Int J Pharm Pharm Sci, Vol 6, Issue 10, ??-??Original Article
HPLC METHOD DEVELOPMENT AND VALIDATION: SIMULTANEOUS DETERMINATION OF ACTIVE INGREDIENTS IN COUGH AND COLD PHARMACEUTICALS
HATİCE ÇAĞLAR, EBRU BÜYÜKTUNCEL
Inonu University, Faculty of Pharmacy, Department of Analytical Chemistry, 44280 Malatya, Turkey.
Email: saliha.buyuktuncel@inonu.edu.tr
Received: 07 Sep 2014 Revised and Accepted: 03 Oct 2014
ABSTRACT
Objective: This study aimed to develope a simple reversed-phase high performance chromatographic method for simultaneous determination of pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr, diphenhydramine HCI in cough and cold pharmaceuticals.
Methods: The separation of these compounds was achieved within 37.9 min on a Nucleodur gravity C18 column (250 x 4.0 mm, 5μm). The chromatographic separation of these compounds performed in a single run by using isocratic mobile phase consisting of methanol:buffer mixture (38:62, v/v) at room temperature, with flow rate of 0.75 mL.min-1.
An ultraviolet absorption at 210 nm was monitored. 2,4,6-trimethoxybenzaldehyde was used as an internal standard (ISTD). The selectivity, linearity of calibration, accuracy, intraday and interday precision and forced degradation studies were examined as parts of the method validation.
Results: The concentration–response relationship was linear over a concentration range of 0.2-250 µg.mL-1 for acetaminophen, 0.5–250 µg.mL-1 for pseudoephdrine HCI and pheniramine maleate, 1–250 µg.mL-1 for guaifenisin, 2.5-250 µg.mL-1 for chlorpheniramine maleate and triprolidine HCI, 5-250 µg.mL-1 for pyrilamine maleate and diphenhydramine HCI, 10-20 µg.mL-1 for dextromethorphan HBr with correlation coefficients better than 0.9993. The relative standard deviations of the intraday and interday were all less than 4%. Conclusion: The proposed liquid chromatographic method was successfully applied for the routine analysis of these compounds in different cough and cold pharmaceutical preparations such as syrups and tablets.
Keywords: High performance liquid chromatography, Active Ingredients Cough and Cold Pharmaceuticals, Validation.
INTRODUCTION
Cough and cold pharmaceutical preparations are one of the most extended formulations in the world and have got many pharmaceutical forms: syrup, suspension, sachets, capsules and tablets [1]. These preparations represent complex formulations containing several active ingredients and a broad spectrum of excipients such as flavoring agents, saccharose or aspartame, acidulants, natural or artificial colorings and flavoring agents, dyes sweeteners and preservatives [2,3]. These compounds are contained in the pharmaceutical form in very different proportions and present chemical forms of very different nature [4].
Acetaminophen (paracetamol) is analgesic and antipiretic [5]. As pain and fever are common, no home should be without some paracetamol, particularly homes with children. Acetaminophen is available in many different pharmaceutical preparations such as tablets, capsules, and liquid suspensions [6]. Chlorpheniramine maleate inhibits the effects of histamine on capillary permeability and bronchial smooth muscles. It is an anti-allergic drug, widely used in cough-cold preparations. The combination of antihistamine such as pyrilamine maleate and chlorpheniramine maleate is used to overcome the allergic effects and reduce or relieve cold symptoms [3]. Pheniramine maleate, dipenhydramine HCI, triprolidine HCI and pseudoephedrine hydrochloride are widely used in combination with other drugs for the clinical treatment of common cold, sinusitis, bronchitis and respiratory allergies [7]. Two common actives in such products are dextromethorphan HBr and guaifenesin. Dextromethorphan HBr is an antitussive which acts through depression of the medullary centers of the brain to decrease the involuntary urge to cough [8-11]. Guaifenesin is an expectorant believed to stimulate receptors that initiate a reflex secretion of respiratory tract fluid, thereby increasing the volume while decreasing the viscosity of mucus in the lungs. This action facilitates removal of mucus and reduces irritation of the bronchial tissue. Dextromethorphan hydrobromide and guaifenisin were used as cough suppressants antitussive for the relief of nonproductive cough and cold preparations [12]. All these components have different polarities and exist in very different proportion. Due to these characteristics and because of diverse properties inherent to their formulation, these preparations offer an analytical problem [13].
