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Validated ultra-performance liquid chromatographic and thin-layer chromatographic–densitometric methods for the determination of paracetamol, pamabrom, and pyrilamine maleate
Abstract
Two chromatographic methods were developed for the simultaneous determination of paracetamol, pamabrom, and pyrilamine maleate in bulk and combined pharmaceutical dosage form. The first method is an ultra-performance liquid chromatographic (UPLC) method, in which separation was carried out by gradient elution using C18 column and a mobile phase composed of solution A (acetonitrile) and solution B (phosphate buffer) (pH 3.5). The elution started with 20% (by volume) acetonitrile ramped up linearly to 100% in 2 min, then kept constant till the end of the run at a flow rate of 1.5 mL min⁻¹ and ultraviolet (UV) detection at 277 nm. The second method depends on the densitometric determination of thin-layer chromatograms of the three drugs. Separation was carried out at 275 nm using chloroform‒acetonitrile (15:35, v/v) as the mobile phase. The proposed methods were validated according to the International Conference on Harmonisation (ICH) guidelines. Beer’s law was obeyed in the range of 5–100 μg mL⁻¹ for paracetamol and 0.5–20 μg mL⁻¹for pamabrom and pyrilamine maleate, respectively, with mean recoveries of 98.40‒100.32% ± 0.551‒0.771 for the UPLC method. Linearity of the thin-layer chromatographic method was achieved in the range of 10‒280, 5‒45, and 1–20 ng per spot of the three drugs with mean recoveries of 98.75‒100.30% ± 0.971‒1.061, respectively. The two methods were successfully applied for the simultaneous determination of the cited drugs in their laboratory-prepared mixtures and pharmaceutical dosage form with good accuracy and precision. The results obtained were compared with those of the reported method and found to be in good agreement.
... Thin-layer chromatography is usually employed as a fast screening method in different stages of monitoring processes including synthesis, isolation and biological studies. Numerous reports dealing with TLC employment in control of drugs quality for inspection of quantity and quality of active compounds [57][58][59][60][61][62][63][64][65][66][67][68][69] but also for control of the presence of accompanying substances [70,71] are available in the recently published literature. As examples, some of papers relating to TLC stability studies of biologically active compounds in pharmaceutical formulations under acidic, alkaline, oxidative, photolytic and dry heat degradation conditions are listed in Table 2. ...
Among widely used chromatographic methods modern thin-layer chromatography is not only the simplest to perform but is also considered as respectable analytical method in various phases of drug discovery and development processes such as monitoring of synthesis, identification of bioactive substances from various natural sources and their isolation and purification, determination of lipophilicity and other physico-chemical parameters, quantitative structure-activity relationship studies, bioautography, as well as qualitative and quantitative analysis of drugs and their metabolites. An overview of recently published papers dealing with application of thin-layer chromatography in medicinal chemistry is presented.
Simultaneous chromatographic determination of both ertapenem (ERP) and paracetamol (PCL) was achieved for the first time via a new HPTLC-densitometric method. The developed method relied on a simple planar chromatographic separation by mixture of acetonitrile and water (35:15, v/v) and dual-wavelength detection at λmax of both drugs (294 and 247 nm for ERP and PCL, respectively). The Rf values were 0.74 ± 0.05 and 0.88 ± 0.05 for ERP and PCL, respectively. Under the optimized conditions, the linearity range was 40.0–600.0 and 15.0–200.0 ng band⁻¹ for ERP and PCL, respectively with good correlation coefficients (0.9997 and 0.9998 for ERP and PCL, respectively). The LOD were 13.08 and 3.65 ng band⁻¹ for ERP and PCL, respectively. The method validation parameters were in compliance with the international standards. The developed method was enough sensitive and selective for the determination of ERP in the presence of its open beta-lactam ring degradation product, which makes it a suitable stability-indicating method. Consequently, it was successfully applied for pharmaceutical formulation, spiked and real plasma samples of healthy rabbits for real pharmacokinetic studies of ERP and PCL.
A new, simple and sensitive spectrophotometric method for the determination of paracetamol has been developed. The proposed method is based on the reaction of paracetamol with iron(III) and a subsequent reaction with ferricyanide in an hydrochloric acid medium to yield Prussian bluish green coloured product with a miximum absorption at 715 nm. There were no interferences observed from the common excipients present in the formulations. The method is successfully employed for the determination of paracetamol in various pharmaceutical preparations and the results have been statistically compared with those obtained by the official method.
