Evaluation of Dextromethorphan HBr and Glyceryl Guaiacolate mixture in syrup preparation by Derivative Spectrophotometry Methods


Muchlisyam*, Yade Metri Permata, Hafid Syahputra

Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Sumatera Utara, Indonesia.

*Corresponding Author E-mail: muchlisyam@usu.ac.id



The derivative spectrophotometric methods are the goal of evaluating the simultaneous level of Dextromethorphan HBr and Glyceryl Guaiacolate mixture in Syrup. The determination of the mixture is used the derivative spectrophotometry method with methanol-water (50:50) for Dextromethorphan HBr and Glyceryl Guaiacolate. The derivative spectrophotometric method on 1st derivatization with ∆λ 2 nm have wavelength 263.6 nm for  Dextromethorphan HBr and  2nd derivatization with ∆λ 2 nm at 285 nm for Glyceryl guaiacolate, The locally producted syrup samples met the level requirements mixture of Dextromethorphan and Glyceryl guaiacolate less than 90.0% and not over 110.0% of the amount stated on the label. The validation test showed that these two methods had met the validation parameter. The derivative spectrophotometry method   was used to determine a mixture of Dextromethorphan HBr and Glyceryl guaiacolate and fulfilling the validation requirements and level requirements according to USP 30.


KEYWORDS: Dextromethorphan HBr, Derivative spectrophotometry, Glyceryl guaiacolate, Validation.




A cough medicine contains various types of medicinal components including Glyceryl guaiacolate and Dextromethorphan HBr.Various combination in cough medicine has synergistic effects as cough suppressants and expectorants. The combination of Dextromethorphan HBr (DMP) and Glyceryl guaiacolate (GG) is a cough medicine combination for adults and children whose composition consists of 3.5 mg DMP and 50 mg GG1,2,3,4.


In the pharmaceutical field, drug preparations consist of more than one active substance and can complicate the quality control of each substance, especially by classical spectrophotometric methods. The overlapping spectrum of the individual substances causes this difficulty, and it can be overcome by applying good simultaneous analysis methods without prior separation5,6,7,8.


Now a day, The HPLC and RP-HPLC was done for the determination of drug mixture including grading Metaxalone in Spiked Human Plasma by HPLC and telmisartan and Chlorthalidone by RP-HPLC9,10.  The development of computer engineering has made the derivatization process in mathematical calculations very easy by using computer software combined with spectrophotometric tools, namely UV-Probe 2.3 or 2.4.  So that derivatization of a mixture of two or three components can be carried out resulting in a separation of the wavelengths of each, and determination of levels for each component with derivatization 1,2,3 and 4 as well as differences in ∆λ of 2,4, 8, and 16 nm. The level of one component can be done when the other components of the absorption curve support the X-axis (zero-intersection )11,12,13.


The literature shows that assay for both drugs can be done individually or in combination. DMP was determined by the development of spectrophotometric methods and potentiometric14,15, While GGwas carried out by spectrophotometry16, HPLC17,18. The Guaifenesin in human plasma with  HPLC methods19. Combination of Guaifenesin and DMP by UPLC20.  However, the mixture of this drug components have not been determinated by the zero intersection derivative spectrophotometry method.


Therefore, the research goal was carried out using zero-intersection  derivative spectrophotometric methods in syrup preparations.



Chemical and Reagent:

The material was used DMP from Indonesian Ministry, Glyceryl Guaiacolate, Methanol pro analysis (E. Merck).



UV Spectrophotometer (Shimadzu), UV probe 2.42 software, Boeco analytical balance, and Branson 1510 ultrasonicator.


Prepare a working solution:

Carefully weighed of 15mg GG and 20mg DMP, then put each into 50ml volumetric flask, dissolved in methanol-water (1: 4) and sufficient to sign-line. Each solution pipette 5ml and inserted each to 25ml volumetric flask, and added methanol solvent: water (1:4) for each flask to the sign-line and  obtain a working solution of 30μg/ml GG for one flask and the other flask a working solution of 80μg/ml DMP2,4.


Construction of the Maximum Absorption Spectra:

The maximum absorption spectra of 180µg/ml DMP  and 35µg/ml GG  are measured at a wavelength of 200-400nm respectively. The maximum wavelength for DMP is 278, nm, and GG is 273.2nm2,4.