A variety of methods exist in the literature for the determination of some of these compounds [6,14-23]. Among them Louhaichi et al., have provided maximum separation that six active ingredients were separated, simultaneously [23]. But the presented study has identified the separation of nine active ingredients simultaneously.
The aim of this study was to develop basic, accurate and selective LC method for the simultaneous determination of pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr, diphenhydramine HCI in cough and cold pharmaceuticals. The method was then subjected to validation. The validation characteristics were evaluated as the selectivity, intraday and interday precision, linearity, accuracy, LOQ and LOD values and stress forced degradation studies.
The proposed liquid chromatographic method was successfully applied for the routine analysis of these compounds in different cough and cold pharmaceutical preparations such as syrups and tablets.
MATERIALS AND METHODS
Instrumentation and Chromatographic Conditions
The integrated high performance liquid chromatography system (LC 1100, Hewlett-Packard, USA) is equipped with a diode-array UV detector, a quarternary pump, a degasser, an autosampler, an injector with 20 µL loop, and a column oven. Different columns were tested for analysis and pseudoephedrine HCI and acetaminophen peaks was observed to overlap in the other columns. Therefore separation was carried out using Nucleodur gravity C18 column (250 x 4.0 mm, 5μm).
The mobile phase was a mixture of 38% methanol, 62% of 80 mM KH2PO4 aqueous solution adjusted to pH 3.0, to which was added 10% (v/v) orthophosphoric acid. The mobile phase was vacuum-filtered through a 0.45 μm nylon filter and degassed on-line by micro vacuum degasser. The chromatographic separation of these compounds performed at room temperature. Analysis was run at flow rate of 0.75 mL.min-1 with 37.9 min run time. The analysis was carried out at 210, 220, 254 and 280 nm and the best separation and high peak area have been monitored at 210 nm. The injection volume was 20 μL.
Reagents and Chemicals
Pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr, diphenhydramine HCI, HPLC grade methanol, sodium benzoate, 1,2-propylene glycol, citric acid, sorbitol and sodium saccharin were purchased from Sigma-Aldrich. Sodium carboxymethyl cellulose, pseudoephdrine HCI, 2,4,6-trimethoxybenzaldehyde, sunset yellow, and orthophosphoric acid were purchased from Fluka. Potassium dihydrogenphosphate and glycerol were obtained from Riedel-de Haën. Orange and cinnamon flavor were purchased from Eurofragance.
Water was purified (18 MΩ cm−1 quality) from New Human Power I (Korea).
The commercialized pharmaceutical products used are detailed below:
Actidem syrup (10 mg dextromethorphan HBr, 30 mg pseudoephedrine HCI and 1.25 mg/5 mL triprolidine HCI for 150 mL) was manufactured by GlaxoSmithKline, France.
Actifed syrup (30 mg pseudoephedrine HCI and triprolidine HCI 1.25 mg/5 mL for 150 mL) was manufactured by GlaxoSmithKline, France.
Aferin plus pediatric syrup (160 mg acetaminophen, 1 mg chlorpheniramine maleate, 15 mg/5 mL for 100 mL) was manufactured by Hüsnü Arsan, Turkey.
Benical syrup (10 mg dextromethorphan HBr, 20 mg pseudoephedrine HCI, 2 mg/5 mLchlorpheniramine maleate for 100 mL) was manufactured by Bayer, Germany.
Corsal syrup (120 mg acetaminophen, 2 mg chlorpheniramine maleate, 5 mg/5 mL phenylephrine HCI for 120 mL) was manufactured by İ.E Ulagay, Turkey.
Katarin pediatric syrup (120 mg acetaminophen, 50 mg oxolamine citrate, 1 mg/5 mL chlorpheniramine maleate for 100 mL) was manufactured by Biofarma, Turkey.
