Liquid Chromatographic Method Development and Validation for the Simultaneous Determination of Phenylephrine Hydrochloride, Paracetamol, Caffeine and Diphenhydramine Hydrochloride in Pure and Formulations
Nandeesha IM1, Basappa C. Yallur1, Manjunatha D. Hadagali1,2*
*Corresponding Author E-mail: manjunathdh@gmail.com, manjunathdh@davangereuniversity.ac.in
ABSTRACT:
The objective of the proposed work was to develop and validate a simple and sensitive RP-HPLC method for the simultaneous determination of four drugs viz., Phenylephrine hydrochloride (PNY), Acetaminophen (ACN) or Paracetamol (PAR), Caffeine (CFN) and Diphenhydramine Hydrochloride (DPH) in cough syrup combination. The Flourishing separation of four analytes were found by using a high performance liquid chromatography (HPLC) Shimadzu LC 2010 CHT and mobile phase consisting of methanol: buffer (0.1% v/v triethylamine pH-3.0 - adjusted with 0.1N Orthophosphoric acid) in the ratio of 45:55, waters sunfire C18 column (250mm, 4.6 mm and 5μ particle size). An isocratic elution with a flow rate was fixed at 1.0mLmin-1 and ambient temperature. Detection was carried out with wavelength at 220nm. The retention times of all analytes were stable at 2.2, 3.6, 4.5 and 6.42 minutes for PNY, ACN, CFN and DPH respectively. For these four drugs, the lower limit of detection (LOD) was 1.0, 0.5, 1.5 and 2.5μgmL-1 and the lower limit of quantification (LOQ) was 3.3, 1.7, 5.0 and 8.3μgmL-1, respectively. The calibration curves for PNY, ACN, CFN and DPH were found linear over the concentrations of 2.0-7.0, 1.5-7.5, 3.0-9.0 and 6.0-16.0μgmL-1 respectively. The inter- and intra-day of Relative Standard Deviation (RSD) were less than 2.0%. The developed method is hasty and stable for a combination of drugs, PNY, ACN/PAR, CFN and DPH. The proposed method was applicable for routine analysis of PNY, ACN, CFN and DPH quantification of these selected drugs for their assay in pure and wet cough syrup formulation.
KEYWORDS: Phenylephrinehydrochloride, Acetaminophen, Caffeine, Diphenhydramine Hydrochloride, RP-HPLC, Method development, Simultaneous determination.
INTRODUCTION:
Phenylephrine hydrochloride (PNY) is chemically known as (1R)-1-(3hydroxy-phenyl)-2-(methylamino) ethanol hydrochloride and is used as sympathomimetic (decongestants)1-4. It is an alpha-adrenergic as well as vasoconstrictor with little effect on the myocardium or the central nervous system2.
Acetaminophen (ACN) is N-(4-hydroxyphenyl) acetamide, is also called as paracetamol (PAR) and widely used as analgesicas well as antipyretic4. Caffeine (CFN) is chemically known as (1,3,7-trimethyl-1H-purine-2,6(3H,7H)-dione and acts as stimulant forcentral nervous system4. Diphenhydraminehydrochloride (DPH) is 2-(diphenylmethoxy)-N, N-dimethylethanamine hydrochloride, is a reversible H1 antagonist which is used in the symptomatic treatment of allergic diseases5. The structures of all the molecules are given in Fig. 1.
