STABILITY INDICATING GREEN ANALYTICAL GAS CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF DIPHENHYDRAMINE IN PURE AND MARKETED FORMULATIONS

STABILITY INDICATING GREEN ANALYTICAL GAS CHROMATOGRAPHY METHOD FOR THE DETERMINATION OF DIPHENHYDRAMINE IN PURE AND MARKETED FORMULATIONS

T. S. Vishwas, B. M. Gurupadayya ^* and Rupshee Jain

Department of Pharmaceutical Chemistry, JSS College of Pharmacy, JSS Academy of Higher Education & Research, Mysuru - 570015, Karnataka, India.

ABSTRACT: The current work defines development of a simple, rapid and precise method based on gas chromatography coupled with Flame Ionization Detector (GC-FID) for the quantitative estimation of diphenhydramine (DPH) present in the marketed formulations following the principles of green analytical chemistry. The analysis was performed using Zebron Phase: ZB-DRUG-1 column with Nitrogen as a carrier gas having a flow rate of 1 ml/min. The injection volume was 1 µL and with the split ratio of 1:10. The retention time of the drug was found to be 4.5 minutes. The developed method was validated according to the ICH guidelines, proving the method to be sensitive towards the analyte with accuracy (mean recovery = 98%), linearity in the range of 2-10 µg/ml (r² 0.993), precision (intraday = 1.118% RSD, interday = 1.16 % RSD), LOD 0.2 µg/ml and LOQ 0.5 µg/ml. The degradation study showed that the drug remains stable in acid, base, neutral, oxidation, and photolytic method. The developed method can be effectively employed for the day to day analysis of DPH in pure and marketed formulations by industries and not so well equipped laboratories, as the method employs direct analysis of DPH without any derivatization technique by gas chromatography with simple flame ionization detection.

Diphenhydramine, Degradation study, Flame ionization, Gas chromatography, Green analytical chemistry, ICH Guidelines

INTRODUCTION: Diphenhydramine (DPH) Fig. 1 is an antihistamine which comes under the class of the first-generation antihistamine. It is a derivative of ethanolamine with sedative and anti- allergic properties and acts by binding and antagonizing histamine H1 receptor ¹. Diphenhydramine competitively inhibits the histamine-1 (H1) receptor, thereby alleviating the symptoms caused by endogenous histamine on bronchial, capillary and gastrointestinal smooth muscles, mechanism of the CNS depressant effects of diphenhydramine is unknown ².

The literature survey disclosed, titrimetric methods for the estimation of DPH has been described in European Pharmacopoeia ³ and British Pharmacopoeia ⁴ where has HPLC has been described in United States Pharmacopoeia ⁵. Several unofficial analytical techniques such as fluorometry ⁵, spectrophotometry ^{7, 8, 9, 10, 11, 12} and electrochemical analysis ^{13, 14} has also been reported for the estimation of DPH in pharmaceutical preparations. Atomic absorption spectroscopy ^{15, 16} and capillary electrophoresis ^{17, 18, 19} was also used previously. Some of the chromatographic techniques such as HPLC ^20,21 LC-MS ^{22, 23}, HPTLC ^{24, 25} and GC ^{22, 26, 27}. Comparison of other methods available for the estimation of DPH in references with the present developed method is as depicted in Table 1.

This paper is directed towards the development of a method for estimation of DPH present in the formulation and degradation study of DPH following the principles of green analytical chemistry ²⁸. The method involves the direct analysis of DPH without any derivatization and use of no harmful chemicals which poses any harm to analytical chemists; it also minimizes the generation of analytical wastes during the process. The method was validated according to ICH guidelines ²⁹.

FIG. 1: DIPHENHYDRAMINE (DPH)

TABLE 1: COMPARISON OF OTHER METHOD AVAILABLE FOR ESTIMATION OF DPH WITH PRESENT METHOD

MATERIALS AND METHODS:

Chemicals and Reagents: Diphenhydramine pure drug (API) was procured from TCI Chemicals (India) Pvt. Ltd., Tamil Nadu, India. HPLC analytical grade methanol was procured from specialties, private limited, Mumbai, India. The formulations were procured from the local pharmacy store.

GC (Analytical) Instrumentation and Chromatographic Conditions: Shimadzu GC-2014 Coupled with flame ionization detector (FID) was used for method development, validation, and degradation study of DPH. GC solution software was used to control the instrument. A 10 µL syringe made by Hamilton CO., Reno, Nevada, USA was used for the injection of samples.