Kongest syrup (160 mg acetaminophen, 2.5 mg chlorpheniramine maleate, 1 mg/5 mL phenylephrine HCI for 100 mL) was manufactured by Eczacıbaşı, Turkey.
Peditus syrup (120 mg acetaminophen, 50 mg guaifenesin, 6.25 mg pyrilamine maleate, 5 mg/5 mL phenylephrine HCI for 100 mL) was manufactured by Sandoz, Turkey.
Sudafed syrup (30 mg/5 mL pseudoephedrine HCI for 150 mL) was manufactured by GlaxoSmithKline, France.
Benical cold film tablet (500 mg acetaminophen, 30 mg pseudoephedrine HCI, 20 mg dextromethorphan HBr for one tablet) was manufactured by Bayer, Germany.
Corsal capsule (300 mg acetaminophen, 2 mg chlorpheniramine maleate, 5 mg phenylephrine HCI for one tablet) was manufactured by İ.E Ulagay, Turkey.
Gerakon fort tablet (650 mg acetaminophen, 10 mg phenylephrine HCI, 4 mg chlorpheniramine maleate for one tablet) was manufactured by Münir Şahin, Turkey.
Kongest forte tablet (650 mg acetaminophen, 4 mg chlorpheniramine maleate, 10 mg phenylephrine HCI for one tablet) was manufactured by Eczacıbaşı, Turkey.
Sudafed syrup (60 mg pseudoephedrine HCI for one tablet) was manufactured by GlaxoSmithKline, France.
Theraflu forte film tablet (650 mg acetaminophen, 10 mg phenylephrine HCI, 4 mg chlorpheniramine maleate for one tablet) was manufactured by Novartis, Switzerland.
All these medicines were purchased by local pharmacy.
Standard solutions and sample preparation for quantification
Stock standard solutions of pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr and diphenhydramine HCI were prepared in ultrapure water. The calibration curves were prepared by diluting the stock solution in the mobile phase to furnish solutions with final concentrations of 0.2-250 µg.mL-1 for acetaminophen, 0.5–250 µg.mL-1 for pseudoephdrine HCI and pheniramine maleate, 1–250 µg.mL-1 for guaifenisin, 2.5-250 µg.mL-1 for chlorpheniramine maleate and triprolidine HCI, 5-250 µg.mL-1 for pyrilamine maleate and diphenhydramine HCI, 10-250 µg.mL-1 for dextromethorphan HBr.
The syrup placebo was prepared wherein: Citric acid was dissolved in glycerin. Sodium benzoat, sorbitol and sodium saccharin were dissolved in ultra pure water. Sodium carboxymethyl cellulose was kept in water for swelling. Then propylene glycol, flavors and colouring agent were added to this mixture and diluted with ultrapure water to 100 mL [24]. The excipients of syrup placebo were shown in Table 1.
Table 1: Excipients of placebo syrup
| Excipients | Amount |
| Citric Acid | 0.3 g |
| Glycerin | 10 g |
| Propylene glycol | 10 g |
| Sodium benzoat | 0.02 g |
| Sodium carboxymethyl cellulose | 0.1 g |
| Sorbitol | 20 g |
| Sodium saccharin | 0.04 g |
| Sunset yellow | enough amount |
| Orange aroma | 1 drop |
| Cinnamon aroma | 1 drop |
The amounts of the commercial cough and cold liquid were depending on the drug concentration of various products. The commercial cough and cold syrups were diluted with mobile phase according to linear range of standards. The resulting solutions were vortexed for 15 min and a portion of the sample was filtered through a 0.45 µm filter before injection in the HPLC.
The mean weight of finely powdered tablets were accurately transferred into 50 mL calibrated flask and ultrapure water was added. The mixtures were extracted in the ultrasonic bath for 15 min at room temperature and diluted with ultrapure water to the mark. The solutions were filtered through a 0.45 µm filter. Then the solutions were diluted with mobile phase depending on the drug concentration of various products. All preparations were performed in three replicates.