Construction Zero Intersection at X-Axis of Derivatives Absorbance Spectrum:

The zero-intercept on the X-axis is determined by overlapping the absorbance spectra for each derivative within a given solution concentration range. Absorbance spectra were prepared with 35g/ml GG and 180g/ml DMP, and absorbance spectra were measured at wavelengths of 200-400nm, respectively. The absorption spectra of DMP and GG were respectively converted into a derived absorption spectrum using UV Probe 2.42 software into a derived spectrum. The selected wavelength is the wavelength when the absorption of the first substance gives a zero value on the X-axis, while the second substance has an absorption spectrum.5.


Preparation of calibration graphs:

A different aliquot of the standard solution of (40- 120) µg/ml DMP, and (20-50)µg/ml GG, was transferred into 50 ml volumetric flask respectively. Then measured the second derivative absorbance (Δλ=2nm) in predetermined wavelength analysis 285.8nm for GG and second derivative absorbance (Δλ = 4nm) in predetermined wavelength at 269.8nm for DMP. Conducted analysis of the relationship between concentration and absorbance values thus obtained by linear regression equation y = ax + b21,22.


Validation Test:

Method validation was carried out with parameters of accuracy, precision, the linearity of LOD, and LOQ23,24.



Determination of accuracy in this analysis, with the addition of 30% standard on 70% of the sample with three treatments, namely 80%, 100%, 120%. Then analyzed by the same procedure as sample testing. The following is the accuracy calculation formula:23,24


Accuracy  = X 100%

CF=Number of analytes measured

CA = Amount of analyte in the sample

CA* = Default number added



Precision is calculated from a series of measured calibration data. To find the RSD calculation using the formula 23,24:


RSD =  X 100%


Limit of Detection and Limit of Quantification:

The lowest limit of detection (LOD) and the lowest measurable limit (LOQ) were obtained based on the absorbance value at the analytical wavelength calculated by the formula23,24:




RSD    = Relative standard deviation

SD       = Standard deviation

X         = The data have been average


Determination simultaneous drug mixture in syrup preparations

Thoroughly pipette 2.5ml of syrup solution and transfer it to a 25ml volumetric flask, and add solvent to the marked line. homogenized with a sonicator for ten minutes. Filtered, then carefully pipetted as much as 2.5 ml into a 25ml volumetric flask, added 180 ppm of DMP working solution as an enhancer. Enough with the solvent up to the marked line. The absorption is measured at a wavelength of 200-400 nm, and is calculated by the regression equation5.


Statistical test of  data:

The test of data is the t-test. Data distribution is calculated using a formula21,22


The actual level with a 99% confidence level is calculated using the formula23,24:



µ = actual level interval

X ̅ = the average level of the sample

X = sample level

t = price t table according to dk = n-1

dk = degree of freedom (n-1)

α = level of trust

SD = standard deviation

N = number of replications



Absorption spectrum  of GG and DMP by UV Spectrophotometry:

The determination curve absorption spectrum each using a working solution with a concentration of 50 µg/ml GG, and 3.5 µg/ml DMP4,5,6. The result of overlapping spectrum of GG and DMP can be seen in Figure 1.


Fig. 1: Overlapping  a absorption spectrum of 35 ppm GG at 278 nm and 80 ppm DMP at 273,2nm


The figure 1  above,  it can be seen that the maximum absorption spectrum of DMP is 273.2nm and GG 278 nm2,3, overlap with each other, so it cannot be determined by ordinary spectrophotometry because the resulting absorbance is already a mixed productt5. Therefore, the mixture of these two components is determined by using the spectrophotometry derivative method by zero-intersection, so that the derivative of the two components can be determined simultaneously without any separation5.


Zero intersection  derivative spectrophotometric method:

The absorption spectrum of  50μg/ml GG, and 120 μg/ml DMP  was transformed into the second derivative with Δλ 2nm, 4nm, 8nm, and 16nm to get a zero-intersection  point. The zero-intersection  point was obtained by overlapping the second derivative spectrum of each component5. Zero-intersection  in the derivative spectrum of each component was indicated by the wavelength and  Δλ of the spectrum, which zero absorption4,5. The overlapped absorption spectrum of DMP and GG can be seen in Figure 2.


