Journal of   Research Article 
 Research in Pharmacy  www.jrespharm.com 
 
  
Development and validation of first and zero order derivative 
spectrophotometric methods for the determination of rilmenidine 
in pharmaceutical preparations and forced degradation study 
 
Ayça KARASAKAL 1 * İD  , Elif ÖZDEMİR 2 İD   
 
1  Department of Chemistry, Science and Letters Faculty, Namık Kemal University, Tekirdağ, 59030, Turkey. 
2  Department of Analytical Chemistry, Pharmacy Faculty, İstanbul Yeni Yüzyıl University, İstanbul, 34010, Turkey. 
* Corresponding Author. E-mail: akarasakal@nku.edu.tr (A.K.); Tel. +90-282-250 26 58. 
Received: 05 January 2019 / Revised: 11 February 2019 / Accepted: 05 March 2019 
ABSTRACT: Zero and first order derivative spectrophotometric methods were developed for the analysis of 
rilmenidine  in pharmaceutical preparations. Absorbances of rilmenidine were measured at 224 nm for the zero order 
by measuring height of peak from zero and at 223.20 and 230.20 nm for the first order derivative spectrophotometric 
method by measuring peak to peak height. The linearity ranges were found to be 50-250 µg/mL for zero and first order 
spectrophotometric method. The developed methods were validated and the analytical parameters of linearity, limit of 
detection, limit of quantification, accuracy, precision and recovery were evaluated. In addition, Rilmenidine exposed to 
the stress conditions of acid, base and oxidative in order to calculate to degradation % by developed zero and first order 
derivative spectrophotometric method. 
KEYWORDS: Rilmenidine; derivative spectrophotometric method; forced degradation study; validation. 
 1.  INTRODUCTION 
Currently, the high worldwide prevalence of hypertension in adults is a major public health problem 
Hypertension has become a leading cause of cardiovascular disease, which accounts for a high proportion of 
premature mortality and contributes to 7 million deaths each year. [1-7] Today, there are various drug 
treatment classifications used in the treatment of hypertension, commonly referred to as antihypertensive 
drugs. [8-11] Antihypertensive drugs were categorized into six separate drug groups: Angiotensin converting 
enzyme (ACE)‐inhibitors, Angiotensin‐receptor blockers (ATR‐blockers), diuretics, beta‐blockers, calcium‐
channel blockers and other antihypertensive drugs. [12] Rilmenidine is an antihypertensive agent with 
selectivity for I1 imidazoline receptors that acts both centrally by reducing sympathetic overactivity and in the 
kidney by inhibiting the Na+/H+ antiport. Rilmenidine provides antihypertensive efficacy comparable with 
that of diuretics, beta-blockers, calcium channel blockers, and angiotensin-converting enzyme (ACE) 
inhibitors. [13] Rilmenidine phosphate (RIL)’s chemical formula is RN-(dicyclopropylmethyl)-4,5-dihydro-1,3-
oxazol-2-amine phosphoric acid. [14] Rilmenidine is not subjected to pre-systemic metabolism and is rapidly 
and extensively absorbed with time to peak plasma concentrations between 1.5 to 2 h. [15-16] It isn’t reported 
in literature determination of RIL by zero and first order derivative spectrophotometric methods but there is 
only one method described for quantification of rilmenidine by LC-MS/MS in human serum. [17] The 
derivative spectrophotometric method is one of the advanced modern spectrophotometric techniques that 
supply a functional means for extracting both qualitative and quantitative information from the spectra 
composed of overlapped bands. It is based on using the first- or higher-order derivatives of absorbance with 
respect to wavelength from parent zero-order ones. Because derivatization can lead to the separation of 
unresolved signals and reduction of spectral background interferences, this technique permits the 
quantification of one analyte in the presence of others without initial separation or purification. [18] The 
derivative spectrophotometry consists of calculating and plotting one of the mathematical derivatives of a 
spectral curve. [19] There are several studies based on derivatization in the literature for analysis of drug 
substances. [19,21-25] Force degradation studies were performed on the placebo and drug products to show 
the stability-indicating nature of the method. These studies were performed in accordance with established 
 
