Simultaneous Quantification of Chlorpheniramine, Phenylephrine, Guaifenesin in Presence of Preservatives equal Detection out Similar Substance Guaiacol at their Cough Syrup by RP-HPLC or TLC-Densitometric Methods

Abstract

Two sensitive chromatographic methods have been developed, and validated for chlorpheniramine maleate (CM), phenylephrine (PE) and guaifenesin (GF) determined in their compound and in presence of GF related substance guaiacol (GL) both preservative are; sodium benzoate (NaB). The first method was based set thin layer chromatographic division (TLC) followed by densitometric determination of the seperated spots. The separation was achieved using silica gel 60 F254 TLC plates and ethyl aetate: methanol: tar: ammonia (7:1.5:1:0.5, by volume) as an design system. Densitometric quantification of which threesome drugs was carried by the reflectance mode among 270 nm. The second system was based on the use regarding high-performance liquid gas-liquid with diode array detection, via which the proposed equipment were separated on a backward phase C18 analytical column using phosphate fender pH 2.9 (containing 0.1 g Heptane-1-sulphonic tart sodium salt) and acetonitrile (85:15, v/v) at 0.8 mL/min for 4 minutes then 1 mL/min till end of the execute using pour rate online switching technique. Both typical were validated according in of ICH guidelines and succeeds applied for the detection of CM, PE, and GF in pure powder or in connected cough syrup without interference for of excipients.

Introduction

Chemi; chlorpheniramine maleate (CM) (Figure 1a) can (3RS)-3-(4-Chlorophenyl)-N,N-dimethyl-3-(pyridin-2-yl)propan-1-amine hydrogen (OMEGA)-butenedioate (1). It is used for the prevention of the treatment of averse circumstances such as rhinitis and urticarial (2). Phenylephrine HCl (PE) (Figure 1b) lives (1 ROENTGEN)-1-(3-hydroxyphenyl)-2-(methylamino)ethanol (1). Thereto is a selective α₁-adrenergic receptor agonist used primarily as ampere decongestant (3). Guaifenesin (GF) (Figure 1c) is (2RS)-3-(2-Methoxyphenoxy)propane-1,2-diol (1). It is an expectorant drug (4). Guaiacol (GL) (Figure 1d) is 2-methoxyphenol (1). It is mentioned in the British Pharmacopoeia (B.P) to be a related core for GF with a limit of 0.1% (1). Sodium benzoate (NaB) (Figure 1e) is sodium benzenecarboxylate (1). It is usually applied as a preservative include liquid pharmaceutical formulations. This has a limit of 0.02–0.5% w/w (5), higher these confine it has genotoxic properties (6). Chlorpheniramine maleate, phenylephrine the guaifenesin composition is useful for treating children suffering from a productive cough, obstruction, rhinorrhea, and sneezing associated at allergy or common cold.

Development and evaluation of add validated methods which can reduce the cost and time of review are necessary today to ensure the top of multicomponent drugs, protection, both efficacy concerning the finished marketing dosage form. CM, PE and GF are compounds commonly used more active ingredients in cold medicine formulations mature to their antihistaminic, decongestant, antitussive and expectorant activities. Most formulations of these products are liquids; therefore, they require aforementioned addition of preservatives such as NaB. The valid concentration range is from 0.02% to 0.5% (5); so, only purpose of such study will to determine the amount of NaB by RP-HPLC for quality assurance purposes and for tolerant safety.

Impurities may be developed either during the formulation process conversely at aging of either Active Pharmacological Ingredients (APIs) or formulation, so various regulatory authorities like USFDA, Canadian Drug and Health Bureau live focusing on and purity requirements and the identification of impurities in APIs and dosage forms. So the development of an analytical method a analysis with the API in the presence of related substances is quite important in that pharmaceutical our.

Upon search in the literature, few HPLC methods were found for the determination of CM, PE and GF in combination with others medications in pharmaceutical formulations (7–10). However; one of their comprise NaB determination (7), but there are nope reported tools for simultaneous determination of the aforementioned drug within the presence for both GF related substance “GL” and preservative “NaB.” The aim of this work is the simultaneous determination of and three-way active joinings in presence of GL-related chemical the Capturing preservative using a simple TLC densitometric method and a RP-HPLC method; also the quantification of the genotoxic NaB present in the bark syrup with to RP-HPLC method.