Thorough literature survey revealed that none of the articles were found with regard to simultaneous determination of these four drugs. Various publications have been reported on the simultaneous determination of either two or three combination of these four drugs in analytical methods. Dewani and co-workers1 were worked on determination of PNY, PAR and CFN by RP-HPLC method in the tablet formulation. They used gradient program mobile phase, consist of 10mM phosphate buffer (pH 3.3) and acetonitrile. The elution was taken place by three step gradient elution program and linearity ranges were of 5-100, 100-1000 and 10-200μgmL-1 for PNY, PAR and CFN respectively. A spectrophotometric method was developed and validated for the simultaneously determination of PAR and PNY in tablet formulation2. Another method revealed the HPLC method development and validation of ACN, PNY and CFN in tablet formulation with wavelength at 214nm and the mobile phase used was laborious like mobile Phase A (Dissolved 1gm of sodium salt of heptane sulphonic acid in 1liter of purified water. pH was adjusted to 3.0 with orthophosphoric acid) and mobile Phase B (Acetonitrile)3. Another method reported an isocratic RP-HPLC-DAD method for the simultaneous determination of PNY, PAR and CFN. These were eluted with mobile phase consisting of acetonitrile, methanol and 10mM phosphate buffer 16:22:62 (v/v) (pH 2.5±0.02 adjusted with orthophosphoric acid) and wavelength was fixed at 280 nm4. One more method was developed for rapid derivative spectrophotometric method for the simultaneous determination of ACN and DPH in tablets formulation5. Heydari has developed a HPLC method for the simultaneous determination of ACN and PNY in pharmaceutical formulations6. In this method the mobile phase consists of methanol and buffer in the ratio of 95:5% (v/v). Buffer was prepared by 6.0g of ammonium acetate and 10ml of triethylamine per liter, pH 5.0 was adjusted with orthophosphoric acid. Detection was carried out using a variable wavelength UV detector fixed at 254, 220nm for ACN and PNY respectively6. A spectrophotometric method for the simultaneous determination of PAR, PNY and chlorpheniramine maleate (CPM) in pharmaceutical preparations by using multivariate calibration was reported in the literature7. The HPTLC method was developed for the determination of ketorolac tromethamine (KTLT) with (PNY) (Mixture-1) and with febuxostat (FBXT) (Mixture-2) drug in the form of bulk as well as combined dosage forms by high-performance thin-layer chromatography (HPTLC)8. A derivative spectrophotometric method for the simultaneous analysis of CPM, PNY and phenylpropanolamine HCl in ternary mixtures and pharmaceutical dosage forms has been developed by Kazemipour and Ansarib9. The method reveals that PNY absorbed at 286.5nm and linearity range found 5-30µgmL-1. Deconincket. al.,10 have developed an ultra-high pressure liquid chromatographic method for quantification of paracetamol, acetyl salicylic acid and/or antihistaminics. In this method the resolution between PNY, CFN and Acetylesalicylic acid peaks were not good. And also the PNY was eluted with the void volume only, which is not acceptable. The reported UPLC method10 lacks with speed, simplicity, good specificity and excellent precision and accuracy like our method. The proposed method completes in 10 min, whereas the UPLC method completes in 11 min (a single minute is also accountable in chromatographic methods, where high cost mobile phases are in use). More over the sophisticated equipment like UPLC cannot be in the reach of most laboratories and small-scale industries. The literature survey revealed that many analytical methods are reported for the simultaneous determination of various combination drugs by employing different analytical instruments like HPLC and UV-visible spectroscopy11-20
Keeping the drawbacks of the reported methods in mind and also no single method reported in the literature for the simultaneous determination of PNY, ACN/PAR, CFN and DPH, we planned to develop a RP-HPLC method for the simultaneous determination of PNY, ACN/PAR, CFN and DPH drugs. The proposed method is simple, sensitive, accurate and has quick run time.
Fig. 1: Chemical structure of (A) Phenylephrine HCl (PNY), (B) Acetaminophen / Paracetamol (ACN/ PAR), (C) Caffeine (CFN), (D) Diphenhydramine HCl (DPH).
MATERIALS AND METHODS:
Instrumentation:
The method development and validation was performed by employing Shimadzu LC-2010 CHT. The instrument consists of deuterium lamp as source of light in UV detector, auto injector and quaternary pump. LC solution software was used to monitor and processed output signal. The pH was measured by using Eutech pH meter. Ultrasonic sonicator bath was used to degas all the solvents. Mobile phase was filtered through 0.45μm nylon membrane filter paper.