Zebron phase: ZB-DRUG-1 (dimethylpolysiloxane backbone with a special phenyl) column procured from Phenomenex private Ltd., Hyderabad, India, with an internal diameter 0.25 mm with a length of 30 meters was used for the analysis. The chromatographic conditions were as depicted in Table 2.

TABLE 2: CHROMATOGRAPHIC CONDITIONS

Preparation of Solutions:

Preparation of DPH Standard Solutions: A Stock solution containing 100 µg/ml of DPH was prepared by using methanol as a diluent. Working standards having a concentration of 2, 4, 6, 8, 10 µg/ml was prepared by pipetting out 0.2, 0.4, 0.6, 0.8, 1 ml of stock to 5 different volumetric flasks and the volume was made up with methanol. All the solutions were sonicated for 5 min to remove the entrapped air.

Preparation of Sample Solution (Formulations):

Syrup: Syrup solution having a concentration of 5.63 µg/ml was prepared by making necessary dilutions using methanol, and it was sonicated and filtered through syringe filter having a pore size of 0.20 µm.

Tablet: Powdered tablet equivalent to 25 mg of DPH was dissolved in methanol, and further dilutions were made to get a final concentration of 5 µg/ml. The solution was sonicated and filtered through a syringe filter, having a pore size of 0.20 µm.

Method Validation: The method was validated according to ICH guidelines for the following parameters: Linearity, accuracy, precision, the limit of detection (LOD) and limit of quantification (LOQ), and system suitability.

Preparation of Solution for Degradation Studies: 5 ml of the standard DPH solution was transferred into five, 25 ml volumetric flasks and treated with 5 ml of 1M Hydrochloric acid, 1M Sodium hydroxide, 30% Hydrogen peroxide, Water and UV rays respectively for 30 min.

The acid and alkali samples were neutralized. 5 ml of water was added to a UV stability sample and kept in a UV chamber for 4 h. The degradation studies for DPH areas depicted in Table 3.

TABLE 3: PREPARATION OF SAMPLE FOR DEGRADATION STUDIES

RESULTS AND DISCUSSION:

System Suitability: After establishing the method conditions as described, the method was validated as per ICH guidelines for accuracy, precision, linearity, the limit of detection (LOD), the limit of quantitation (LOQ). Precision of the method was established by injecting the middle concentration 6 times intraday and inter-day. Accuracy, in terms of percentage recovery, was established by spiking the formulation at 80%, 100% & 120% (n=3). Linearity was established by injecting a series of dilutions in increasing concentrations (n=5). LOD & LOQ was established using standard deviation and slope. The selectivity of the method was assessed by comparing the retention time of standard and formulation; Quantitative analysis of DPH was performed under the established conditions. GC validation report of DPH is as depicted in Table 4. Blank, overlay, and standard chromatograms of DPH are shown in Fig. 3, 4 & 5, respectively.

TABLE 4: GC VALIDATION REPORT OF DPH

Linearity: The linearity of DPH was established by injecting 5 different concentrations, in the range of 2-10 µg/ml. The calibration curve was constructed by concentration vs. peak area. The regression equation (r² = 0.993) demonstrated good linearity of the method. The linearity of DPH is as depicted in Table 5 and shown in Fig. 2

TABLE 5: LINEARITY OF DPH

FIG. 2: CALIBRATION CURVE OF DPH

Precision: The precision was established by injecting the 6 replicates of middle concentration on the same day (intra-day) and the consecutive day (inter-day). The % RSD values were found to be less than 2% as depicted in Table 6.

TABLE 6: PRECISION STUDY OF DPH

Accuracy: The accuracy, in terms of percentage recovery was established by spiking the formulation at 80%, 100% & 120% and injecting each spiked sample 3 times (n=3) finding out the amount of the drug recovered. The mean percentage recovery was found to be (mean) 98.64% as depicted in Table 7.

Limit of Detection and Limit of Quantitation: LOD and LOQ was calculated with the aid of standard deviation (σ) and slope (s) by using the formula LOD = 3.3 σ/s LOQ = 10 σ/s, LOD and LOQ of DPH was found to be 0.2 µg/ml and 0.5 µg/ml respectively as depicted in Table 4.