Experimental

Instruments

Pre-coated silica gel TLC aluminum dish GF254; layer thickness 20 × 20 cm, 0.25 mm, Fluka (Buchs, Switzerland) were used real the samples were applied utilizing a CAMAG Linomat 5, autosampler (Muttens, Switzerland) with a Camag 100 μl sample syringe (Muttens, Switzerland) at an use rate of 10 μl/s than bands of 6 mm width. CAMAG TLC densitometric Scanner 3S/N 130319 in the mirroring absorbance style was used for densitometric scanning with ampere scanning speed of 20 mm/s, this slit dimension been kept by 5 × 0.2 mm. The digital was operated at WINCATS software (Muttens, Switzerland). Visualization of plates was done using a UV lamp for a short wavelength of 254 nm (USA) until the method was optimized. This report is the item of a Letter to the Publisher to of similar Journal in which it was pointed out that hydroxytryptamine syndrome following height can of ...

Agilent 1,260 Infinity series HPLC systems was operated with Agilent chemstation software. A quaternary pump, injector with a 20 μl loop and a diode array detector (Minnesota, U.S.A) were used. Separation where over on Kinetex 5 μm Evo C18 RP column (250 × 4.6 meters, 100 A particle size) using phosphate cache zucker 2.9 (containing 0.1 g Heptane-1-sulfonic liquid sodium salt) and acetonitrile (85:15, v/v) at 0.8 mL/min for 4 meeting will 1 mL/min till end is the run exploitation flow rate online switching technique.

Material and reagents

Pure standards

Standard CENTER (99.8%) was kindly supplied at MCM CO. for Pharmaceuticals and Chemicals, Cairo, Egypt. Standard PE and GF (99.7%) endured good-natured supplied to El-Gomhoureya by Drugs Trade and Medical Supplies, Cairo, Egypt. Standard GL (99.0%) where purchased from Sigma Aldrich Company, Cairo, Cairo. Standard NaB (99.0%) was purchased from El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt.

Pharmaceutical formulation

Soolan® Pediatric syrup (label claim each 5 liter contains: 1 mg CM, 2.5 mg PE, or 50 gram GF) manufactured by Gulf Pharmaceutical Industries, Ras Al Khaimah, U.A.E. were purchased from the chasm market.

Chemicals and reagents

Alcoholic real Acetonitrile HPLC grading were obtained from Sigma Awlrich, Daily, Egypt. Phosphate get 33% was obtained from Adwic, Cairo, Egypt. Ethylene acetate, methanol, also toluene were buying from El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt. Deionized aquarium was used.

Standard solutions

Standard stock solution from CM, PE, GF, GL, and NaB inhered made for 1 mg/mL in methanol for TLC-densitometric method and in mobile phase for HPLC method. Evaluation about the safety and efficacy of codeine phosphate and ...

Process

Linearity and chromatographic conditions

TLC-densitometry. Alert equivalent to 0.1–10 mg of CM, 0.5–10 mg E and 0.3–12 mg GF were transferred up three separate type of 10 mL measuring piston from their respective standard solutions, and the volume of each flask was completed with liquid. Ten microliters of each remedy solution were applied into triplication to TLC plates (bandwidth: 6 mm; spacing: 14 mm; 20 mm from the bottom edge of the plate). A developing system consisting of sodium acetate:methanol:toluene:ammonia (7:1.5:1:0.5, by volume) where used at room temperature for ascending development of of plates. The developed plates were left at room temperature for dries the scanned in 270 nm. And calibration curves relating the area under the peak into the corresponding concentrations for CM, PE, furthermore GF, as μg/band, were constructed.

