEA) 27 mmol and correspondingly initiator 2, 2-azobisisobutyronitrile (AIBN) 0.32 mmol (0.0775g) were then added to the tube to be mixed with the solution. The mixture dipped in water bath with a shaker for a period of 30 minutes, and then nitrogen passes through the mixture to remove the oxygen from the mixture solution. After complete removing oxygen from the mixture, the solution put in 45°C water bath to permit starting the polymerization reaction which continued 24 hr.

After the polymer matrix has been formed the particles of CYPH should be removed from the matrix using soxhlet extraction with MeOH/ acetonitrile (90/10, v/v) until the CYPH particles removed completely. To remove residual acetonitrile it must be washed with pure methanol and dried at 55 ◦C. The resulting bulk rigid polymer was crushed and sieved which obtained particles have size 125 μm used for synthesis the sensing membrane of the electrodes.

Preparation and Measurements of Membrane Electrode In order to prepare a membrane capable of sensing drug, calculated quantities were mixing; 0.34 g PVC, 0.72 g

of the plasticizer (DIA, DIBM, NPDE, and TMTM) and 0.08 g of the MIP. After mixing 7-8 mL of tetrahydrofuran (THF) was added as solvent Follow good stirring in order to obtain homogenization solution. The mixture was poured in a petri dish of 10 cm2 in diameter. To evaporate the THF, a mixture was left at room temperature for 24 hours. A certain quantities of MIP were used to prepare flexible membranes. The sensor membranes must be soaking in 10-1 M of CYPH for electrode conditioning. Electrode potential was measured after an equilibrium was achieved in a steady state.

Pharmaceutical Samples Ten grains were taken and weighed thoroughly after they were well grinded by using the mortar and then

taking the equivalent of 50 mg of the powder into 100 mL volumetric flask and diluted to 50 mL with the same solvent using magnetic stirrers for 15 min. The solution was filtered by whattmann filter paper, and transferred into a 100 mL volumetric flask to get concentration of 1.0 x 10-3 M CYPH.

RESULTS AND DISCUSSION

Scanning Electron Microscopy (SEM) To know the shapes and sizes of MIP particles a scanning electron microscopy (SEM) was used. The morphology

of MIP and NIP membranes for cyproheptadine hydrochloride before and after washing is showed by electron microscope in Figure 2.

(a) (b) Figure 2. SEM photograph of the surface of MIP, a) after washing b) before washing

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Figure 2a showed a conglomerate consistent of particles on the surface about 5 μm may indicate that the polymer bond is consistent. Figure 2b showed clear holes about 10 μm after removed the particles from template using soxhlet extraction. Micro analysis shows very small particles and spherically shaped polymeric particles with small sizes around (2.50-4.16) µm.

Measurements and Analysis To ascertain the formation of the template, a spectral identification must be performed, Fourier-transform

infrared spectroscopy (FTIR) spectra of CYPH-MIP before template removal and after template removal were recorded in the range of 400–4000 cm-1 by the KBr disk method as shown in Figures 3, 4 and 5.

Figure 3. FTIR of (CYPH) drug

Figure 4. FTIR of CYPH-MIP(NDMAT) before the removal (CYPH)

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From the FTIR spectrum of CYPH-MIP(NDMAT) before template removal showed five variation absorption bands at 3010,2923 cm-1 (C-H) aromatic,1299 cm-1 (C-N) stretching,747 and 711 cm-1 for out of plane-mono-sub when comparing with the FTIR spectrum of CYPH-MIP (NDMAT) after template removal show disappearance the four bands which proved that CYPH was removed from template, shown in Table 1.

Different plasticizers di-isodecyl adipate (DIA), di-isobutylmaleate (DIBM), 2-nitrophenyldodecylether (NPDE) and 3-trimethyltrimellitata (TMTM) used for preparation four electrodes of different compositions based upon the different viscosities. The results of electrode specification were obtained from the calibration curves are shown in Figure 6 and the characteristics listed in Table 2.