System Suitability Tests
As system suitability test is an integral part of chromatographic methods development and it is used to verify that the system is adequate for the analysis to be performed, the parameters for pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr and diphenhydramine HCI were evaluated. Several parameters may be used to demonstrate that the chromatographic system as a whole continues to be fit for the intended purpose. As well as monitoring the column performance, we can monitor the performance of the injector, pumps, and detector and so together provide an overview of system suitability. The user may define the minimum performance values or acceptance criteria according to local needs or business requirements [25].
System suitability test parameters were checked to ensure that the system was working correctly during the analysis [26]. Parameters which are typically used in suitability evaluations are capacity factor (k’), selectivity factor (α), resolution (R), number of theoretical plates (N) and tailing factor (T). For an optimum separation, capacity factor should be in the range of 0.5 < k’ <10. A value of 1.5 for resolution implies a complete separation of two compounds. The number of theoretical plates must be higher than 2000. The calculated values of tailing factor should be in the range of 0.5 ≤ T ≤ 2.
Validation
A full validation of assay consisting of selectivity, linearity, lower limit of detection and quantitation (LOD and LOQ), intraday and interday accuracy and precision of the method was performed according to the ICH description [27].
Selectivity
Selectivity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present.
Linearity and Range
The linearity of the assay was performed with a six point calibration curve prepared by diluting stock analyte solution in placebo syrup sample for five consecutive days. Six point calibration curves of each analyte were obtained by linear regression analysis.
The lowest concentration that can be quantified (LOQ) with acceptable accuracy and precision was evaluated at a signal-to-noise ratio of 10. Limit of detection (LOD) was evaluated at a signal-to-noise ratio of 3.
Accuracy and Precision
The accuracy of the proposed procedure was evaluated by means of recovery experiments. Recoveries were calculated as peak area ratios of reference standard/analyte (spiked placebo) at different concentration.
The precision was expressed as relative standard deviation of a series of measurements. Three different concentrations of standard solutions were analyzed five consecutive days and five times within the same day.
Forced Degradation Conditions
Forced degradation studies was carried out to demonstrate that the method was stability indicating. Solutions were prepared containing each substance at working standard concentration. They were treated with the following conditions:
a) Acid conditions: Solutions were acidulated with 37% HCl to reach a final concentration of 0.1 M HCl and heated for 2 h and 8 h at 80ºC, respectively.
b) Basic conditions: Solutions were treated with 1 M NaOH to reach a final concentration of 0.1 M NaOH and heated for 2 h and 8 h at 80ºC, respectively.
c) Oxidation with H2O2: Solutions were treated with 3% (v/v) H2O2 for 2 h and 8 h, respectively.
d) UV radiation: Solutions were exposed under a UV light at 254 nm during 35 h.
e) Thermal conditions: Solutions were heated for 2 h, 8 h and 24 h at 60 ºC and 80 ºC, respectively.
RESULTS AND DISCUSSION
Method Optimization
The first step of the study was the optimization of the chromatographic conditions. Various mobile phase combinations were tried initially to separate pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr and diphenhydramine HCI on C18 column. The mixture of methanol and phosphate buffer (38:62, v/v) was capable of a good separation and sensitivity (Figure1).
Fig. 1: Effect of methanol and phosphate buffer composition on separation of the mixture
The effect of phosphate buffer concentration (20, 40, 60, 80 ve 100 mM ) on the retention time of mixture was investigated in methanol-buffer (38:62) mobile phase. The concentration of the phosphate buffer solution was chosen as 80 mM for optimum separation (Figure 2).
Fig. 2: Effect of phosphate buffer concentration on separation of the mixture.
In order to find suitable buffer pH, the effect of pH on retention and resolution was investigated over the range of 2.0 and 6.0, using 10% (v/v) ortho-phosphoric acid solution. pKa values of these compounds are higher than 8. The changes in retention time as a function of pH result from changes in the ionization form of these solutes. Therefore, a pH value of 3.0 was selected because of optimum resolution. As shown in Figure 3, the resolution of all active compounds under the optimum conditions was adequate.