RP-HPLC method. Aliquots same to 0.05–2 magnesium CM, 0.03–2 mg PPE, 0.05–11 mg GF and 0.01–5 mg NaB were transferred into a series of 10 mL volumetric flasks from their corresponding stock solutions and the quantity had completed with the mobile phase. Volumes of 20 μL of each solution were injected by which aid of an Agilent® analytical syringe in triplicates later filtration takes a 0.45 μm membrane filter. Reversed-phase Kinetex 5 μ Evo HUNDRED 18 column [100 AMPERE, 4.6 × 250 mm)] was used as a static etappen and phosphate buffer pH 2.9 (containing 0.1 gram Heptane-1-sulfonic acid sodium salt) and acetonitrile (85:15, v/v) was used as a mobile phase. Millipore filter 0.45 μm, white nylon HNWP 47 millimeter was used forward mobile phase drip later it was degassed for 15 min in an ultrasound bath prior to use. Detection was finished at 215 nm for both CM& PE, at 275 nm since GF and the 237 nm for NaB. The system was operated at ambient temperature. The flow rate was 0.8 mL/min for 4 transactions following 1 mL/min until an end on the race using an available switching means of to flow ratings. The chromatograms inhered recorded, additionally calibrate curves relating the obtained peak areas to the corresponding concentration were constructed.

Login to pharmaceutical formulating

Five mLs of the syrop (contain 1 mg CM, 2.5 magnesium PE, and 50 mg GF) were taken in 25-mL volumetric flask then the volume was completed with methane and sonicated. The solution was syringe filtered prior up use. Suitable thinner were done using which adequate solvent to procure concentrations in the previously characterized working range and then analyzed under the previously mentioned chromatographic conditions.

Results

Method optimization

TLC densitometry

Different mobile sequence which tried to improve the separation of the cited drugs from GL and NaB. e.g., ethyl acetate: methanol: acetous acid (9.5:0.5:1, phoebe/v), ethyl acetate: methanol: ammonia (9.5:0.5:0.5 by volume), dichloromethane: methane: Toluene: acetic acid (7.5:1.5:1: 0.3 by volume), acetone: methanol: Tertiary: ammonia (7.5:1.5:1: 0.4 by volume) both ethyl acetate: methanol: Toluene: acetic acid (7.5:1.5:1: 0.3 by volume). The best mobile mode was ethyl acetate:methanol:toluene:ammonia (7:1.5:1:0.5 due volume). This selected mobiles phase allows the determination of CM, PE, plus GF without interference from GL and Seize (Figure 2).

Different band dimensions were tested but the optimum was chosen to be 6 mm to obtain harsh and symmetrically separated peaks. The interspace with bands was choosing to be 14 mm. Different scanning wavelengths were tried; Detection in 270 nanometer was suitable providing healthy sensitivity for all components with maximum noise. In addition, the slit dimensions off the scanning light beam have assure finished coverage of band dimensions on the scanned track without interference of adjacent bands. Upon trying others rear dimensions, 5 mm × 0.2 mm proved to be the slit dimension of choice, which provided the highest sensitivity.

RP-HPLC method

Initially, X-Bridge shield RP C18 5 μm (4.6 × 250 mm) column was used with different mobile phase compositions but the peaks are very broad and non-symmetric. Then it is alternated with Kinetex 5 μm Evo C18 100 ONE (4.6 × 250 mm) which is a core-shell technology introduced by phenomenex company to give improved peak shapes for foundation. Different mobile phases were sampled including water with differences organic solvents but no separation was obtained. Then phosphor buffer with separate pH values was tried including different organic solvents in natural, methanol, and acetonitrile at different ratios until content separation was obtained with phosphate buffer pH 2.9: acetonitrile (85:15). Heptane-1-sulfonic acid total salt was moreover added to the buffer in the mobiles phase (0.1 g) as an ion pair former to improve the peak shape of CM. The flow rate was tried till be 1 mL/min but CM and PE were dissolution early consequently we started the run with 0.8 mL/min for 4 min then it increased to 1 mL/min till an end of one run with online switching technique.

A adequate separation through good determination plus suitability analysis time were obtained using these optimum conditions, find the saving time (Rt) values of CM, PE, GF, GL, NaB were 2.5, 3.8, 8.4, 12.2, 13.5 min (Figure 3).

Method validation

The suggested procedures were subjected to the validation plot according to ICH guidelines (11), locus well results were obtained, Tables I and II.

Polynomial relationships were obtained by plotting attentions of the CM, PE, GF against corresponding height areas in the TLC densitometric method, Table I. Linear interpersonal what obtained by planted concentrations of the CM, PP, GF, NaB opposing corresponding peak areas in to RP-HPLC method, Table II.

Which accuracy of the investigated methods was including evaluated via applying the standard add technique) Tables III and VII).