Figure 5. FTIR of CYPH-MIP(NDMAT) after the removal (CYPH)

Table 1. The identified peaks of FT-IR spectra for CYPH-imprinted polymer using N, N-(diethylaminoethyl methacrylate) (NDMAT) as a functional monomer

No. Functional Group CYPH CYPH-MIP (NDMAT) before template removal

CYPH-MIP (NDMAT) after template removal

1 C-H aromatic. (cm-1) 2925 3010,2923 ---- 2 C-Nstr.ester.(cm-1) 1265 1299 ---- 3 Out of plane-mono-sub 754,651 747,711 -----

Figure 6. Potentiometric calibration of CYPH-MIP selective sensors

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The results from Table 2 show best characteristics appeared for the sensor with plasticizers DIA and DIBM which have a near-Nernstian with slopes of 58.4 and 55.8 mV/decade and a detection limit of 3.3x10-5 and 2.4x10-5 M, respectively.

Effect of pH These electrodes with different concentrations 1.0 × 10-2, 1.0×10-3 and 1.0 × 10-4 M CYPH solutions. In this work

used (1.0 M HCl and 0.1 M NaOH) for control to the acidic and basic medium by adding small volumes and the potential measured. From the figure Note that the range of the pH of the different concentrations is allowed 3.1-9.7 this is an indication of the work of electrodes in a wide range of medium.

Response Time and Reversibility of the Electrode Electrode response time is an important feature that must be studied in order to reach the time required for ion

exchange, and then the potential is measured taking into consideration the difference in concentration. The CYPH concentrations range 1.0 × 10-5 to 1.0 × 10-4 M with about 10 - 30 seconds as shown in Figure 8. In this work, it was observed that the response time decreases in the case of high concentrations and increases in the case of low concentrations because access to the equilibrium state in high concentration shorter than the low solutions this proves the response time dependent upon the concentration of CYPH.

Table 2. The characteristics of CYPH-MIP ISE selective electrode using (NDMAT) as a functional monomers and different plasticizers Electrode no. I II III IV

Type of Electrode CYPH- MIP(NDMAT)+ EBMAA + DIA

CYPH- MIP(NDMAT) + EBMAA + DIBM

CYPH- MIP(NDMAT)+ EBMAA+ NPDE

CYPH- MIP(NDMAT)+ EBMAA+TMTM

*Slope mV/decade 58.4 55.8 49.4 52.3

Limit of detection (M) 3.3×10-5 2.4×10-5 3.1×10-5 3.5×10-5 Correlation coefficient 0.9998 0.9993 0.9874 0.9977

linearity (M) 1×10-1-3×10-4 1×10-1-2×10-4 1×10-1-5×10-4 1×10-1-6×10-4 Life time(day) ~30 ~48 ~18 ~21

Figure 7. pH effect on CYPH electrodes at different concentrations (●10-4, ▲10-3 and ■ 10-2) M

0

50

100

150

200

250

300

350

0 2 4 6 8 10 12

Res

pons

e m

V

PH

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Interfering Ions Affection Two different methods were applied to determine the selectivity coefficient, separate solution method (SSM),

which based on Nickolsky-Eisenman equation and matched potential method (MPM) when the involved ions have unequal charges. From the results in Table 3, show the preparation electrodes were selective to CHPY comparing with interfering ions used as inorganic ions, sugars, amino acids, and drugs. The calculated values of selectivity coefficient appeared no interfering obtained between and CYPH and interfering ions.