Fig. 3: Effect of phosphate buffer pH on separation of the mixture
Fig. 4: A typical chromatogram of standard active ingredients. 1, maleic acid; 2, acetaminophen; 3, pseudoephedrine HCI; 4, pheniramine maleate; 5, guaifenisin; 6, pyrilamine maleate; 7, 2,4,6-trimethoxybenzaldehyde; 8, chlorpheniramine maleate; 9, triprolidine HCI; 10, dextromethorphan HBr; 11, diphenhydramine HCI.
Method Validation
System suitability
The important parameter t0 was 2.319± 0.062 min in the analysis. This was the time of KBr peak. The capacity factor (k') values were in the range of 0.5 < k’ <10 except dextromethorphan HBr and diphenhydramine HCI. The resolution value for separation of acetaminophen and pseudoephedrine HCI was 1.3801 and dextromethorphan HBr and diphenhydramine HCI was 1.4995. The resolution values of other compounds were higher than 1.5. The therotical plate numbers of all compounds were higher than 2600 and the calculated tailing factors of them were obtained in the acceptable range of 0.5 ≤ T ≤ 2.
Selectivity
The representive chromatogram (Figure 5) of placebo solution constituted by excipient blend showed that there was no interfering peak in the retention times corresponding to the analytes. Therefore, the proposed method was considered to be selective.
Fig. 5: Chromatogram of placebo solution, 1. citric acid; 2. sodium benzoate.
Linearity and Range
To evaluate linearity of the method, six different concentrations of the nine analytes in the range of 0.2-250 mg mL-1 for acetaminophen, 0.5-250 mg mL-1 for pseudoephedrine HCI and pheniramine maleate, 1-250 mg mL-1 for guaifenisin, 5-250 mg mL-1 for pyrilamine maleate and diphenhydramine HCI, 2.5-250 mg mL-1 for chlorpheniramine maleate and triprolidine HCI, 10-250 mg mL-1for dextromethorphan HBr were analysed and the calibration curves for nine active compounds constructed under optimum conditions as the ratio of the peak areas of analysed subtance to internal standard against the concentration and the results are presented in Table 2.
The limit of detection (LOD) and limit of quantification (LOQ) of the method were determined by injecting progressively low concentrations of the standard solutions.
Accuracy and Precision
Accuracy was evaluated with recoveries obtained in the analysis of synthetic sample prepared in the placebo and compared with the corresponding standards. The results indicate good accuracy of the method for the simultaneous determination of the active compounds as revealed by mean recovery data. The results are given in Table 3. For evaluation of the precision estimates, intra-day and inter-day precision were performed for each active compound. The intra-day precision of the method was determined by preparing the standards of nine active compounds at three different concentration and values for each compounds were determined by five repeated analyses. Inter-day precision was check with the same concentration as intra-day assay, and the determination of each active compound was repeated day by day during five days. The obtained results are shown in Table 3.
Table 2: Linearity study results (n=5)
| Acetaminophen | Psudoephedrine HCI | Pheniramine
Maleate |
Guaifenis in | Pyrilamine
Maleate |
Chlorpheniramine Maleate | Triprolidine HCI | Dextromethorphan HBr | Diphenhydramine HCI | Acetaminophen |
| Regression
equation |
y=0.3659x+0.0583 | y=0.3x+0.0134 | y=0.3698x-0.0462 | y=0.2798x+0.0225 | y=0.2236x-0.0015 | y=0.3101x-0.0205 | y=0.4438x+0.0116 | y=0.2556x-0.0351 | y=0.4565x-0.0059 |
| Standard error of intercept | 0.0222 | 0.0047 | 0.0188 | 0.0175 | 0.0011 | 0.0092 | 0.0162 | 0.0046 | 0.0243 |
| Standard error of slope | 0.0244 | 0.0071 | 0.0099 | 0.0162 | 0.0012 | 0.0085 | 0.0058 | 0.0007 | 0.0091 |
| Correlation coefficient (r) | 0.9993 | 0.9997 | 0.9993 | 0.9991 | 0.9999 | 0.9999 | 0.9998 | 0.9990 | 0.9997 |
| Linearity range (mg L-1) | 0.2-250 | 0.5-250 | 0.5-250 | 1-250 | 5-250 | 2.5-250 | 2.5-250 | 10-250 | 5-250 |
| LOD (mg L-1) | 0.1 | 0.1 | 0.2 | 0.2 | 0.2 | 0.5 | 0.75 | 2.5 | 0.75 |
| LOQ (mg L-1) | 0.3 | 0.5 | 0.75 | 1 | 5 | 2.5 | 2.5 | 10 | 5 |
Forced Degradation
The drug substances was found almost stable in peroxide degradation. No degradation was seen in 3% H2O2 at 80 °C up to 2 and 8 hours.