A new high performance liquid chromatographic (HPLC) method was developed for the determination of paracetamol, phenylephrine hydrochloride, oxolamine citrate and chlorpheniramine maleate in combined pharmaceutical formulations and dosage forms. The separation was performed on an Agilent Zorbax SB-CN column with the mobile phase consisting of 0.02 M phosphate buffer (pH:4) and acetonitrile (85:15,v/v) in flow rate 1.5 mL at 22 °C. The overall retention time of the analytes was 3.5 min. The method was validated with respect to linearity, precision, accuracy and recovery. The relative standard deviation for 10 replicate measurements of paracetamol, phenylephrine HCl, oxolamine citrate and chlorpheniramine maleate were 0.12, 0.36 0.18 and 0.59%, respectively. Total recoveries of analytes were 99.99, 100.56, 100.20 and 99.60%, respectively. No chromatographic interference from the tablet excipients was found. The linearity of paracetamol, phenylephrine HCl, oxolamine citrate and chlorpheniramine maleate were in the range of 20-120 μg/mL, 0.4-2.4 μg/mL, 8-48 μg/mL and 0.16-0.96 μg/mL, respectively. This simple, fast, economical and precise high performance liquid chromatographic method can be adopted for routine quality control analysis.
A method is described for the simultaneous determination of paracetamol, tizanidine, and diclofenac in mixtures. The method was based on HPLC separation of the three drugs followed by UV detection at 254 nm. The separation was carried out on a Hypersil ODS, C18 (250 x 4.6 mm id, 10 microm particle size) column using the mobile phase aqueous 0.2% ammonium carbonate-methanol (60 + 40, v/v) at a flow rate of 1 mL/min. The linear regression analysis data were used for the regression curve in the range of 170-10 000 ng/mL for paracetamol, 120-10 000 ng/mL for tizanidine, and 20-10 000 ng/mL for diclofenac. No chromatographic interference from tablet excipients was found. In order to check the selectivity of the proposed method, degradation studies were carried out using hydrolysis (acid, basic, and neutral), thermolysis, and oxidation. The developed method, after being validated in terms of precision, robustness, recovery, LOD, and LOQ, was successively applied to the analysis of pharmaceutical formulations and human serum.
Two simple, rapid, and selective analytical procedures were developed for the simultaneous determination of paracetamol (PR) and tramadol hydrochloride (TR) in a binary mixture using high-performance liquid chromatography with UV detection (HPLC-UV) and gas chromatography with mass spectrometry (GC-MS) techniques. HPLC resolved the two compounds on a Hypurity Advance column using a mobile phase consisting of phosphate buffer pH 6.3 and acetonitrile (90:10, v/v). PR and TR were detected by their UV absorption at 220 nm. GC-MS involved separation of the two compounds using 100% dimethylpolysiloxane (Rtx-1) column with temperature programming. The EI mass spectrum of PR was characterized by [M](+) at 151 and a base peak at m/z 109 while TR mass spectrum was characterized by [M](+) at 263 and a base peak at m/z 58. Quantification of the analytes in both methods was based on measuring the peak areas. The reliability and analytical performance of the proposed methods including linearity, ranges, precision, accuracy, detection, and quantification limits were statistically validated. Calibration curves were linear over the range 10-400 microg/mL for both PR and TR using the HPLC method and over the ranges of 75-500 and 25-350 microg/mL for PR and TR, respectively, using the GC-MS method. The proposed methods were successfully applied for the determination of the two compounds in laboratory-prepared mixtures and in commercially available tablet formulation. No interference peaks were observed from common pharmaceutical adjuvants. The results compared favorably with those obtained by a derivative spectrophotometric method.
A rapid method for determining paracetamol (acetaminophen) in whole blood and liver tissue samples is described. Blank plus
single-point calibration gives reliable quantitation at therapeutic and higher concentrations. Whole blood and liver tissue
samples containing a deuterated internal standard were extracted using Bond Elut Certify columns. Butyl derivatives were formed
using n-iodobutane and tetramethyl ammonium hydroxide under mild conditions and were extracted into ethyl acetate as a cleanup step.
Recovery was better than 90%, and sample preparation time was less than 2 h. Gas chromatograph run time was less than 20 min.
SIM of two ion pairs formed by electron impact ionization resulted in intraday coefficients of variation (CV) less than 3.03%
(7.48% in liver) and interday CVs less than 8.93% (for midtherapeutic concentrations in whole blood). Linearity was observed
from subtherapeutic to high, fatal levels. This method has been applied to forensic cases and has significantly reduced analytical
time while improving casework quality. Results of a case study involving paracetamol are given.
A comparative study of the use of first derivative zero order crossing spectra for the resolution of Paracetamol and Tramadol hydrochloride in mixtures has been achieved showing the success of the first derivative method in resolving and quantifying both compounds. Using the first derivative, rather than the second derivative, results in improved signal to noise ratio. The absorption spectra of prepared mixtures were scanned in the range of 200-500 nm. The linear concentration ranges were 25-112 and 6-48 µg mL -1 for paracetamol and Tramadol hydrochloride, respectively. The method has been successfully used for prediction of concentrations of both compounds in mixtures with good selectivity, high sensitivity and extremely low relative error. Statistical comparison was performed using t-test at 95% confidence level. There was no significant statistical difference between the results obtained by the first derivative method and the accepted values for both compounds. Also, the percentage errors were very low which adds to the merits of our work in terms of both sensitivity and accuracy.