How to cite this article: Karasakal A, Özdemir E. Development and validation of first and zero order derivative spectrophotometric methods for 
the determination of rilmenidine in pharmaceutical preparations and forced degradation study. J Res Pharm. 2019; 23(3): 457-464. 
© 2019 Marmara University Press https://doi.org/10.12991/jrp.2019.152    
ISSN: 2630-6344 
457 
Karasakal and Özdemir Journal of Research in Pharmacy 
Determination of rilmenidine in pharmaceuticals and forced degradation study  Research Article 
 
ICH guidelines. [26-27] In addition, the aim of this work was to develop stability methods for estimation of 
percentage degradation of RIL by zero and first order derivative spectrophotometric methods. The developed 
methods were applied to the determination of RIL in commercially available tablets and were validated for 
accuracy, precision, recovery, linearity and stability to forced degradation studies according to the prescribed 
procedures mentioned in ICH guidelines. [20] 
2. RESULTS  
Figure 1 shows  Rilmenidine phosphate (RIL) chemical formula. In this study to obtain more sensitive 
results, the first, second, third and fourth derivative spectra of RIL was achieved and then it was determined 
that first derivative was the best. In addition, we compared with first and zero order derivative 
spectrophotometric methods. Figure 2 shows first order derivative spectrum of 200 μg/mL RIL. During 
method development, several tests were performed in order to establish the assay parameters. The effect of 
different extraction solvents on the absorbance of RIL was investigated using chloroform, ethyl acetate and 
dichloromethane It was found that dichloromethane was the best solvent for extraction since it gave the 
highest absorbance. Zero order derivative spectrums of RIL shows maximum absorbance at 224 nm (Figure 3) 
and Figure 4 shows calibration curve of RIL for first order derivative spectrophotometric method. The 
proposed methods were successfully applied to pharmaceutical preparations. The determination of RIL 
tablets, Hyperium® 1 mg were analyzed by zero and first order derivative spectrophotometric method. From 
the calibration curves, concentration of drug in % were calculated. The recovery were found to be 105.61% 
±3.61 and 101.73% ±2.65  for zero and first order derivative spectrophotometric method, respectively. As the 
values of % RSD  and RME and the percentage recovery within the limit indicated that the methods were 
accurate, and  when we compared blank solution with RIL's absorption wavelengths in absorption spectrum, 
It was observed that absorption wavelengths didn’t overlay. Because of this, there is no interference from the 
excipients. 
H O
N N . HO P OH
O OH
 
Figure 1. Chemical structure of rilmenidine phosphate (RIL). 
 
 
Figure 2. First order derivative spectrum of 200 μg/mL RIL. 
 
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Determination of rilmenidine in pharmaceuticals and forced degradation study  Research Article 
 
3. DISCUSSION 
3.1. Method validation  
3.1.1. Linearity and Calibration Curve 
The linearity range was found to be 50-250 µg/mL. Optical characteristics and statistical datas  for zero 
and first order derivative spectrophotometric methods are shown in Table 1 and 2.  
 
 
Figure 3. The absorption spectrums of RIL at various concentrations (50-250 µg/mL) by zero order 
derivative spectrophotometric method. 
 
Figure 4. Calibration curve  of RIL for first order derivative spectrophotometric method. 
Table 1. Optical characteristics and statistical data for zero order derivative spectrophotometric method. 
Parameters Value 
Linearity (µg/mL) 50-250 
Regression equation y = 0.0024x+0.2362 
Slope 0.0024 
Intercept 0.2362 
Correlation coefficient 0.9997 
LOD (µg/mL) 0.97 
LOQ (µg/mL) 2.9 
 
