5- Normal Phase High Performance Liquid Chromatographic Determination of Chlorphenoxamine Hydrochloride, Caffeine and 8-Chlorotheophylline
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5- NORMAL PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF CHLORPHENOXAMINE HYDROCHLORIDE, CAFFEINE AND 8-CHLOROTHEOPHYLLINE Y. M. Dessouky, H. H. Hassanein, M. Abdul-Azim Mohammad* and R. S. Hanafy** Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasrel-Aiani, 11562, Cairo, Egypt. Received:14-3-2004 Accepted:16-5-2004 Abstract: INTRODUCTION An isocratic, rapid and reproducible high performance liquid chromatographic (HPLC) Chlorphenoxamine hydrochloride (CPHX), [I] method for quantitative determination of chlor- is a histamine H1- receptor antagonist with anti- phenoxamine hydrochloride (CPHX), caffeine muscarinic properties 1. Caffeine (CAFF), [2] is a (CAFF) and 8-chlorotheophylline (CTHE) has been xanthine derivative with CNS stimulant activity 1-3. adopted. The optimal conditions for separation and 8-Chlorotheophylline (CTHE) [3] is also a xanthine quantitative determination of these compounds derivative that forms with dipenhydramine a were a normal phase mode chromatography and compound named dimenhydrinate that is widely isocratic elution at ambient temperature. A silica used for alleviation of motion sickness 1-3. column and a mobile phase consisted of ethyl Combination of the three drugs is known 1,4. acetate - methanol (50:50 v/v) / triethylamine pH 9, CPHX was determined by titrimetric 2, have been used at a flow rate of 1 ml min-1. The electrochemical 5, spectrophotometric 6-8, derivative components of column effluent were monitored by spectrophotometric 9,10, derivative ratio spectro- 11 11 UV detector at 254 nm. The retention times (tR) for photometric , chemometric and spectro- CPHX, CAFF and CTHE were 6.15, 4.23 and 2.97 fluorimetric 12 methods. Chromatographic methods min, respectively. The linearity range for CPHX, including HPLC 13, TLC 14 and GC15,16 were also CAFF and CTHE were 37.5 - 725 µg ml-1, 0.75 - 48 reported. CAFF was determined in µg ml-1 and 0.1 - 40 µg ml-1, respectively. The mean pharmaceutically dosage forms and also in accuracy ± % RSD for CPHX, CAFF and CTHE biological fluids by titrimetry 3,17,18, 19-21 were 100.06 ± 0.47, 99.43 ± 0.60 and 100.65 ± spectrophotometry , derivative spectrophoto- 10,22 1.25, respectively. The results obtained by the metry , derivative ratio spectrophotometry 11,23,24 11,25,26 27 recovery studies indicated suitable validation , chemometry , near IR spectrometry 28 parameters. The proposed method showed good and NMR spectrometry . Chromatographic efficiency for simultaneous quantitative methods were also reported, such as HPLC determination of CPHX, CAFF and CTHE in bulk 2.13,23,24,29-35, TLC 14,36,37, GLC 38,39and GC 40,41. and tablet dosage from. Statistical comparison CTHE was determined by titrimetry 3,4,42, between the results obtained by the proposed conductometry 4,43, spectrophotometry 44, HPLC method and those obtained by the selected official 13,35, and TLC 14. methods for these drugs was carried out and no significant differences were found. Cl Keywords :chlorphenoxamine, chlorotheophylline, caffeine,pharmaceutical analysis, HPLC. O CH N 3 * Postal address: Faculty of Pharmacy, Cairo CH CH university- Kasrel-Aini- 11562-Cairo-Egypt. 3 3 ** Faculty of Pharmacy, Cairo university, Beni- Suef Branch. [1] Chlophenoxamine Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004) 53 O CH • Ethyl acetate for HPLC (Panreac, 3 Barcelona, Spain). H3C N • Triethylamine (Fluka-Sigma-Aldrich, N Germany). • Reference standards: CPHX, CAFF and O N N CTHE were obtained from the Egyptian International Pharmaceutical Industries CH Co.(EIPICO), 10th of Ramadan City, 3 Egypt. Purity of these standards was 99.82, 99.65 and 99.76% for CPHX, CAFF and [2] Caffeine CTHE, respectively as certified by the dealer. The purity was also checked by the pharmacopoeial methods. R O • Emeral tablets , batch number 020697 H from the Egyptian International Pharmaceutical H3C N N Industries Co. (EIPICO), 10th of Ramadan Cl City, Egypt. It is labeled to contain 30 mg, N 50mg and 20 mg per coated tablet for O N CPHX, CAFF and CTHE, respectively. CH 3 Chromatographic condition The mobile phase consisted of ethyl acetate- [3] 8-Chlorotheophylline methanol (50:50 v/v), adjusted to pH 9 using triethylamine was flowing at a constant flow rate of A pharmaceutically combination dosage forms 1.0 ml min-1. A Shimpack NP- Sil. column (150 of CPHX, CAFF and CTHE used to treat nausea, mm x 6.0 mm i.d., 5 µm) was used as stationary vertigo and vomiting are available on the market. phase. Detection was carried out using an UV The only two stated methods for the determination detector set at 254 nm. of these drugs in mixture are a reported TLC- 14 densitometry and an HPLC manufacturer's Standard preparations 13 procedure . Thus, the purpose of this study was to 1) Stock standard solutions: develop rapid, easy and precise liquid Standard stock solutions of 5000 µg ml-1 CPHX, chromatographic (HPLC) method for the 500 µg ml-1 CAFF and 200 µg ml-1 CTHE were simultaneous determination of the three drugs. prepared by dissolving 250 mg standard CPHX, 25 mg standard CAFF and 10 mg standard CTHE each EXPERIMENTAL in 50 ml ethyl acetate- methanol (50:50 v/v). Instrumentation and Parameters 2) Working standard solutions: A liquid chromatographic system: Shimadzu Each standard stock solution (20 ml) was class Avp Interface Module (Shimadzu-Japan) diluted to 100 ml with the same solvent system. equipped with: This give a concentration of 1000 µg ml-1 CPHX, • An isocratic HPLC pump (Model LC-10 ADVP, 100 µg ml-1 CAFF and 40 µg ml-1 CTHE. These Shimadzu, Japan). solutions were used as the working standards for • UV-visible detector (Model SPD-10A- VP, the analysis. Shimadzu, Japan). • A Rheodyne syringe loading sample injector 20 Laboratory prepared mixtures: µL (Mode l7725i, Catati, California,USA). Accurately weighed 15, 150 mg standard • Column: Shimpack NP-Sil. (150mm x 6mm i.d., CPHX, 25, 250 mg standard CAFF and 10, 100 mg 5µm) , (Shimadzu, Japan). standard CTHE were separately dissolved each in • Ultrasonic Processor Crest, 20 KHz frequency. 100 ml ethyl acetate- methanol (50:50 v/v). This give concentrations of 150, 1500 µg ml-1 CPHX, 250, 2500 µg ml-1 CAFF and 100, 1000 µg ml- 1 Reagents and Chemicals CTHE, respectively. Different aliquots of these • Methanol for HPLC (Riedel de Häen, Sigma- solutions within the linearity ranges of the studied Aldrich, Germany). drugs were mixed together into a series of 1-ml 54 Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004) Eppendorf centrifuge tubes and used as the determination of the studied drugs might be a great laboratory prepared mixtures. need. Sample of pharmaceutical preparation (tablets): Method Development Twenty tablets were weighed, ground to fine The challenge encountered in designing the powder and thoroughly mixed. A quantity of the chromatographic conditions during this study was powder equivalent to one tablet was accurately the separation of CAFF and CTHE, because they weighed in a 50-ml volumetric flask, then 35 ml of have more or less similar structure and polarity. A the solvent system, ethyl acetate- methanol (50:50 previously reported HPLC method for estimation of v/v) were added. The mixture was sonicated for 20 CTHE in the double salt (dimenhydrinate) with min, then cooled, completed to the mark with the diphenhydramine, a congener of chlorphenoxamine same solvent and filtered using 0.45 µm Millipore employed a gradient elution mode 2. Another filter. The first 10 ml of the filtrate was rejected and reported HPLC method for the analysis of CAFF the rest was used for the analysis. and CTHE in mixture with diphenhydramine applied an acidic mobile phase and RP-8 column Procedure and linearity because it has one of the lowest hydrophobic and Accurately measured aliquots of working silanol activity factors35. The presence of silanol standard solutions equivalent to 37.5 - 750 µg, 0.75 blocker such as triethylamine facilitates elution of - 48 µg and 0.1 - 40 µg of CPHX, CAFF and basic drugs without tailing 45. In addition, adjusting CTHE, respectively were transferred into three pH of the eluent was needed to allow differences in series of 1-ml Eppendorf centrifuge tubes and the the degree of ionization, change elution order and volume was brought to 1 ml with the same solvent. modify resolution 35. A volume of 20 µl of each solution was separately On the other hand, silica columns used with non injected into the chromatograph. The peak area of aqueous eluents such as methanol containing ionic each drug concentration was recorded. A calibration modifier provide stable and flexible systems for the graph for each drug was obtained by plotting peak analysis of drugs by HPLC 46. Retention on silica areas against its corresponding drug concentrations. column / non aqueous ionic eluent systems is The regression equations were computed. mediated primarily via cation exchange with surface silanol 47. Assay of laboratory prepared mixtures Different parameters affecting the The laboratory prepared mixtures were chromatographic separation by the proposed analyzed applying the same procedure as under method were studied. Several columns were tested linearity starting from `` the volume was brought and several pH modifiers were tried to cause ……``. The peak areas were recorded and the differences in the degree of ionization of the studied concentration was calculated from the respective drugs and hence, modify their elution order. The regression equation of each drug. composition of the mobile phase was adjusted after varying of the less polar (ethyl acetate) to the polar Assay of pharmaceutical preparation (methanol) ratios and also the nature of the pH Different aliquots of sample preparation within modifier. Meanwhile, increasing polarity and the linearity ranges of the studied drugs were acidity of the mobile phase resulted in bad transferred into a series of 1-ml Eppendorf separation between CAFF, CTHE and tailing of centrifuge tubes.