TABLE 7: ACCURACY STUDY OF DPH

Analysis of Sample:

Syrup: According to the label claim of the formulation every 5 ml of syrup contains 14.08 mg of DPH. 5 ml of syrup was taken in a 25 ml volumetric flask, and the volume was made up to 25 ml with methanol. Then 0.1 ml of the diluted solution was taken in a 10 ml volumetric flask and diluted to 10 ml with methanol to get a final concentration 0f 5.63 µg/ml. The % of label claimed drug found by GC-FID method was found to be 98.43%, which has been depicted in Table 11. The chromatogram of syrup sample is shown in Fig. 6

Tablet: According to the label claim of the formulation each tablet contains 25 mg of DPH. Weight of 10 tablets was noted, and then the tablets were crushed using mortar and pestle. Quantity of the tablet equivalent to 25 mg of DPH was transferred to a 25 ml volumetric flask, and the volume was made up with methanol. Necessary dilutions were made to get a final concentration of 5 µg/ml. The % of label claimed drug found by GC-FID method was found to be 98.08%, which has been depicted in Table 8. The chromatogram of tablet sample is shown in Fig. 7

Degradation Studies: After subjecting the drug solution for respective degradation environment as depicted in Table 2, the drug solution is neutralized, filtered through a syringe filter, and injected to GC. The percentage degradation of DPH in acid, alkali, neutral, oxidation, and photolytic environment was found to be less than 11% as depicted in Table 9.

TABLE 8: ANALYSIS OF FORMULATION

TABLE 9: RESULTS OF DPH DEGRADATION STUDIES

CONCLUSION: A simple, fast, accurate, sensitive, cost-effective and environmentally friendly method has been developed for the estimation of DPH in pure form and formulations along with the stability study of DPH following the principles of green analytical chemistry. This method can be used by industries and laboratories which are not very well equipped for the routine estimation of DPH present in formulations as the method employs direct analysis of DPH without any derivatization by gas chromatography with simple flame ionization detection.

ACKNOWLEDGEMENT: The authors are thankful to the Principal, JSS College of Pharmacy, Mysuru for providing necessary facilities to carry out the research work.

CONFLICT OF INTEREST: Nil

REFERENCES:

