Keywords 1,3,4-oxadiazole derivative - column chromatography - RP-HPLC - forced degradation
Introduction
A novel derivative 1,3,4-oxadiazole (5-(4-bromophenyl)-N-(2-chloro-4-nitrophenyl)-1,3,4-oxadiazol-2-amine
(A3) is a five-membered heterocyclic compound with two nitrogen, one oxygen, and two
carbon atoms ([Fig. 2 ]). This system is crucial not just in medicinal chemistry but also in pesticide,
polymer, and material sciences.[1 ] Because of its broad range and capacity to target various biological targets, 1,3,4-oxadiazole
is a popular scaffold and an exciting pharmacophore for the development of novel drugs.[2 ]
[3 ] As a result, researchers continued to be interested in molecules having this heterocyclic
structure. 1,3,4-oxadiazole derivatives, because of their widespread biological and
pharmacological activity, are found to be a fascinating compound for the researchers.[4 ]
[5 ]
Fig. 1 Chemical structure, infrared (IR), and high-performance liquid chromatographic (HPLC)
spectra of pure compound A3.
In order to accurately measure and identify the analyte in the presence of its degradation
products, process contaminants, and excipients, stability-indicating methods are established
protocols that may detect changes in the physicochemical properties of a compound.
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals
for Human Use (ICH) recommendations strongly advocate forced degradation studies on
new compounds, active ingredients, and formulations to assess the suitability and
applicability of the prescribed analytical techniques in stability testing and routine
quality control assessments.[6 ] In this study, we developed and validated a novel reversed-phase high-performance
liquid chromatography (RP-HPLC) method for the A3 compound. In addition, these studies'
findings provide crucial details about a molecule's inherent stability properties,
which can be used to identify potential degradation products and pathways/mechanisms.[7 ]
Analytical method development plays a crucial role in the exploration, advancement,
and production of pharmaceuticals. RP-HPLC stands out as the most versatile and sensitive
analytical procedure, and it possesses the unique ability to handle mixtures with
multiple components effortlessly. RP-HPLC is an exceptionally effective technique
for purifying a wide array of compounds, including synthetic ones. It is often the
preferred method for analyzing and separating intricate mixtures in order to identify
specific compounds, consistent reproducibility, and efficient recovery.
Materials and Methods
Chemicals and Reagents
Reagents and chemicals were purchased from Merck, Hi-media, India. The mobile phase
was composed of HPLC-grade acetonitrile, water (Millipore water), and methanol in
the ratio 9:0.5:0.5. Syringe filters were made of 0.22-mm nylon. The method development
and validation analysis were carried out by using an HPLC (LC-20AD Prominence) photodiode
array detector with quadruplet solvent system (Shimadzu, Japan). The infrared spectra
were recorded by using Bruker's alpha attenuated total reflectance (ATR) Fourier transform
infrared spectroscopy (FT-IR) spectrometer.
General Method for the Synthesis of 5-substituted-n-aryl-1,3,4-oxadiazol-2-amine analogues
[8 ]
: All the new compounds were synthesized as per the reported procedure ([Fig. 1 ]
[Fig. 2 ]).[9 ]
Fig. 2 Route for synthesis of compound A3.
5-(4-bromophenyl)-N-(2-chloro-4-nitrophenyl)-1,3,4-oxadiazol-2-amine (A3): Yellow solid. Molecular formula: C14 H9 BrClN4 O3 ; percentage yield: 78%, 122 to 126°C; FT-IR (cm−1 ): 3,350.02 (NH), 3,059.46 (CH, Ar-H), 1,627.27 (C = N), 1,561.07 (C = C), 1,112.12
(C–O–C), 7,62.96 (C-Br). Lambda max: 235 by HPLC method; retention time (RT): 3.350 minutes.
Physicochemical Properties of the Synthesized Derivatives
Solubility
Solubility of the synthesized compound A3 was performed by dissolving the sample in
acetonitrile (ACN) and water (50%). The amount of solid dissolved in specified quantity
of solvent will be determined by the RP-HPLC method (purity and RT).
Method Development and Sample preparation
Mobile Phase
ACN, 0.1 N orthophosphoric acid (OPA), methanol in the ratio 90:05:05 is maintained
at pH 7.0 and degassed.[7 ] The working standard solution was prepared by using stock solution, which was diluted
with the mobile phase in the range of 10 to 100 µg/mL.
Working Standard
The compound A3 aliquot (10 µL; Launa 5µ 250 4.80 mm) was passed through the column
(HPLC 272817-7). The proportions of ACN, OPA, and methanol in the mobile phase were
set at 90:05:05. The temperature was kept at 40°C and the flow rate at 1.0 mL/min.