Chemicals and reagents:
The standards of PNY, ACN, CFN and DPH compounds used were above 98% pure. All the standards were provided by Karnataka antibiotics and pharmaceutical Ltd. Bangalore, India as gift samples. Analytical grade Triethylamine procured from Spectrochem Ltd. India. Methanol was procured from Merk Ltd, India. Ortho-phosphoric acid was purchased from SD fine chemicals; India. Ultra-purified water was used for the study. Waters Sunfire C18 column with 250mm length, ID4.6 cm and 5μ particle size column was employed for the study.
Standard Solutions: Preparation and Dilutions:
The preparation of standard solutions were developed as follows: Weighed 100mg of PNY, ACN CFN and DPH accurately and obtained in to the 100mL standard flask, mobile phase (Methanol and 0.1% v/v triethyleamine, pH-3.0, ratio 45:55) used as diluent and made up to the mark. From this solution 0.1mL was pipetted out into another 100mL standard flask and made upto mark with mobile phase. This secondary concentration of the solution gives 1.0μgmL-1. A calibration curve constructed by appropriate different concentrations of the working standard solutions, which were prepared by suitable dilution of the stock solution.
Chromatographic conditions:
The separation of the four peaks was carried out with the following chromatographic circumstances. The mixture of mobile phase consists of methanol and triethylamine (0.1% v/v) buffer pH-3.0 was adjusted with 0.1 M ortho-phosphoric acid in the ratio 45:55. This was used as diluent. The isocratic mixture of mobile phase flow rate was fixed at 1.0mLmin-1at ambient temperature. Stationary phase column with C18, 250mm, ID 4.6mm, 5μ particle size was used. At 220nm wavelength the four peaks were detected, 20μL injection volumes were fixed for the whole method development and validation process.
RESULTS AND DISCUSSION:
The stabilization of isocratic mobile phase was conceded initially along with column (waters sunfire) with C18 by using various concentrations of triethylamine and acetonitrile. In this mobile phase, the peaks (analytes) were established only for PAR and PNY. In an additional trial with 0.1% (v/v) triethylamine and methanol 75:25% (v/v) it was found that the expected analytes were not separated suitably. The various trails were conceded out with different ratios of 0.1% (v/v) triethylamine and methanol. Finally the mobile phase ratio was altered to 45:55% (v/v) the flow rate was fixed at 1.0mLmin-1. At these circumstances the analytes were satisfactorily eluted with good resolution and better chromatographic parameters. In the proposed method the total run time was 10 minutes. The wavelength was fixed at 220nm for the elution of these four peaks. The established method was followed by the ICH procedure21.
System suitability:
In the proposed method, the retention time (RT) was consistent for PNY, ACN, CFN and DPH at 2.2, 3.6, 4.5 and 6.42 minutes respectively. Throughout the analysis RT were not varied drastically. Percentage related to standard deviation for all six spikes given less than 2%of individuals at least concentrations. The outcomes obtained were 0.24, 0.68, 0.16 and 0.34% for PNY, ACN, CFN and DPH, respectively. The parameters of system suitability tabulated in Table 1. The representative chromatograms are given in Fig. 2. Limit of detection (LOD) were 1.0, 0.5, 1.5 and 2.5μgmL-1 and limit of quantification (LOQ) 3.3, 1.7, 5.0 and 8.3 μgmL-1 for PNY, ACN, CFN and DPH respectively. For these parameters all the four drugs values were found with better sensitivity. The outcomes are shown in Table 2.