Sample Analyses The electrodes prepared were applied to pharmaceuticals but in the first the synthetic solutions were tested

used four potentiometric techniques included direct (DM), standard addition (SAM), multiple standard solution

Figure 8. Response time for step changes in concentration of cyproheptadine hydrochloride

Table 3. Selectivity coefficients of various interfering ions for (CPH- MIP(HEMA)+EBMAA + DOA) electrode Interfering ion SSM MPM

Li+ 9.71×10-4 6.75 ×10-6 Na+ 6.92×10-4 6.84×10-4 K+ 4.36×10-4 7.21×10-5

Cd2+ 6.11×10-4 6.35×10-6 Mg2+ .71×10-82 6.81×10-7 CO2+ 3.73×10-3 9.79×10-8 Al3+ 5.83×10-5 6.88×10-8 Ce3+ 3.55×10-6 7.71×10-8 Fe3+ 6.82×10-6 3.95×10-2

D-Froctose 7.88×10-3 5.79×10-5 D-Galactose 4.33×10-3 6.42×10-8

Maltose 6.87×10-3 7.72×10-6 Sucrose 7.84×10-3 7.99×10-5 Gluctose 1.86×10-4 6.12×10-6 Captopril 4.98×10-3 6.31×10-3

Spiramycine 3.88×10-2 5.97×10-1 Ephidrine 7.34×10-5 6.67×10-1 Lidocaine 5.76×10-4 2.06×10-6

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(MSAM), and titration (TM) methods, respectively. Different concentrations (10−3 and 10−4M) were test and the recovery percentage (%RC), relative standard deviation (%RSD), and relative error (%RE) were calculated and are listed in Table 4.

To remove the effect of interference using the method of standard additions is the best method. The method includes an addition of five times different volumes of standard CYPH to the synthetic solution and the measurements were repeated several times, these methods appear low relative error (%RE) when used to determine the concentration of CYPH.

Classic analysis to determination CYPH was done by used potentiometric titration Technique by used a 10-4 M N-bromo succinamide (NBS) as a titrant. A typical titration plot was shown in Figure 10.

From the results in Tables 2 and 4 shows the synthesis electrodes I and II were proved to be useful in the potentiometric determination of CYPH in pharmaceutical preparations using different analysis methods.

Table 4. Determination of cyproheptadine hydrochloride ion standard solutions by different potentiometric technique

Electrode No. Concentrations (M)

Sample Measurements using different methods

DM SAM MSAM TM

CYPH- MIP(NDMAT)+

EBMAA + DIA (I)

1×10-3 0.997×10-3 0.977×10-3 0.999×10-3 0.96×10-3 RSD% 2.623* 1.399* - 3.925* RC% 102 97.7 99.9 96 RE% 2 -2.3 -0.1 -4

1×10-4 0.96×10-4 0.978×10-4 1.019×10-4 0.94×10-4 RSD% 0.979* 0.872* --- 3.885* RC% 96 97.8 101.9 94 RE% -4 -2.2 1.9 -6

CYPH- MIP(NDMAT) +

EBMAA + DIBM (II)

1×10-3 0.998×10-3 0.992×10-3 0.999×10-3 0.976×10-3 RSD% 2.856* 1.328* - 3.477* RC% 99.8 99.2 99.9 97.6 RE% -0.2 -0.8 -0.1 -2.4

1×10-4 1.019×10-4 0.993×10-4 1.007×10-4 1.07×10-4 RSD% 3.284* 2.692* - 2.195* RC% 101.9 99.3 100.7 107 RE% 1.9 -0.7 0.7 7

* Each measurement was repeated three times

Figure 9. Calibration curve of estimation of cyproheptadine hydrochloride solution( 10-4M) by MSAM using (CYPH- MIP(NDMAT)+EBMAA + DIA ,DIBM) electrodes

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Figure 10. Titration curve of 10-4M CYPH used using (CYPH- MIP(NDMAT)+EBMAA + DIA ,DIBM) electrodes with 10-3 M of NBS as titrant solution

Table 5. Analysis of pharmaceutical cyproheptadine hydrochloride samples using (CYPH- MIP(NDMAT)+EBMAA + DIA) electrode

Pharmaceutical Germany DM SAM MSA TM (NBS)

Concentration Prepared 1×10-3 1×10-3 1×10-3 1×10-3

Found 1.041×10-3 1.031×10-3 1.010×10-3 0.930×10-3 RC% 104.1 103.1 101.0 93.0 RE % 4.1 3.1 1.0 -3.0