1. Goodman LS: Goodman and Gilman's the pharmacological basis of therapeutics. New York: McGraw-Hill 1996.
2. Thomas Kraemer, Hans H. Maurer: Handbook of Analytical Separations, First Edition, Vol. II, 2008.
3. European Pharmacopoeia, 5^th Main Volume, European Pharmacopoeia Commission, European Directorate for the Quality of Medicines & Healthcare: Maisonneuve, France, 2005.
4. British Pharmacopoeia, Her Majesty’s Stationary Office, London, 1988.
5. The United States Pharmacopoeia, United States Pharmacopoeial Convention, Rockville, MD 2002.
6. Wirz DR, Wilson DL and Schenk GH: Fluorescence and phosphorescence of antihistamines having the diphenylmethane chromophore. Effect of halide ions on luminescence. Analytical Chemistry 1974; 46: 896-900.
7. Tipparat P, Lapanantnoppakhun S, Jakmunee J and Grudpan K: Determination of diphenhydramine hydrochloride in some single tertiary alkylamine pharmaceutical preparations by flow injection spectrophotometry. Journal of Pharmaceutical & Biomedical Analysis 2002; 30: 105-12.
8. El-Didamony AM and Moustafa MA: Spectrophotometric determination of diphenhydramine hydrochloride in pharmaceutical preparations and biological fluids via ion-pair formation. Arabian Journal of Chemistry 2010; 3: 265-70.
9. Woo D, Yen JK and Heimlich KR: Determination of diphenhydramine hydrochloride in elixir. Journal of Pharmaceutical Sciences 1973; 62: 1993-4.
10. De Fabrizio F: Spectrophotometric determination of diphenhydramine hydrochloride in an antiallergic cream. Journal of Pharmaceutical Sciences 1970; 59: 1470-1.
11. Davidson AG and Hassan SM: Assay of benzenoid drugs in tablet and capsule formulations by second derivative ultraviolet spectrophotometry. Journal of Pharmaceutical Sciences 1984; 73: 413-6.
12. Al Bratty M: Visible spectrophotometric determination of chlorpheniramine maleate and diphenhydramine hydro-chloride in raw and dosage form using potassium permanganate. Oriental Journal of Chemistry 2016; 32: 885-94.
13. Erdem A, Ozsoz M, Kirilmaz L, Kilinc E and Dalbasti T: Diphenhydramine selective plastic membrane sensor and its pharmaceutical applications. Electroanalysis 1997; 9: 932-5.
14. Frag EY, Mohamed GG and El-Sayed WG: Potentiometric determination of antihistaminic diphenhydramine hydrochloride in pharmaceutical preparations and biological fluids using screen-printed electrode. Bioelectrochemistry 2011; 82: 79-86.
15. Nerin C, Garnica A and Cacho J: Indirect determination of nitrogenated drugs by atomic absorption spectrometry. Analytical Chemistry 1986; 58: 2617-21.
16. El-Ries MA and Khalil S: Indirect atomic absorption determination of atropine, diphenhydramine, tolazoline, and levamisole based on the formation of ion-associates with potassium tetraiodometrcurate. Journal of Pharmaceutical and Biomedical Analysis 2001; 25: 3-7.
17. Gomez MR, Olsina RA, Martı́nez LD and Silva MF: Simultaneous determination of dextromethorphan, diphenhydramine and phenylephrine in expectorant and decongestant syrups by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 2002; 30: 791-9.
18. Marchesini AF, Williner MR, Mantovani VE, Robles JC and Goicoechea HC: Simultaneous determination of naphazoline, diphenhydramine and phenylephrine in nasal solutions by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 2003; 31: 39-46.
19. Dong Y, Chen X, Chen Y, Chen X and Hu Z: Separation and determination of pseudoephedrine, dextromethorphan, diphenhydramine and chlorpheniramine in cold medicines by nonaqueous capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis 2005; 39: 285-9.
20. Ye L and Stewart JT: HPLC determination of an ondan-setron and diphenhydramine mixture in 0.9% Sodium chloride injection. Journal of Liquid Chromatography & Related Technologies 1996; 19: 711-8.
21. Walters-Thompson KM and Mason WD: Method for the determination of diphenhydramine in rabbit whole blood by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection in conjunction with gas chromatography (GC) with mass selective detection (MSD). Pharmaceutical Research 1992; 9: 929-32.
22. Jianshe MA, Zhang M, Shentu Y, Zhang Y, Fan X and Gong Y: Determination of diphenhydramine hydrochloride in rabbit plasma by LC-MS/MS and its application to a pharmacokinetic study. Latin American Journal of Pharmacy 2011; 30: 1372-76
23. Ferrer I, Heine CE and Thurman EM: Combination of LC/TOF-MS and LC/ion trap MS/MS for the identification of diphenhydramine in sediment samples. Analytical Chemistry 2004; 76: 1437-44.
24. Muller EE and Sherma J: Quantitative HPTLC determination of diphenhydramine hydrochloride in tablet, gelcap, and capsule antihistamine pharmaceuticals. Journal of Liquid Chromatography & Related Technologies 1999; 2: 153-9.
25. DiGregorio D and Sherma J: Determination of the sleep aid ingredients diphenhydramine hydrochloride and doxylamine succinate in pharmaceutical products by quantitative HPTLC. Journal of Liquid Chromatography & Related Technologies 1999; 22: 1599-06.
26. Raj SV, Kapadia SU and Argekar AP: Simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup by gas chromatography (GC). Talanta 1998; 46: 221-5.
27. Meatherall RC and Guay DR: Isothermal gas chromatographic analysis of diphenhydramine after direct injection onto a fused-silica capillary column. Journal of Chromatography B: Biomedical Sciences and Applications 1984; 307: 295-04.
28. Anastas PT and Warner JC: Green Chemistry: Theory and Practice, Oxford University Press: New York, 1998: 30.
29. International Conference on Harmonization (ICH). Guidance for Industry, Q1A (R2): Stability Testing of New Drug Substances and Products. IFPMA. Geneva, 2000.
    

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    How to cite this article:

    Vishwas TS, Gurupadayya BM and Jain R: Stability indicating green analytical gas chromatography method for the determination of diphenhydramine in pure and marketed formulations. Int J Pharm Sci & Res 2019; 10(9): 4280-86. doi: 10.13040/IJPSR.0975-8232. 10(9).4280-86.

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