Fig. 2: Chromatogram showing the clear resolution of PNY, CAN, CFN and DPH respectively
Table 1: System Suitability Parameters
|
Parameter |
PNY |
CAN |
CFN |
DPH |
Limit |
|
Number of theoretical plates |
3361.688 |
5455.637 |
6806.779 |
6992.998 |
NLT* 2000 |
|
Retention time (tR) in min |
2.20 |
3.68 |
4.50 |
6.42 |
- |
|
Resolution |
- |
8.42 |
3.91 |
7.38 |
NLT* 2.0 |
|
Capacity factor (k’) |
0.00 |
0.672 |
1.043 |
1.926 |
- |
|
Peak asymmetry (AS) |
1.489 |
1.322 |
1.275 |
1.264 |
NMT* 2.0 |
|
% RSD |
0.24 |
0.68 |
0.16 |
0.34 |
NMT* 2.0 |
*NLT – Not less than
*NMT – Not more than
Calibration curve:
In the calibration curve straight lines were produced by using peak area against six different concentrations. All these four peaks were eluted at six different concentrations of standard solutions. The results are as shown in Fig. 3. For all the four drugs regression coefficient (R2) values were found to be more than 0.999. The calibration concentrations of the solutions were 2.0-7.0, 1.5-7.5, 3.0-9.0 and 6.0-16.0 μgmL-1for PNY, ACN, CFN and DPH respectively. The outcomes are tabulated in Table 2.
Table 2: Calibration Curve Results, Limit of Detection And Limit Of Quantification
|
Parameter |
PNY |
ACN |
CFN |
DPH |
|
Linear dynamic range (μgmL-1) |
2.5-8.5 |
1.0-7.0 |
4.0-14.0 |
4.5-14.5 |
|
Slope (b) |
545654 |
577572 |
999171 |
1425030 |
|
Intercept (c) |
241364 |
125633 |
170986 |
170901 |
|
Correlation coefficient (r) |
0.9992 |
0.9999 |
0.9995 |
0.9993 |
|
LOD (μgmL-1) |
1.0 |
0.5 |
1.5 |
2.5 |
|
LOQ (μgmL-1) |
3.3 |
1.7 |
5.0 |
8.3 |
|
% RSD |
0.24 |
0.68 |
0.16 |
0.34 |
Ya = mX + c, where X is drug concentration in μgmL
Fig. 3: Calibration curves of PNY, CAN, CFN and DPH
Recovery: (Accuracy)
The recovery parameter was accomplished by the study of accuracy estimation. The concentrations of standard solutions were kept at lower, middle; upper and blank were spiked at 60, 80, and 120% against 100%. Recovery values were generated by the standard procedure 10 and established adequate results for all the four drugs well within the limit (98-102%). The consequences are shown in Table 3. The outcomes indicated that the method is perfect and precise.
Precision:
The validation was carried out for all the four drugs in combination by simultaneous determination. Results of precision parameter showed that the recognized procedure had improved values. The outcomes obtained from the studies were shown in Table 3. The repeatability experiment was conducted by spiking six individuals. The inter-day and intra-day analysis of the results showed that percentage of relative standard deviation for all the four drugs were found to be less than 2.0%. There were no much deviation and the results were obtained 0.24, 0.68, 0.16 and 0.34% RSD for PNY, ACN, CFN and DPH respectively. The outcomes are revealed in Table 3. The established results showed that method is more precise.
Robustness studies:
To examine the robustness of a proposed method, some of the parameters like flow rate, composition in the mobile phase and pH of the buffer were knowingly varied from unlike lots were taken and injected the known concentration solution. The distinct conditions like, modification in the mobile phase, change in the flow rate of the mobile phase to 0.9mLmin-1 and 1.1 mLmin-1, change in the column temperature at 250C and 300C resulted acceptable data for the analysis these four drugs. Robustness results were found to be satisfactory and there were no considerable difference in outcomes. The outcomes were shown in Table 4.
