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Guaifenesin and dropropizine were analyzed through oxidation with periodic acid to give formaldehyde which was allowed to condense with 4-Amino-5-hydrazino-4H [1,2,4]-triazole-3-thiol (AHTT). The condensation product was further oxidized to yield a purple colored compound with maximum absorption at 550nm. Beer's law was obeyed in the range of 5–45μg mL−1 for guaifenesin and 10–80μg mL−1 for dropropizine. Both drugs were also successfully determined in their dosage forms.
Guaifenesin (GF), 3-(2-Methoxyphenoxy)-1,2-propanediol; is reported to increase the volume and reduce the viscosity of tenacious sputum and is used as expectorant for productive cough . Different methods have been reported for the analysis of GF including HPLC [2–8], GC [9, 10], capillary electrophoresis mass spectrometry , X-ray diffraction , voltammetry .
Dropropizine (DP), 3-(4-Phenyl-1-piperazinyl)-1,2-propanediol, is a cough suppressant reported to have a peripheral action in nonproductive cough . Only two GC-mass spectrometry methods have been reported for the determination of DP in biological fluids [14, 15] in addition to a manufacturer procedure that involves the determination of dropropizine by measuring its UV absorbance at 237nm in 0.05N HCl (personal contact):
Bromhexine HCl (BR), 2-Amino-3,5-dibromo-N-cyclohexyl-N-methylbenzylamine hydrochloride; N-(2-Amino-3,5-dibromobenzyl)-N-methylcyclohexylamine hydrochloride:
The aim of the present paper is to develop a simple and accurate method for the determination of dropropizine and guaifenesin that permits their analysis in dosage forms without interference from excipients and other coformulated drugs.
Purpald or 4-Amino-5-hydrazino-4H [1,2, 4]-triazole-3-thiol reagent (AHTT) (Sigma-Aldrich) was prepared as 0.5% in 0.5M hydrochloric acid. Periodic acid (Winlab, UK.) was prepared as 1mgmL−1 solution in 0.2M potassium hydroxide. HPLC grade acetonitrile was from Fisher Scientific, UK. Potassium dihydrogen phosphate was from Sigma-Aldrich Chemie, Germany. All other chemicals used were of analytical grade and water was freshly distilled.
Reference standard guaifenesin (GF), dropropizine (DP) and bromhexine HCl (BR) were kindly supplied by Rameda Co. for pharmaceutical industries and diagnostic reagents, EVA Pharma for pharmaceutical and medical appliances and EVA Pharma for pharmaceutical and medical appliances, respectively.
It is a product of Rameda Co., Batch No. 08455, claimed to contain 100mg guaifenesin and 8mg bromhexine HCl.
It is a product of EVA Pharma, Batch No. 602170, claimed to contain 20mg dropropizine.
0.1mgmL−1 solution of GF was prepared in distilled water. Aliquots of this solution were diluted to produce working solutions of 5–45μg mL−1.
0.1mgmL−1 solution of DR was prepared by in distilled water. Aliquots of this solution were diluted to produce working solutions of 10–80μg mL−1.
0.1mgmL−1 solution of BR was prepared in methanol. Aliquot of this solution was diluted to produce working solution of 50μg mL−1.
One mL of each working solution of both drugs was transferred in a test tube, then 1mL periodic acid was added. The mixture was left at room temperature for 15 minutes for GF and 20 minutes for DR, 0.5mL 5M KOH solution was then added followed by 1mL of AHTT solution. The mixture was shaken and allowed to stand for about 15 minutes for GF and 20 minutes for DR. Absorbance of the resulting solution was measured at 550nm, against blank experiment. Calibration curves relating the absorbance at 550nm to GF or DR concentrations were plotted and regression analysis of the results was computed.
The well mixed powdered content of five capsules was used in the assay. An amount equivalent to 10mg of GF was transferred into 100mL volumetric flask, dissolved in distilled water then adjusted to volume and treated as previously mentioned under calibration procedure.
Five tablets were grounded to a homogenous fine powder, weighed and the average mass per tablet was determined. The amount of powder equivalent to 10mg of DR was dissolved into 70mL of distilled water. The solution was sonicated for about five minutes then filtered to separate insoluble excipients. Afterwards, the filtrate was accurately collected into 100mL calibrated flask and diluted to volume with water. The obtained solution labeled to contain 0.1mgmL−1 of each drug was analyzed by the proposed method as detailed under calibration.
s-Triazoles have been utilized to produce reagents that can react with drugs containing carbonyl group or susceptible to oxidation with periodic acid to produce carbonyl function such as diols and amino alcohols. In the present work, guaifenesin and dropropizine were converted into formaldehyde and the corresponding carboxylic acids by the selective oxidizing effect of periodic acid. The liberated aldehyde was allowed to react with 4-Amino-5-hydrazino-4H [1,2, 4]-triazole-3-thiol, which is a specific reagent for aldehydic functional group .