*RSD% 1.275* 0.847* - 2.573* F experimental 13.812 10.229 - 14.619 F theoretical 19.0

Pharmaceutical Turkey

DM SAM MSA TM (NBS) Concentration Prepared 1×10-3 1×10-3 1×10-3 1×10-3

Found 1.041×10-3 1.028×10-3 1.020×10-3 0.950×10-3 RC % 104.1 102.8 102.0 95.0 RE % 4.1 2.8 2.0 -5

*RSD% 3.639* 1.226* - 3.454* F experimental 12.648 10.847 - 11.252 F theoretical 19.0

* Each measurement was repeated three times

Table 6. Analysis of pharmaceutical cyproheptadine hydrochloride samples using CYPH- MIP(NDMAT) +EBMAA + DIBM electrode

Pharmaceutical Germany

DM SAM MSAM TM (NBS) Concentration Prepared 1×10-3 1×10-3 1×10-3 1×10-3

Found 1.061×10-3 0.984×10-3 0.988×10-3 0.55×10-3 RC % 106.1 98.4 98.8 95.5 RE % 6.1 -1.26 -1.2 -4.5

*RSD% 1.735* 0.639* - 3.291* F experimental 12.836 8.349 - 13.533 F theoretical 19.0

Pharmaceutical Turkey DM SAM MSAM TM (NBS)

Concentration Prepared 1×10-3 1×10-3 1×10-3 1×10-3

Found 1.044×10-3 1.036×10-3 1.028×10-3 0.95×10-3 RC % 104.4 103.6 102.8 95.0 RE % 4.4 3.6 2.8 -5.0

*RSD% 1.762* 0.348* - 3.464* F experimental 14.163 11.742 - 14.439 F theoretical 19.0

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CONCLUSION The preparation of sensors for electrodes based on MIP- cyproheptadine using N,N-(diethylaminoethyl

methacrylate) as monomer with different plasticizers gave excellent results for determination of the drugs in pharmaceutical samples. Good results were obtained showed high sensitivity, reasonable selectivity, fast static response, long-term stability and low detection limit.

REFERENCES 1. Vendamme R, Eevers W, Kaneto M, Minamizaki Y. Influence of Polymer Morphology on the Capacity of

Molecularly Imprinted Resins to Release or to Retain their Template. Polymer Journal. 2009;41: 1055–66. https://doi.org/10.1295/polymj.PJ2009098

2. Lingxin C, Shoufang X, Jinhua L. Recent advances in molecular imprinting technology: Current status, challenges and highlighted applications .Chemical Society Reviews. 2011;40(5):2922-42. https://doi.org/10.1039/c0cs00084a

3. Adamski S. Extractive spectrophotometric determination of cyproheptadine hydrochloride using bromocresol green. Acta Pol Pharm. 1965;22:311-4.

4. Al-Bayati YK, Aljabari FI. Synthesis of Ibuprofen-Molecularly Imprinted Polymers Used as Sensors to Determine Drug in Pharmaceutical Preparations. Asian J. of Chemistry. 2016;28(6):1376-80. https://doi.org/10.14233/ajchem.2016.19715

5. Al-Bayati YK, Al-Saidi KH, Hussain MA. Liquid Selective Electrodes for Warfarin Sodium Based on Poly (vinyl chloride) Matrix Membrane. Asian J. of Chemistry. 2016;28(9):1962-6. https://doi.org/10.14233/ajchem.2016.19852

6. Al-Bayati YK, Al Khafaji IH. Potentiometric Determination of Mebeverine Hydrochloride Using Imprinted Molecular Polymer in PVC Matrix Membrane. Iraqi Journal of Science. 2016;57(4):2790-9.

7. Basavaiah K, Charan VS. The use of chloranilic acid for the spectrophotometric determination of three antihistamines. Turk J Chem. 2002;26(5):653–61.