Under the acidic conditional at the end of 2h and 8 h, large amount of fall in active compounds peaks area was observed except guaifenisin.
Under the alkaline conditional at the end of 8 h, same active compounds peaks was disappeared.
For photo degradation studies, all standards solution exposed to UV-light for 35 h: in these condition no a large amount of fall in peaks area.
For thermal degradation studies, all standard solutions were heated for 2 h and 8 h at 60 ºC and 80 ºC, respectively: in these condition no a large amount of fall in peaks area.
The all results were given Table 4.
Analysis of samples
Chromatograms of the some samples were shown below (Fig.6-12). The amount of each active compound was appointed using calibration curve method. The results demonstrate that the label claims of drugs close with obtained results which confirms the good accuracy of the proposed method (Table 5, Table 6).
Table 3: The intra-assay (intra-day) and between-assay (inter-day) precision and accuracy results (n=5)
| Intra-day | Inter-day | ||||
| Compound | Added (mg L-1) |
Precision (RSD%) |
Accuracy (Recovery%) |
Precision (RSD%) |
Accuracy (Recovery%) |
| Acetaminophen | 50 | 1.4420 | 101.6520 | 1.4658 | 101.9980 |
| 100 | 0.6876 | 100.3878 | 0.6902 | 101.2141 | |
| 250 | 1.0201 | 100.5995 | 1.1025 | 102.3410 | |
| Pseudoephedrine HCI |
50 | 3.3608 | 98.7927 | 3.7541 | 96.3060 |
| 100 | 1.3449 | 100.0609 | 1.3457 | 100.2120 | |
| 250 | 0.3759 | 99.9519 | 0.3854 | 100.5387 | |
| Pheniramine Maleate |
50 | 2.3691 | 99.1892 | 2.3499 | 100.1165 |
| 100 | 1.2286 | 101.1458 | 1.2427 | 103.1933 | |
| 250 | 1.0122 | 99.4358 | 1.0065 | 100.0570 | |
| Guaifenisin | 50 | 0.9491 | 100.8194 | 0.9569 | 101.0235 |
| 100 | 0.7833 | 100.2711 | 0.7854 | 100.3749 | |
| 250 | 2.2236 | 101.6682 | 2.5607 | 103.1516 | |
| Pyrilamine Maleate |
50 | 3.9870 | 101.2402 | 4.0364 | 103.0688 |
| 100 | 3.3658 | 100.8789 | 3.3954 | 101.0122 | |
| 250 | 3.9282 | 99.0023 | 3.8890 | 100.0521 | |
| Chlorpheniramine Maleate |
50 | 2.5739 | 101.8374 | 2.6212 | 102.056 |
| 100 | 0.5632 | 100.9189 | 0.5684 | 100.9465 | |
| 250 | 1.4962 | 101.3465 | 1.5163 | 102.9471 | |
| Tripirolidine HCI | 50 | 2.2186 | 100.0368 | 2.9564 | 101.5857 |
| 100 | 1.6736 | 102.1042 | 1.7089 | 103.2254 | |
| 250 | 2.0286 | 101.1976 | 2.6815 | 103.8761 | |
| Dextromethorphan HBr |
50 | 2.0466 | 99.5917 | 2.9157 | 102.0081 |
| 100 | 2.0346 | 101.4256 | 2.0636 | 101.9875 | |