Fig. 2: Zero Intersection  2nd Derivative Absorption Spectrum of a) GG and b) DMP


Based on Figure 2 can be seen, there has been a change in wavelength, due to derivatization of the second derivate, GG from a wavelength of 278 nm to a wavelength of 285.8 nm at Δλ 2 nm, and DMP in zero condition. Meanwhile, DMP changes from a wavelength of 273.2 nm to 269.8 nm at Δλ 4 nm and GG in zero intersection s. That's mean the two components can be determined by the derivatization process on a sample12,13.


A Calibration curve of zero-intersection  derivative spectrophotometric:

Standard solutions (40-120) g/ml DMP, and (20-50) g/ml GG, were transferred to a 50 ml volumetric flask. Then the absorbance of GG in the 2nd derivative was measured with Δλ 2 nm at 285.8 nm, and the absorbance of DMP in the 2nd derivative with 4 nm at 269.8 nm. The component analysis data is calculated and a linear regression equation is obtained. 21,22 Results can be seen in Figure 3.


Fig. 3: Absorption Spectrum in 2nd derivative spectrophotometric with zero-intersection  in various concentration a) DMP and   b) GG


Based Fig. 3 shows that the measured absorbance result of (20-50)µg/ml GG at the 2nd with Δλ 2nm has the same absorption spectrum at 285.8nm, and (40- 120)µg /ml DMP at the 2nd derivative with Δλ 4nm, also has the same absorption spectrum at 269.8nm. The results are followed by the construction of the calibration curve so that the regression equation for each component is obtained.


Fig. 4: Calibration Curve of a) DMP and b)GG after treatment derivative spectrophotometric with zero-intersection


The figure 4 shown, that the results of the regression equation construction with a linear line for DMP with the regression equation Y = 0.00002 X + 0.00006 with  r value are 0.9973, while GG is Y = 0.00032 X + 0.00006 and r value 0.9990. Both of the regression equations were used to determine the DMP and GG components in the syrup sample21,22.


The Validation Parameter Test:

The test of the validation parameter is carried out by the accuracy-test utilizing the recovery test. While the precision, linearity, LOD, and LOQ tests are obtained from the data obtained when the calibration curve construction is carried out, and the calculation of the regression equation 23.24.25. The results can be seen in Table 1 below.


Table 1: Result of Regression Characteristitics and Validation Parameter of DMP and GG

S. No





Lambert and Beer Law

(40– 120) µg/ml

(20 - 60 )

(µg/ ml


Regression Equation

Y = 0.00002 X + 0.00006

Y = 0.00032 X + 0.00006


Correlation coeffisien








Presicion (%)




Linierity (%)




LOD (µg/ml)




LOQ (µg/ml)



Based on Table 1, it can be seen that the results of the parameter validation test on the derivative spectrophotometric method with zero-intersection  have according to the validation requirements for validation parameters24,25. Tlinearity value has a very good relationship or correlation between concentration and absorbance. There is a very good correlation between concentration and its amplitude22. The accuracy value used in a range of 80%, 100%, and 120%,  consists of 70% sample solution, and 30% raw material. The accuracy value meets the requirements of method validation24,25.


Precision was tested in several replications and met the validation requirements with RSD <3.9% 23,24,25. So that this method is used to determine the levels of DMP and GG in the form of syrup.


Fig. 5: Zero- Intersection  of Derivative absorption spectrum a. DMP  and b. GG in local production syrup


Figure 5 shows that the syrup sample can be determined by this method because the two components provide a spectrum of unequal wavelengths. After all, when one component has a spectrum and the other in the zero-intersection  region is zero, although DMP has to be done with standard additives with a solution. DMP standard 180 ppm because the sample content is too small from LOD 5,23,24.