Before starting the experiment, the system was also adjusted with the solvent system
to obtain a smooth baseline.[10 ]
System Suitability and Method Validation
Some of the key parameters like theoretical plates (TP; N ), area under the curve (AUC), height equivalent to the theoretical plate (HETP),
and percentage assay were all calculated using the chromatogram.[11 ] By using ICH methodologies, the technique's accuracy and limit of detection (LOD)
were confirmed. Capacity is shown by limit of quantification (LOQ), linearity, precision,
robustness, and stability.
Linearity and Beer's Range
By using the chromatographic conditions described earlier, the working standard solutions
(10–100 µg/mL) were analyzed in triplicates, independent repeats, and not repetitions
at the same readings. The model's linearity was visually assessed using the concentration
versus peak area (mAU*s) plot and confirmed using the linear regression equation (I ) and Pearson's correlation coefficient (R
2 : 0.990–1.000). The linearity investigations yielded the Beer range:
Y = a + bX (I ),
where X , Y , a , and b represent explanatory variable (concentration), dependent variable (peak area), intercept,
and slope, respectively.[7 ]
[12 ]
Specificity, Sensitivity, and Selectivity
The two main parameters of specificity are selectivity and forced degradation. The
researchers were able to measure selectivity by delivering a 10-µL solution of the
test sample solution.[13 ] It is carried out using the LOQ and LOD. By evaluating a number of solutions with
concentrations ranging from 1 to 100 mg/mL, the LOD and LOQ of the sample were evaluated.[14 ]
[15 ]
Forced Degradation
Stress testing is the major use of forced degradation. Different parameters, including
heat degradation, humidity, acidic, basic, and oxidative degradation, were used to
execute forced degradation. Samples were subjected to thermal deterioration by being
held at 60°C for up to 24 hours. The samples' moisture deterioration was held at 35°C
for 7 days. While 0.1N NaOH is added to the samples and 0.1N HCl was used for neutralization
for basic degradation, they were treated with 0.1N HCl for acid degradation for 5 hours.
H2 O2 is commonly used for oxidative breakdown and the samples were given at 3% H2 O2 treatment and kept at room temperature for 24 hours.[7 ]
[10 ]
[14 ]
Forced degradation studies are essential in the development of analytical methods,
specification setting, and formulation design within the quality by design (QbD) framework.
Forced degradation is also known as stress testing and intentional degradation.
Accuracy and Precision
The method's precision and accuracy (repeatability, reproducibility, and robustness)
were assessed using the same concentration levels used in recovery experiments. By
analyzing the solutions three times in 1 day and once every day for 3 days straight,[16 ] the solution's repeatability and reproducibility (intra- and interday accuracy and
precision) were evaluated.[17 ] The accuracy and precision measurements used were relative standard deviation (RSD)
and percent recovery.[18 ]
[19 ]
Stability of Analytical Solution
The stability of the sample was tested by preserving the samples for 0, 24, and 48 hours
at room temperature (37 ± 2°C) and monitoring for changes.[20 ]
[21 ]
Filter Interference
This tests whether aliquots can be prepared using the filter accomplished using a
0.45-µm nylon filter and a test sample that has been centrifuged at 5,000 rpm.[22 ]
Results
To conduct the test, a specific volume of the sample was injected. The RSD was less
than 1.0% for the peak and RT area, and the tailing factor (TF) was less than 1.2.
Theoretical plates for test samples were 1646. The assessment on system appropriateness
is shown in [Table 1 ]. The sample solution was added to the chromatographic apparatus, and an AUC value
was noted for each peak. Percent assay was used to calculate the amount.
Table 1
System suitability characteristics derived from the chromatogram of compound A3
Parameters
Values
Retention time
3.35 ± 1.25 min
Capacity factor
0.109
Tailing factor
1.20
Number of theoretical plates (N )
1,646
Height equivalent to theoretical plates
91.138 µm
Using linear regression (R
2 ) analysis, the calibration's linear standard curve was evaluated. R
2 was Y = 58,607x + 118,188 for the test sample. The observed correlation coefficient (r ) for equality was discovered to be 0.9953. With an R -value of 0.9953, [Fig. 3 ] shows how the method is linear under experimental conditions for the concentration
range of 10 to 100 µg/mL.
Fig. 3 Calibration curve of compound A3.
Throughout the specificity analysis, the diluent had no impact on the test sample
RT. The stress conditions and absolute% deterioration were computed in relation to
the control sample. While heat and humidity degradation revealed 47.58 ± 1.25 and
56.28 ± 2.58 of degradation, respectively, oxidative degradation exhibited 41.58 ± 1.58.