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Table 2. Optical characteristics and statistical data for first order derivative spectrophotometric method.  
Parameters Value 
Linearity (µg/mL) 50-250 
Regression equation y = 0.0008x+0.0627 
Slope 0.0008 
Intercept 0.0627 
Correlation coefficient 0.9977 
LOD (µg/mL) 0.78 
LOQ (µg/mL) 2.7 
3.1.2. Limit of detection and limit of quantification 
Linearity range, regression eq. , correlation coefficient, LOD and LOQ (n=3) from the eq. of (standard 
deviation of intercept)/(slope of regression eq.) by multiplying 3.3 and 10, respectively. LOD and LOQ values 
were calculated as 0.97 and 2.9 µg/mL, respectively for zero order spectrophotometric method and 0.78 and 
2.7 µg/mL, respectively for first order derivative spectrophotometric method. [26] 
3.1.3. Precision and accuracy 
250 µg/mL of RIL were analyzed in six independent series in the same day (intra-day precision) and 3 
consecutive days (inter-day precision).  The accuracy and precision of the method was expressed by relative 
mean error (RME %) and by relative standard deviation (RSD %), respectively. Intra- and inter-day precision 
and accuracy were calculated  by measuring according to first order derivative spectrum of RIL was measured. 
The intra-day accuracy ranged from 0.1 % and the inter-day accuracy ranged from 0.12%. The intra-day 
precision ranged from 0.38, and the inter-day precision ranged from 0.21%. 
3.1.4. Recovery 
The recovery studies were performed by adding known amounts of the compounds studied to a known 
concentration of the commercial pharmaceutical tablets (standard addition method). The % recovery of the 
added pure drug were calculated as  
 
%Recovery = [(Cv – Cu)/Ca] × 100 (Eq. 1) 
 
where Cv is the total tag concentration Measured after standard addition; Cu drug concentration in the 
formulation; Ca, drug concentration added to formulation. The recovery were found to be 99.54% ±0.47 and 
99.33% ±0,21  for zero and first order derivative spectrophotometric method, respectively.  
3.2. Forced degradation studies 
RIL exhibited extensive degradation under acidic (0.1 M HCl) condition, as about 28.83 and 28.97 % 
degradation occurred after 8 h by zero and first order derivative spectrophotometric method, respectively 
(Table 3-4). The drug was degraded in acidic conditions; however, the absorbance (height) of the RIL peak 
decreased. First order derivative spectrum of  acidic degraded RIL was shown in Figure 5. The drug was 
relatively degraded under basic and oxidative conditions (0.1 M NaOH and 3% v/v H2O2 solutions). It was 
observed that there was 100 % degradation after 0 h by both of spectrophotometric methods. The drug was 
comparatively stable to acid hydrolysis more than basic and oxidative degradation. There was severe 
decomposition of the drug on basic and oxidative degradation. As first order derivative spectrophotometric 
method is more sensitive, asidic degradation results analysed by using first order derivative 
spectrophotometric method are higher than zero order derivative spectrophotometric method, relatively. 
 
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Figure 5. First order derivative spectrum of acidic degraded RIL. 
Table 3. Percent of decomposed of degraded RIL by zero order derivative spectrophotometric method 
according to peak to peak amplitudes. 
Time Acidic degradation, A % Basic and Oxidative Degradation, A % 
0 h 0.562 0 Decomposed 100 
1 h 0.525 6.59   
2 h 0.473 15.84   
4 h 0.469 16.55   
6 h 0.469 16.55   
8 h 0.400 28.83   
Table 4. Percent of decomposed of degraded RIL by first order derivative spectrophotometric method 
according to peak to peak amplitudes. 
Time Acidic degradation, A % Basic and Oxidative Degradation, A % 
0 h 0.145 0 Decomposed 100 
1 h 0.135 6.9   
2 h 0.130 10.34   
4 h 0.121 16.55   
6 h 0.121 16.55   
8 h 0.103 28.97   
4. CONCLUSIONS 
There is no publication concerning the UV spectrophotometric determinations of RIL as finished 
product in the current literature.In this study, RIL has been determined to determine using zero and first order 
derivative spectrophotometric methods. In addition, we worked forced degradation study of RIL for the first 
time. Proposed methods were sensitive, rapid, accurate, and reliable for the determination of RIL in pure and 
tablets. The method developed can well be proposed for both routine pharmaceutical analyses. 
5. MATERIAL AND METHODS 
5.1. Reagents and chemicals 
All chemicals and reagents used were of analytical grade. RIL was kindly provided by Servier (Istanbul, 
Turkey). Hyperium® tablets (1 mg) (Servier, İstanbul, TR) was purchased from a local pharmacy. 
5.2. Preparation of standard and working stock solution 
Stock solution was prepared in water at a concentration of 5 mg/mL RIL.Working solution is prepared 
at a concentration of 500 μg/mL RIL by diluting the stock solution with distilled water. 
 