Table 2: Calculation result of DMP and GG in Syrup Preparation.

a. DMP

Volume (ml)


Concentration in etiquette (μg/ml)

Concentration in research (μg/ml)

Percentage level (%)

































Olume (ml)


Concentration in etiquette (μg/ml)

Concentration in research (μg/ml)

Percentage level (%)
































The spectrophotometric derivative method with zero-intersection  can be applied to determine the DMP and GG levels in the syrup preparation. After performing statistical calculations in Table 2 a. and b, it shows that the level of is 93.2±0.40μg/ml and the level of DMP is 100.79±2.97μg/ml in the syrup tested. So that the syrup preparations on the market meet the requirements where the substance content is in the range 90-110% for DMP and nicotinamide according to USP 30. The results given by the zero-intersection  derivative spectrophotometric method in terms of both the value generated in the validation test and the results given for the analysis of samples on the market indicate that this method is a potential method for use, especially for drugs containing a combination of several drugs 3,5,23,24.



Based on the research conducted, it can be concluded: that the Derivative spectrophotometry method can be used for determination syrup preparation containing GG and DMP in their local production preparations and the levels of these two components met the requirements of USP XXX.



Thank you to the Chancellor of the Universitas Sumatera Utara and Chairman of the USU Research Institute, for his approval of the research of Basic Program at the Universitas Sumatera Utara, through in the Talent research program in 2020  with the number 141 / UN5. 2.3.1 / PPM / SPP-TALENTA USU/2020.



The authors declare that there is no conflict of interest.



1.      G1 oodman and Gilman’s The Pharmacological Basis of Therapeutics.Goodman, GA, Hardman JG, and Limbird LE. Editor.  10th Ed, The McGraw-Hill Companies, Inc, Medical Publishing Division, 2006; p.117. DOI: 10.1036/0071422803

2.      Indonesian Pharmacopoeia, Fifth edition, Jakarta, Ministry of Health of the Republic of Indonesia, 2014; p. 498, 848, 1077.

3.      The United States Pharmacopeia, USP 30.The National Formulary, NF XXII, Rockville, MD: U.S Pharmacopeial Convention, Inc. 1989; p. 1293.

4.      Analysis of Drugs and Poisons,   Moffat AC, Osselton MD, Widdop B, and Galichet  LY. Editors.  Clarke’s 4th ed, London, Pharmaceutical Press. 2011. p. 1087. 1468, 1856.

5.      Method Development and Validation for Simultaneous Estimation of Telmisartan and Ramipril by UV-Spectrophotometric Method in Pharmaceutical Dosage Form. Vanaja N, Preethi Ch, Manjunath SY, Krishanu Pal. Asian J. Pharm. Ana. 2015;5(4):, 2015; Page 187-194. DOI: 10.5958/2231-5675.2015.00030.7

6.      Development and Validation of RP-HPLC method for simultaneous Estimation of Metformin HCl and Gliclazide. Pawar J, Sonawane S, Chhajed S , Kshirsagar S. Asian J. Pharm. Ana. 2016; 6(3): p. 151-154. DOI: 10.5958/2231-5675.2016.00024.7

7.      Development and Validation of UV Spectrophotometric Method for The Simultaneous Estimation of Rosuvastatin and Ezetimibe in Pharmaceutical Dosage Form.Sireesha. D, Monika ML, Bakshi V. Asian J. Pharm. Ana. 2017; 7(3): pp 135-140. DOI: 10.5958/2231-5675.2017.00021.7.

8.      Validated Derivative Spectrophotometric method for simultaneous estimation of Levocetirizine Dihydrochloride and Phenylephrine Hydrochloride from tablet formulations.Dyade GK. Asian J. Pharm. Ana. 2019; 9(1):01-04. DOI: 10.5958/2231-5675.2019.00001.29.  

9.      Development and Validation of HPLC Method for the Estimation of Metaxalone in Spiked Human Plasma.   Gangurde P, Sonawane S, Kshirsagar S, and Chhajed S. Asian J. Pharm. Ana. 2019; 9(4): p.210-214. DOI: 10.5958/2231-5675.2019.00035.8

10.   Method Development and Validation for Simultaneous Estimation of Telmisartan and Chlorthalidone by RP-HPLC in Pharmaceutical Dosage Form.   Vanaja N,  Preethi CH, Manjunath SY, and  Krishanu Pal K. Asian J. Pharm. Ana. 2015;  5(4): p 171-177. DOI: 10.5958/2231-5675.2015.00027.7