8. Basavaiah K, Charan VS. Ion-pair complexometric determination of cyproheptadine hydrochloride using bromophenol blue. Sci Asia. 2004;30:163–70. https://doi.org/10.2306/scienceasia1513-1874.2004.30.163

9. Basavaiah K, Charan VS, Chandrashekar U, Nagegowda P, Somashekar BC. Isocratic high-performance liquid chromatographic determination of cyproheptadine hydrochloride in tablet. Bulgarian. Chem Comm. 2004;36(2):112–6.

10. Basavaiah K. Application of bromate-bromide mixture and methyl orange in the titrimetric, spectrophotometric and kinetic assay methods for cyproheptadine in pharmaceuticals. Indian J Chem Technol. 2006;13(4):360–6.

11. Burrows GW, Alliger CL. High-performance liquid chromatographic determination of cyproheptadine hydrochloride in tablet formulations. J Pharm Sci. 1983;72(10):1212–3. https://doi.org/10.1002/jps.2600721026

12. Carducci CN, Barcic G, Mascaro A. Spectrophotometric determination of antihistamines with benzyl orange. Application to Drugs. SAFYBI. 1979;19:1358–61.

13. El-Gindy A, El-Yazby F, Mostafa A, Maher MM. HPLC and chemometric methods for the simultaneous determination of cyproheptadine hydrochloride, multivitamins, and sorbic acid. J Pharm Biomed Anal. 2004;35(4):703–13. https://doi.org/10.1016/j.jpba.2004.02.027

14. Emmanuel J, Yegnanarayan TV. Colorimetric estimation of cyproheptadine hydrochloride. Indian Drugs. 1982;19(12):505–7.

15. Feas X, Ye L, Hosseini SV, Fente CA, Cepeda A. Development and validation of LC-MS/MS method for the determination of cyproheptadine in several pharmaceutical syrup formulations. J Pharm Biomed Anal. 2009;50(5):1044-9. https://doi.org/10.1016/j.jpba.2009.06.006

16. Lingya Z. Determination of cyproheptadine hydrochloride tablets by HPLC. China Pharmacis 09. 2009. 17. Mao AD, Wang BF. Determination of cyproheptadine hydrochloride in tablets by reversed-phaseHPLC.

Yaowu Fenxi Zazhi. 2001;21(1):60–6. 18. Moffat AC. Clarke's analysis of drugs and poisons. Pharmaceutical Press, London, Electronic version.

2006. 19. Rao GR, Raghuveer S. High-pressure liquid-chromatographic determination of cyproheptadine

hydrochloride in dosage forms. Indian Drugs. 1985;22(7):377–80. 20. Sane RT, Karkhanis PP, Anaokar PG. Gas chromatographic estimation of cyproheptadine in

pharmaceuticals. Indian J Pharm Sci. 1981;43(3):111–2. 21. Sane RT, Vaidya UM, Nayak VG, et al. Use of three dyes for the spectrophotometric determination of

cyproheptadine hydrochloride. Indian Drugs. 1982;19(12):398–403.

Eurasian J Anal Chem

11 / 11

22. Sweetman SC. Martindale: the complete drug reference. Pharmaceutical Press, London, Electronic version. 2007.

23. The United States Pharmacopoeia 30, the National Formulary 25. US Pharmacopeial Convention; Electronic version. 2007.

24. Williams PA. Compendial monograph evaluation and development: cyproheptadine hydrochloride. Report of findings and recommendations on the monographs for cyproheptadine hydrochloride, cyproheptadine hydrochloride syrup, and cyproheptadine hydrochloride tablets. Pharmacopeial Forum. 1988;14(1): 3463–72.

25. Yang C, Men Q. Determination of the content of cyperheptadine hydrochloride tablets by gas chromatography. Yaowu Fenxi Zazhi. 1991;11(2):113–8.

26. Ying C, Yin J, Wengang Z. Determination for cyproheptadine in feedstuff by HPLC. China Feed 02. 2011.

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