| 250 | 1.2113 | 100.8714 | 1.2218 | 101.0159 | |
| Diphenhydramine HCI |
50 | 1.1565 | 100.6600 | 1.1641 | 101.1572 |
| 100 | 0.4162 | 100.2087 | 0.4170 | 100.8704 | |
| 250 | 0.4766 | 99.4162 | 0.4738 | 99.9141 | |
Table 4: The results of stress tests
| HCIb | H2O2b | NaOHb | Photolitic Thermal Degradation b | ||||
| 2 h 80 ºC | 8 h 80 °C | 2 h 80 °C | 8 h 80 °C | 2 h 80 °C | 8 h 80 °C | UV Lamp 35 h 2 h 60 °C 8 h 60 °C 24 h 60 °C 2 h 80 °C 8 h 80°C 24 h 80 °C |
|
| Acetaminophen | 56.12 | 0 | 99.5 | 86.3 | 53.8 | 0 | 94.5 99.1 98.6 98.7 98.9 96.6 9\=7.3 |
| Pseudoephedrine HCI | 73.07 | 65.8 | 97.3 | 95.6 | 80.5 | 74.6 | 91.1 98.9 97.3 96.9 98.3 96.5 95.9 |
| Pheniramine Maleate | Poor peak shape | Poor peak shape |
93.1 | 84.3 | 70.9 | 57.2 | 72.7 96.5 95.7 89.1 94.1 88.2 85.4 |
| Guaifenisin | 94.1 | 91.9 | 98.2 | 95.7 | 83.5 | 63.8 | 90.5 99.8 99.2 98.6 99.3 98.9 98.0 |
| Pyrilamine Maleate | 76.7 | 36.3 | 89.9 | 86.2 | 46.8 | 0 | 74.4 96.8 95.2 89.7 89.2 85.9 82.9 |
| Chlorpheniramine Maleate |
90.4 | 78.2 | 94.7 | 75.9 | 54.9 | 20.9 | 54.3 95.8 94.3 90.1 93.5 90.5 87.3 |
| Triprolidine HCI | 81.1 | 71.7 | 90.4 | 79.4 | 23.3 | 0 | 82.0 95.1 93.0 89.4 92.4 88.7 85.8 |
| Dextromethorphan HBr | 84.7 | 63.9 | 98.8 | 92.7 | 93.0 | 36.4 | 71.5 99.2 98.5 96.5 98.8 96.1 92.7 |
| Diphenhydramine HCI | 18.9 | 6.6 | 97.5 | 85.9 | 30.3 | 0 | 72.5 99.5 97.6 88.7 98.6 90.8 87.2 |
aRecovery(%) =[(mg.mL-1) after stres/(mg.mL-1) initial analyze]x100, bStress conditions
Fig. 6: The chromatogram belongs to corsal syrup, 1, acetaminophen; 2, sodium benzoate; 3, ISTD; 4, chlorpheniramine maleate.
Fig. 7: The chromatogram belongs to actidem syrup, 1, pseudoephedrine HCI; 2, sodium benzoate; 3, ISTD; 4, triprolidine HCI; 5, dextromethorphan HBr.
Fig. 8: The chromatogram belongs to actifed syrup, 1, pseudoephedrine HCI; 2, sodium benzoate; 3, ISTD; 4, triprolidine HCI
Fig. 9: The chromatogram belongs to aferin syrup, 1, acetaminophen; 2, pseudoephedrine HCI; 3, sodium benzoate; 4, ISTD; 5, chlorpheniramine maleate.
Fig. 10: The chromatogram belongs to benical syrup, 1, pseudoephedrine HCI; 2, sodium benzoate; 3, ISTD; 4, chlorpheniramine maleate; 5, dextromethorphan HBr.
Fig. 11: The chromatogram belong to peditus syrup, 1, acetaminophen; 2, guaifenisin; 3, pyrilamine maleate; 4, sodium benzoate; 5, ISTD.
Fig. 12: The chromatogram belongs to kongest tablet, 1, acetaminophen; 2, ISTD; 3, chlorpheniramine maleate.