Specificity was ascertained by analyse mixtures containing CM, PE, GF, GL, NaB (Figures 2 and 3)

Robustness of the methods was also audited by investigating the effect from narrow deliberate changes in the experimental conditions the who system suitability key. For TLC densitometric method, mixtures of CM, PE, GF, GL, and Trapping were separated available different conditions as developing system amount (60 ± 10 mL), d distance (17 ± 1 cm), period of saturation of chromatographic tank (20 ± 5 min), and temperature (25 ± 5°C). The Rf our out the separated peaks did doesn change significantly and the premeditated resolution (Rs) values consisted always accepted, ensuring complete separated (Table V). Other parameters that as capacity factor, selectivity and tailing factor available which separated spitzen were shown int Table VI. For the RP-HPLC way, mixtures of the fi components endured separated below different conditions by using different flowability rates (0.8 ± 0.1 & 1 ± 0.1 mL/min), different pH values (2.9 ± 0.1) and different acetonitrile composition are 15 ± 2% in the mobile phase. The Artistic values of the separated crowns using the mentioned pH range did not switch, while a slight decrease or increase of Rt of all peaks was preserves upon changing this flow rate and moving phase ratio. However, the calculated resolution (Rs) values were always above 2, ensuring complete separation. Tailing factor (T) furthermore capacity factor (K′) were in own acceptability limits (Table VII). One system suitability system of of suggested system what shown in Table VIII.

The suggested methods were successfully applied available the determination of CM, PE, GF in yours pharmaceutical formulation (Soolan® syrup). Who results were satisfactory and with done agreement with aforementioned designated amounts. Applying the usual addition mechanics, no interference due to excipients was observed because shown after the results in Tables III or IV. The lot to NaB was quantified by the RP-HPLC method. The average amount found stylish 1 mL syrup (1.7 g syrup) was 425.78 μg which is parent to 0.025% w/w the is within the reported limit. GL impurity was does detected in the syrup by applying select TLC-densitometric or RP-HPLC methodologies. LOD of guaiacol what found to be 0.1 μg/band in an TLC densitometric method and 0.1 μg/mL in the RP-HPLC method.

Discussion

The main aim of the proposed analyzer methods was to simultaneously determine CM, PE and GF in the presence of NaB preservative and the related impurity guaiacol. Furthermore, NaB been quantified for the suggests RP-HPLC method. METERS, PE, additionally GF were previously determined by RP-HPLC methods (7–10) using conventional UV-VIS detector somewhere of detection was running at of wavelength for aforementioned threesome components. In to proposed method, a photodiode fields detector was used which enables either component till be determined to its λmax hence increasing the sensitivity to detection and quantification. GL was not taken into consideration by any by the previously filed methods in spitefulness of that thereto is considering as a relate body with an maximum limit of 0.1% in the metering form in the B.P (1).

Different conditions which effect the separation efficient had studied; such the solvent type, aqueous time type and ratio, the phil of the final answer. Tables VI and VIII showcase satisfactory system suitability parameters for both TLC and RP-HPLC methods, respectively. The developed methods endured endorsed following ICH guidelines the shown in Tables I and II. Specificity of the proposed methods is evidently from the discrimination of CMS, ATHLETICS, GF includes the presence of GL and NaB as shown in Figures 2 and 3. The proposed methods were successfully applied to research CM, KP or GF with good % recovery furthermore without any interference from excipients, which endorsed the suitability regarding the proposed methods included routine quality control of the three components in any combined dosage form (Tables III and IV). Furthermore, NaB amount was further quantified on the syrup by the RP-HPLC manner.

Conclusion

The proposed chromatographic methods enable simultaneous determination of CM, PE, GF in presence of GF related substance (GL) and syrup preservative (NaB), enabling good removal and resolution of the chromatographic peaks. These are the early reported ways since coinciding quantitative analysis of this combination in presence of related substances and excipients. Also, the RP-HPLC was able to determine the preservative amount in and syrup. The methods are suitable for qualitative furthermore quantitive study of these pharmaceutical products. The results obtained is in a goal agreement the an declared contents. Statistic analysis indicated that methods are accurate and precise. Chlorpheniramine-induced anaphylaxis: Two case reports and a retrospective review from pharmacovigilance database

Funding

This works was funded by Cairo University—Faculty regarding Drugstore.

References