When AHTT was allowed to condense with formaldehyde followed by treatment with periodic acid and alkali addition, [1,2, 4]-triazolo-[1,2, 4,5] tetrazine-3-thiol colored product was obtained as shown in Scheme 1.
As reported by Jacobsen and Dickinson , the reaction involves the addition of alkaline solution of AHTT to the aldehyde solution and aerating the reaction mixture to give a purple-colored product. Mimura et al.  modified the procedure of color development by the use of periodic acid as oxidizing agent instead of aeration.
In the present study, periodic acid has a dual function. It acts as a selective oxidizing agent for polyhydroxy compounds to convert them to formaldehyde and corresponding carboxylic acids and help in the development of the purple colored product according to Mimura et al.  modification. It is important to emphasize that Jacobsen and Dickinson  used alkaline solution of AHTT (1% in 1M NaOH) for color production with aldehydes. However, this procedure was modified by using acidic solution of AHTT (0.5% in 0.5M HCl) which offers two advantages, the first was the use of lower concentration of the reagent; the second was the higher stability of AHTT solution as mentioned by Mimura et al. .
As reported for colorimetric determination of some diol-containing drugs , solution of guaifenesin and dropropizine was left for some time, then 5M KOH and AHTT solutions were added whereby a purple color was developed with maximum absorption at 550nm (Figures (Figures1 and1 and and2).2). Maximum color intensity was obtained when periodic acid solution was made to react with guaifenesin for 15 minutes and dropropizine for 20 minutes.
The effect of periodic acid concentration was also studied, it was found to be critical the use of 1mgmL−1 solution of periodic acid in 0.2M KOH produces maximum color intensity. Excess acid concentration causes a great decrease in the intensity of the produced color which may be attributed to the strong oxidizing effect of periodic acid on both drugs which may proceed to give further oxidation products.
The effect of AHTT concentration was also studied where maximum intensity was obtained upon using AHTT solution of 0.5% in 0.5M HCl.
Volume of KOH added was found to be critical; 0.5mL of 5M alkali solution was the optimum volume.
Shaking of the reaction mixture for 4-5 minutes was essential and produced maximum color intensity after addition of AHTT solution and waiting period of 15 minutes for GF and 20 minutes for DR. The obtained color remained stable for about 40 minutes with both drugs.
Calibration curves representing the relation between each drug concentrations and absorbance of colored products were constructed. Results show linear relationship in the range of 5–45μg mL−1 for GF and 10–80μg mL−1 for DR; in triplicate measurement from which linear regression equations were calculated. Correlation coefficient, slope and intercept were listed in Table 1. Results indicate high sensitivity of the proposed method.
According to ICH recommendation , the approach based on the S.D. of the response and the slope was used for determining the detection and quantitation limits. The theoretical values were assessed practically and given in Table 1.
Accuracy of the measurements was determined using the calibration standards of two drugs, where mean percentage of 100.58 for GF and 100.22 for DR were obtained, results are shown in Table 1. Accuracy was also assessed by the recovery of added standard, three concentrations each in duplicate to know concentration of dosage forms using the proposed colorimetric method. Results of mean % recovery for added standards in each formulation are reported in Table 2.
Repeatability and reproducibility of the instrumental response (absorbance of the formed color) were checked during method development and they were assessed from five replicate determinations of sample solutions of GF and DR at the concentration of 30μg mL−1.
The proposed method was applied for the determination of both drugs in their pharmaceutical formulations; results presented in Table 2 revealed that there is no interference from excipients, additives or coformulated drugs such as bromhexine HCl present in Muclear capsules along with guaifenesin. In addition the recoveries of the studied drugs from their formulations were almost the same as the recoveries of the pure added when applying the standard addition technique.
Results obtained by the proposed method were statistically compared with those obtained from the reported HPLC method for GF  and UV manufacturer method for DR. The calculated t and F values are less than the tabulated ones indicating no significant difference between the proposed and reported methods with respect to accuracy and precision at 95% confidence limit (Table 2).
The proposed colorimetric method is selective for polyhydroxy aliphatic compounds, simple and rapid as it takes from 15 to 20 minutes for the sample to be ready for measurement. Validation of the proposed method was carried out according to the ICH guidelines. The short duration of the assay and its specificity were clear bonuses for routine analysis of guaifenesin and dropropizine in their pharmaceutical formulations and in-process quality control.