Acid and alkali hydrolysis reduced the sample by 29.36 ± 1.25 and 65.28 3.65, respectively,
as shown in the [Table 2 ].
Table 2
Force degradation study of compound A3
Sl. no.
Stress condition
Stressor
% absolute degradation in assay
1
Control
Not applicable
100
2
Thermal degradation
60°C for 24 h
47.58 ± 1.25
3
Humidity degradation
7 d at room temperature
56.28 ± 2.58
4
Acid degradation
0.1 N HCl
65.28 ± 3.65
5
Alkali hydrolysis
0.1 N NaOH
29.36 ± 1.25
6
Oxidative degradation
3% H2 O2
41.58 ± 1.58
The LOD and LOQ were calculated statistically and found to be 0.740 and 0.2242 µg/mL,
respectively, between 10.0 and 100 mg/mL of A3. The method was able to determine concentrations
of 1 mg/mL with a sufficient amount of accuracy and precision ([Table 3 ]). The average recovery for the test sample was calculated to be 96.25% with a precision
RSD of 0.632% based on the measurement of recovery at LOQ.
Table 3
Results of calibration, limit of detection (LOD), and limit of quantification (LOQ)
of compound A3
Standard curve
Concentration (µg/mL)
Slope
Intercept
1
10–100
0.8799
0.8250
2
10–100
0.6528
0.7859
3
10–100
0.7852
0.9856
4
10–100
0.6325
0.658
5
10–100
0.7652
0.5362
Mean (n = 6)
0.743
0.758
Standard deviation
0.101
0.170
LOD (3.3*SD/S)
0.740 µg/mL
LOQ (10*SD/S)
2.242 µg/mL
The analysis of intraday and interday precision used the triplicates obtained on the
same day (intraday precision) and 3 days later (interday precision). According to
[Table 4 ], the test compound A3 achieved whole% RSD of 98.95 and 98.95%. The sample solution's
recovery was found to be within the established limits, and the RSD% results in [Table 5 ] reveal that it was 100, 96.25, and 99.25%. [Table 4 ] shows that there was no change in the filter interference data and no appreciable
change in the aliquot's assay result after 2 days. Peak area percentage RSD, TF, TP,
and RT all fell within the acceptable limit during the trial, which was 2%.
Table 4
Solution stability study of compound A3
Time point
%Assay of drug
Cumulative
Average
STDEV
%RSD
Day 0
100
NA
NA
NA
Day 1
98.5623
98.4215
0.2589
0.2630
Day 2
99.3514
98.5236
0.6234
0.6327
Abbreviations: RSD, relative standard deviation; STDEV, standard deviation.
Table 5
Recovery, intraday, interday, and intermediate-day accuracy and precision of compound
A3
Concentration (µg/mL)
%recovery ± SD (n = 3)
Intraday; %RSD (n = 6)
Interday; %RSD (n = 6)
Intermediate; %RSD (n = 6)
10
100 ± 1.25
99.51 ± 3.69
96.25 ± 2.35
99.91 ± 3.69
50
96.25 ± 0.39
98.71 ± 2.58
98.35 ± 1.36
98.52 ± 1.10
100
99.25 ± 1.59
98.62 ± 2.25
98.95 ± 1.99
98.32 ± 2.81
Abbreviations: RSD, relative standard deviation; SD, standard deviation.
Discussion
The present study was described to develop the analytical method of synthesized compounds
5-(4-bromophenyl)-N-(2-chloro-4-nitrophenyl)-1,3,4-oxadiazol-2-amine (A3) by the RP-HPLC
method. A C18 column was used, which was kept for activation and then stabilized by
maintaining the temperature at 40°C with flow rate of 1 mL/min. The optimum mobile
phase ratio in the HPLC method (ACN; water; methanol) is 90:05:05 and the RT was found
to be 3.35 minutes. Once the parameter was optimized, then the developed method was
optimized for different parameters such as accuracy, precision, robustness, selectivity
and forced degradation, sensitivity stability. Therefore, the purpose of this research
is method development by RP-HPLC and validation of the compound A3.
Conclusion
The findings of the present research demonstrated that the method development of compound
5-(4-bromophenyl)-N-(2-chloro-4-nitrophenyl)-1,3,4-oxadiazol-2-amine (A3) was a reliable,
economical, reproducible, and simple technique by RP-HPLC. The newly synthesized compound
A3 showed lambda max at 235 nm with optimum mobile phase ratio and the RT was found
to be 3.35 minutes. The purpose of the study was to show the quality of eluting and
separating of compound is efficient in accuracy and shortest feasible run time.