Table 5: Content of commercial cough syrup with respect to label amount claimed
| Samples | Compounds | Labeled Amount
(mg.L-1) |
Found Amount±SD
(mg.L-1) |
Relative error
(%) |
| Actidem | Pseudoephedrine HCI
Triprolidine HCl Dextrrmethorphan HBr |
6000
250 2000 |
5961.2±1.9
250.9±2.6 2016.1±3.9 |
-0.65
+0.36 +0.81 |
| Actifed | Pseudoephedrine HCI
Triprolidine HCl |
6000
250 |
6275.6±3.5
251.8±4.3 |
+4.59
+0.72 |
| Aferin | Acetaminophen
Pseudoephedrine HCI Chlorpheniramine maleate |
32000
3000 200 |
32351.2±3.7
3072.8 ±1.9 207.41±1.2 |
+1.10
+2.43 +3.71 |
| Benical | Pseudoephedrine HCI
Chlorpheniramine maleate Dextromethorphan HBr |
4000
400 2000 |
4021.8±1.9
398.5±4.9 2076.5±3.9 |
+0.55
-0.38 +3.83 |
| Corsal | Acetaminophen
Chlorpheniramine maleate |
24000
400 |
24067.8±0.9
402.6±1.7 |
+0.28
+0.65 |
| Katarin | Acetaminophen
Chlorpheniramine maleate |
24000
200 |
24982.4±0.3
216.4±3.4 |
+4.09
+8.20 |
| Kongest | Acetaminophen
Chlorpheniramine maleate |
32000
500 |
32842.3±6.1
501.55±0.9 |
+2.63
+0.31 |
| Peditus | Acetaminophen
Guaifenisin Pyrilamine maleate |
24000
10000 1250 |
24412.4±6.1
10690.3±3.1 1254.4±3.01 |
+1.72
+6.90 +0.35 |
| Sudafed | Pseudoephedrine HCI | 6000 | 6003.1±2.1 | +0.05 |
Table 6: Content of commercial cough tablet with respect to label amount claimed.
| Samples | Compounds | Labeled Amount
(mg.L-1) |
Found Amount±SD
(mg.L-1) |
Relative error
(%) |
| Benical | Acetaminophen
Pseudoephedrine HCI Dextromethorphan HBr |
10000
600 400 |
10010.7±1.25
600.2±1.62 397.9±1.38 |
+0.11
+0.33 -0.53 |
| Corsal | Acetaminophen
Chlorpheniramine maleate |
6000
40 |
5993.0±0.98
40.1±0.71 |
-0.12
+0.25 |
| Gerakon | Acetaminophen
Chlorpheniramine maleate |
13000
80 |
13080.1±2.21
79.8±2.76 |
+0.62
-0.25 |
| Kongest | Acetaminophen
Chlorpheniramine maleate |
6000
40 |
6016.6±1.45
40.67±1.92 |
+0.28
+1.67 |
| Theraflu | Acetaminophen
Chlorpheniramine maleate |
13000
80 |
13049.7±1.51
80.1±1.27 |
+0.38
+0.18 |
| Sudafed | Pseudoephedrine HCI | 1200 | 1205.6±1.78 | +0.47 |
The obtained results were satisfactory for each compound. Because they show that the content of drug corresponds to the drug label. Therefore they confirm the good accuracy of the proposed method.
CONCLUSION
A basic and reliable HPLC method has been developed and validated for the determination of pseudoephdrine HCI, pheniramine maleate, acetaminophen, guaifenisin, pyrilamine maleate, chlorpheniramine maleate, triprolidine HCI, dextromethorphan HBr and diphenhydramine HCI in cough and cold pharmaceuticals. Compared to the other reported ones, the developed method offers separation of a large number of the active ingredients simultaneously. Thus large number of cough and cold pharmaceuticals could be analyzed by only one method. Forced degradation studies led to understand the chemical properties of drug molecules. Stability-indicating nature of the method was demonstrated on the experimental cough and cold syrup preparation under oxidation, acidic and alkaline, UV-light and thermal stress conditions. Although there is no acceptance criteria concerning degradation products, stability study provided to predict usage and storage conditions of the syrup.
ACKNOWLEDGMENT
I thank Dr. M. Sinan Kaynak from Department of Pharmaceutical Technology for preparation of placebo syrup. This research was supported by the İnönü University Research Council (Project no: 2010/131).
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