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Determination of rilmenidine in pharmaceuticals and forced degradation study  Research Article 
 
5.3. Optimization  
Liquid-liquid extraction experiments were carried out in basic medium to convert the phosphate salt of 
rilmenidine to base and extract into an organic solvent. For this purpose 0.1 M NaOH, 0.1 M NH4OH and 
dichloromethane, chloroform, ethylacetate solvents were used. In order to determine the optimal condition, 
spectra of this rilmenidine were taken at the maximum absorbance wavelength that these solutions showed. 
The highest absorbance value was obtained by extraction with dichloromethane of rilmenidine, which was 
converted to its base with 0.1 M NaOH solution 
5.4. Methods 
5.4.1. Zero and first order derivative spectrophotometric method 
Aliquots of RIL working solution were added, so that the final concentration was in the range 50–250 
μg/mL. 1 mL of  0.1M NaOH was added. The solution was mixed vigorously. The content of the tube was 
extracted three times with 1.5 mL of dichloromethane.For zero order derivative spectrophotometric method, 
absorbances of RIL were measured at 224 nm and the distances between two extremum values (peak-to-peak 
amplitudes), 223.20-230.20nm (=2nm), were measured in the first derivative (dA/dλ) spectra of standard 
solutions of  the concentration ranges of 250–500 μg/mL. The calibration curves were plotted both of 
spectrophotometric methods.We calculated the amount % of degraded in all degradation studies to provide 
an indication of the stability-indicating property and specificity of the proposed methods.  
5.4.2. Acidic and basic degradation 
50.0 mg of RIL was waited in 5 mL of 0.1 M HCl (acidic degradation) and 5 mL of 0.1 M NaOH (basic 
degradation) at room temperature for 8 hours. The first order derivative spectrum of the RIL was measured 
in between two extremum values (peak-to-peak amplitudes),  223.20-230.20nm (==2nm) for first order 
derivative spectrophotometric method and the absorbances of RIL  were measured at 224 nm for zero order 
derivative spectrophotometric method. We calculated % of the amount of degraded. 
 5.4.3. Oxidative degradation 
To study hydrogen peroxide-induced degradation, initial studies were performed in 3% hydrogen 
peroxide at room temperature immediately for all spectrophotometric methods. 
5.5. Procedure for tablets 
Ten tablets of RIL were accurately weighed. A portion of the powder equivalent to 50.0 mg RIL was 
transferred into a 100 mL calibrated flask and dissolved in 50 mL of dichloromethane. The contents of the flask 
were sonicated for 30 minutes and then completed to volume dichloromethane. This solutions were prepared 
three times and the absorbance of each solution were determined at 224 nm and 223.20-230.20nm (==2nm) 
with all spectrophotometric methods. All determinations were conducted in triplicate. 
5.6. Method validation 
Method validation was performed by following the International Conference on Harmonization ICH 
guidelines for analytical method validation [26]. 
Acknowledgements: This work was supported by the Research Fund of the University of Namik Kemal (Project no: 
NKUBAP.00.GA.16.026). 
Author contributions: Concept – A.K. ; Design – A.K., E.O.; Supervision – A.K.; Resource – This work was supported 
by the Research Fund of the University of Namik Kemal; Materials – This work was supported by the Research Fund 
of the University of Namik Kemal; Data Collection &/or Processing – E.O.; Analysis &/or Interpretation – E.Ö.; 
Literature Search – A.K.; Writing – A.K.; Critical Reviews – A.K., E.O. 
Conflict of interest statement: The authors declared no conflict of interest 
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