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
ABSTRACT:
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 1^{st} derivatization with ∆λ 2 nm have wavelength 263.6 nm for Dextromethorphan HBr and 2^{nd} 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.
INTRODUCTION:
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 GG^{1,2,3,4}.
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 separation^{5,6,7,8}.
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-HPLC^{9,10}. 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 potentiometric^{14,15}, While GGwas carried out by spectrophotometry^{16}, HPLC^{17,18}. The Guaifenesin in human plasma with HPLC methods^{19}. Combination of Guaifenesin and DMP by UPLC^{20}. 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.
MATERIAL AND METHODS:
Chemical and Reagent:
The material was used DMP from Indonesian Ministry, Glyceryl Guaiacolate, Methanol pro analysis (E. Merck).
Instrumentation:
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 DMP^{2,4}.
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 273.2nm^{2,4}.
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 + b^{21,22}.
Validation Test:
Method validation was carried out with parameters of accuracy, precision, the linearity of LOD, and LOQ^{23,24}.
Accuracy:
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%
Precision:
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 formula^{23,24}:
Note:
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 equation^{5}.
Statistical test of data:
The test of data is the t-test. Data distribution is calculated using a formula^{21,22}
The actual level with a 99% confidence level is calculated using the formula^{23,24}:
Note:
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
RESULT AND DISCUSSION:
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 DMP^{4,5,6}. 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 nm^{2,3}, overlap with each other, so it cannot be determined by ordinary spectrophotometry because the resulting absorbance is already a mixed productt^{5}. 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 separation^{5}.
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 component^{5}. Zero-intersection in the derivative spectrum of each component was indicated by the wavelength and Δλ of the spectrum, which zero absorption^{4,5}. The overlapped absorption spectrum of DMP and GG can be seen in Figure 2.
Fig. 2: Zero Intersection 2^{nd} 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 sample^{12,13}.
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 2^{nd} derivative was measured with Δλ 2 nm at 285.8 nm, and the absorbance of DMP in the 2^{nd} 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 2^{nd} 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 2^{nd} with Δλ 2nm has the same absorption spectrum at 285.8nm, and (40- 120)µg /ml DMP at the 2^{nd} 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 sample^{21,22}.
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 |
Parameter |
DMP |
GG |
1. |
Lambert and Beer Law |
(40– 120) µg/ml |
(20 - 60 ) (µg/ ml |
2. |
Regression Equation |
Y = 0.00002 X + 0.00006 |
Y = 0.00032 X + 0.00006 |
3. |
Correlation coeffisien |
0.9973 |
0.9990 |
2. |
Accuracy(%) |
99.34 |
100.02 |
3. |
Presicion (%) |
1.0042 |
1.0212 |
4. |
Linierity (%) |
0.9984 |
0.9992 |
5. |
LOD (µg/ml) |
26 |
12 |
6. |
LOQ (µg/ml) |
84 |
42 |
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 parameters^{24,25}. Tlinearity value has a very good relationship or correlation between concentration and absorbance. There is a very good correlation between concentration and its amplitude^{22}. 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 validation^{24,25}.
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) |
Absorption |
Concentration in etiquette (μg/ml) |
Concentration in research (μg/ml) |
Percentage level (%) |
2.5 |
0.000129 |
3.5 |
3.25 |
99.26 |
2.5 |
0.000132 |
3.5 |
3.6 |
103.58 |
2.5 |
0.000132 |
3.5 |
3.65 |
103.51 |
2.5 |
0.000129 |
3.5 |
3.45 |
99.26 |
2.5 |
0.000128 |
3.5 |
3.4 |
97.82 |
2.5 |
0.000131 |
3.5 |
3.65 |
101.31 |
b.GG
Olume (ml) |
Absorption |
Concentration in etiquette (μg/ml) |
Concentration in research (μg/ml) |
Percentage level (%) |
2.5 |
0.01412 |
50 |
46.87 |
93.27 |
2.5 |
0.01406 |
50 |
46.67 |
92.87 |
2.5 |
0.01414 |
50 |
46.93 |
93.53 |
2.5 |
0.01402 |
50 |
46.53 |
92.59 |
2.5 |
0.01411 |
50 |
46.83 |
93.19 |
2.5 |
0.01410 |
50 |
46.80 |
93.13 |
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}.
CONCLUSION:
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.
ACKNOWLEDGEMENT:
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.
CONFLICT OF INTEREST:
The authors declare that there is no conflict of interest.
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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