11.   Implementation UV-Spectrophotometry approach for Determination of Tamsulosin HCl in tablets using area under Curve Technique.  Prajakta D. Mhaske, Burhan A. Bohari, Suraj R. Chaudhari, Pritam S. Jain, Atul A. Shirkhedkar. Asian J. Pharm. Ana. 2020; 10(2): 86-90. DOI: 10.5958/2231-5675.2020.00015.0

12.   Simultaneous Spectrophotometric Estimation of Naphazoline Nitrate and Hydrocortisone in Nasal drops. Jetal Patel, Dhara Patel, Sharav Desai. Asian J. Pharm. Res. 2016; 6(2): 61-66. DOI: 10.5958/2231-5691.2016.00011.3

13.   Validated First Order Derivative Spectrophotometric Method for Simultaneous Estimation of Lansoprazole and Aspirin in Tablet G. Kumaraswamy, Repudi. Lalitha, D. Sudheer Kumar . Dosage Forms. Asian J. Res. Pharm. Sci. 2016; 6(3): 185-190. DOI: 10.5958/2231-5659.2016.00025.4.

14.   New potentiometric and spectrophotometric methods for the determination Dextromethorphan in pharmaceutical preparations. Elmosallamy MA, and Amin ASAnal Sci 2014;30:pp. 419-425.

15.   A Review on UV Spectrophotometric Method for Estimation of Dextromethorphan in Bulk and Syrup Formulation by Area Under Curve Method.  Kamal AH, El-Malla SF, and Hammad SF.  International Journal of Pharmaceutical ang Chemical Science. 2016; 2(4): pp. 348-360

16.   Development of UV spectroscopic method for the determination of guaifenesin in bulk and formulation  Pushpalatha E, Tejaswini P, Najboonbi M, Vineesha S, Madhanna MD, and Kumar TA.. Int J Pharm Res Anal 2015;5: pp. 90-95.

17.   Analytical Method Development and Validation of Stability Indicating assay method of analysis for Dolutegravir/Lamivudine/Tenofovir Disoproxil Fumarate tablets using High Performance Liquid ChromatographySaravanan. R, Somanathan. T, Gavaskar D, Tamilvanan M. Research Journal of Pharmacy and Technology. 2021; 14(5):2434-9. DOI: 10.52711/0974-360X.2021.00428   Available on: https://rjptonline.org/AbstractView.aspx?PID=2021-14-5-12

18.   High performance liquid chromatographic method for the determination of guaifenesin in pharmaceutical syrups and in environmental samples.  Ahmed NR, and Lottfi SN. Baghdad Sci J 2012;10:pp. 1014-1021.

19.   Determination of guaifenesin in human plasma by liquid chromatography in the presence of pseudoephedrine Aluri JB, and Stavchansky. Journal of Pharmaceutical and Biomedical Analysis. 1993;11(9): pp. 803-808. DOI:10.1016/0731-7085(93)80072-9

20.   Determination of Guaifenesin and Dextromethorphan by UPLC.  Suneetha G, Venkateswarlu P, and  Prasad PSS Der Pharma Chemica. 2012;  4 (3): pp.1019-1025.

21.   Pharmaceutical Chemistry Analysis. Rohman A, First print, Yogyakarta: Student Library.Yogyakarta: Student library. 2007; p. 220-262

22.   Bioanalytical Method Validation.  Kadam AS, Pimpodkar NV, Gaikwad PS, and Chavan SD.. Asian J. Pharm. Ana. 2015;5(4): p219-225

23.   Implementation Guidelines and the Calculation Method Validation. Harmita F. Jakarta: Pharmaceutical Science Magazine; 2004, pp. 118-20.

24.   An Overview of Concept of Pharmaceutical Validation Raul SK, Padhy GK, Mahapatra AK, and Charan SA.. Research J. Pharm. and Tech. 2014; 7(9): p. 1081-1090.

25.   A Guide to Best   Practice: Method Validation in Pharmaceutical Analysis.  Ermer J. and Miller JH. Weinheim: Wiley-Vch Verlag GmBH & Co. KGaA. 2005;






Received on 11.01.2021            Modified on 19.04.2021

Accepted on 28.06.2021           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(1):171-176.

DOI: 10.52711/0974-360X.2022.00028