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 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.
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 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.
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%
Note:
CF=Number of analytes measured
CA = Amount of analyte in the sample
CA* = Default number added
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 formula23,24:
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 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:
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 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 |
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 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.