Table 3: Recovery and Precision Data
|
Concentrations |
Amount of drug taken |
Recovered Drug in Percentage |
||||||||
|
PNY |
%RSD* |
ACN |
%RSD* |
CFN |
%RSD* |
DPH |
%RSD* |
Limit |
||
|
60% |
60 mgmL-1 |
100.5 |
0.15 |
101.0 |
0.30 |
100.6 |
0.33 |
100.1 |
0.45 |
98-102% |
|
80% |
80 mgmL-1 |
101.0 |
0.12 |
99.5 |
0.44 |
100.2 |
0.47 |
99.2 |
0.31 |
98-102% |
|
120% |
120 mgmL-1 |
99.5 |
0.95 |
99.0 |
0.41 |
100.5 |
0.32 |
99.8 |
0.54 |
98-102% |
|
Intra-day |
|
0.63 |
|
0.53 |
|
0.34 |
|
0.62 |
NMT- 2.0 |
|
|
Inter-day |
|
0.51 |
|
0.61 |
|
0.49 |
|
0.39 |
NMT- 2.0 |
|
*RSD: Relative Standard deviation
Table 4 Robustness Evaluation Parameters
|
Parameters |
Deliberate Variations |
PNY Retention time |
ACN Retention time |
CFN Retention time |
DPH Retention time |
|
Flow rate |
0.9Ml min-1 |
2.49 |
3.97 |
4.77 |
7.01 |
|
|
1.0mL min-1 |
2.26 |
3.60 |
4.32 |
6.50 |
|
|
1.1mL min-1 |
2.04 |
3.29 |
3.95 |
5.62 |
|
Temperature |
25°C |
2.05 |
3.29 |
3.95 |
5.50 |
|
|
30°C |
2.04 |
3.28 |
3.95 |
5.54 |
Table 5: Assay Results
|
Name of the drugs |
Label claims of market sample in 5 mg mL-1 |
Obtained result in mg 5 mL-1 |
Assay values (%) |
Limit |
|
PNY |
5.0 |
4.95 |
99.0 |
98.00-102% |
|
ACN |
500.0 |
498.0 |
99.5 |
98.00-102% |
|
CFN |
30.0 |
30.2 |
100.6 |
98.00-102% |
|
DPH |
25.0 |
24.9 |
99.8 |
98.00-102% |
Ruggedness:
In this parameter the HPLC method used for the analysis of standard working solutions was examined with the same chromatographic system on different days. Tiny differences in area ratios, good consistency and there were no much diverges in retention time. The RSD values for areas were found less than 2.0% for four drugs. The data is mentioned in Table 1. Data showed the ruggedness of the method. On different days, the same detector responses obtained adequately that the method is able to generate results with high precision on different days. Different HPLC instruments were examined the ruggedness of the method by injecting the known concentration of solution. The retention time and good reproducibility of the detector response showed that the proposed method is experimentally rugged.
Specificity:
The specificity of the method was carried out by the chromatogram where successive resolution of PNY, ACN, CFN and DPH were developed against potential interferences in the presence of placebo (diluents i.e., acetonitrile and buffer (0.1% triethylamine). In the assay, there were no interventions of all the four peaks obtained and they were sharp and well separated at the baseline. In formulation, excipients were not interfering at the retention times of PNY, ACN, CFN and DPH in sample solution. Hence the proposed method was found specific. The outcome data were revealed that the values were found to be acceptable and are tabulated in Table 5.
DISCUSSION:
Most of the cough syrup formulations contain PNY, ACN, CFN and DPH to relieve from the cough, cold and ache. Some of the liquid chromatographic methods were found in the literature for the determination of PNY, ACN, CFN and DPH. The proposed RP-HPLC method for the simultaneous determination of these drugs is isocratic, accurate, simple, sensitive and robust. The proposed method is economic compared to other HPLC methods, having highly sensitive and short run time. The precision of intra and inter-day data were acceptable and recovery values were within the range of limit. All the parameters of proposed method results were acceptable and can adopt in academic as well formulation analysis of these drugs in combination. The result of all the parameters obeys with ICH guidelines. Since the method is accurate, precise, simple and economic, it can be adopted for academic as well as in pharmaceutical industries for the assay of PNY, ACN, CFN and DPH.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 04.02.2021 Modified on 24.12.2021
Accepted on 07.06.2022 © RJPT All right reserved
Research J. Pharm. and Tech 2022; 15(9):3993-3998.
DOI: 10.52711/0974-360X.2022.00669