Two chromatgraphic methods were developed for determination of Paracetamol (PCM) and Pamabrom (PAM) in presence of P-aminophenol (PAP) and Theophylline (THEO) as potential impurities of both drugs respectively. First method is HPTLC which depends on separation and quantitation of the studied drugs on aluminum plates pre-coated with silica gel 60 F254 as a stationary phase using chloroform:methanol:ethyl acetate:glacial acetic acid (8:0.8:0.6:0.2, v/v/v/v) as mobile phase followed by densitometric measurement of the bands at 254nm. Second method is RP-HPLC which comprises separation of the studied drugs on a Phenomenex C8 column by gradient elution using mobile phase consisting of sodium dihydrogen phosphate buffer (0.05M): methanol:acetonitrile (85:10:5, v/v/v) at a flow rate of 1mL/min for first 7.5min and (70:20:10, v/v/v) at a flow rate of 1.5mL/min for the next 5min. The proposed methods were successfully applied for determination of the potential impurities of PCM and PAM after resolving them from the pure drugs. The developed methods have been validated and proved to meet the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated methods were successfully applied for determination of the studied drugs in their pharmaceutical formulation. The results were statistically compared to those obtained by the reported RP-HPLC method where no significant difference was found; indicating the ability of proposed methods to be used for routine quality control analysis of these drugs.
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Purpose: To develop a stability indicating RP-HPLC method for a combination drug product containing a high dose of paracetamol (PR) and low doses of domperidone (DM) and tramadol HCL (TR).Methods: The analytes are well separated by a reverse phase column and an isocratic mobile phase consisting of 0.1 %v/v trifluoroacetic acid: acetonitrile: methanol in the ratio 70:25:5 (v/v) with a flow rate of 1.0 mL/min. The effluent was monitored at 272 nm. The drug products were subjected to stress conditions of acid, base, peroxide, thermal and photolytic degradation and peak homogeneity of PR, TR and DM were obtained using photo diode array detector.Results: The degradation products were well resolved from PR, TR and DM peaks, thus indicating the stability-indicating nature of the method. The assay was linear from 165 – 495 μg/mL for PR, 18.75 – 56.25 μg/mL for TR, and 5 – 15 μg/mL for DM. Although the tablet contained high and low doses of the drugs, the intra- and inter-day variations were < 2.0 %.Conclusion: The proposed method was validated according to the ICH guidelines and proved suitable for stability and homogeneity testing, as well as for quality control of the combined drugs in pharmaceutical preparations.
A fast, simple, specific and accurate stability indicating liquid chromatographic method is described for simultaneous determination of piroxicam and paracetamol in bulk drugs and pharmaceutical formulations. Optimum chromatographic separations among the piroxicam, paracetamol and stress-induced degradation products were achieved within 6 min by using Hypersil BDS C8 column as stationary phase with acetonitrile and 0.02 M phosphate buffer pH 3.0 (60:40, v/v) as mobile phase at a flow rate of 1.0 mL min with detection using diode array detector at 254 nm. ICH guidelines were used to validate the developed method. Linearity was from 1.6-6.4 µ g mL for piroxicam and 26-104 µ g mL for paracetamol. All the analytes including the degradation products were separated with acceptable peak tailing and resolution. The peak purity index for both the analytes after all types of stress is > 0.999 indicating complete separation of both analytes peaks from the stress induced degradation products. The developed method can be successfully used for simultaneous determination of piroxicam and paracetamol in pharmaceutical formulations and stability studies.
A validated HPLC method was developed for the analysis of paracetamol, psedoephedrine, triprolidine, methylparaben, propylparaben, sodium benzoate and their related substances including p-aminophenol, triprolidine Z-isomer, 4-hydroxy benzoic acid and 4-chloracetailide in syrup using symmetry C18 column at 25°C with UV detection at 214 nm. A linear gradient elution was employed starting with 100% mobile phase A and 0% mobile phase B for 5 min to reach 40% mobile phase A and 60% mobile phase B at 16 min then 0% mobile phase A and 100% mobile phase B at 28 min. Total run time is 30 min using solution of 28 mM sodium dihydrogen phosphate containing 2.6 mM hexanesulphonic acid sodium salt and adjusted to apparent pH 3.0 with phosphoric acid–acetonitrile in ratios of (90:10 v/v) and (60:40 v/v) as mobile phase A and mobile phase B, respectively. All mentioned compounds have been successfully separated and quantified using the developed method. The developed method was linear with (r = 0.9999) for all compounds. The proposed method was completely validated.
Combined high-performance liquid chromatography/thermospray mass spectrometry (HPLC/TSMS) and HPLC/thermospray tandem mass spectrometry (HPLC/TSMS/MS) were utilized for the analysis of rat urine for metabolites of pyrilamine. The sample was analyzed via HPLC/TSMS/MS in the parent ion mode in order to identify potential metabolites of pyrilamine. Then HPLC/TSMS/MS analysis in the daughter ion mode was performed to provide additional analytical selectivity plus enhanced fragmentation of suspected protonated molecules. By this methodology, suspected pyrilamine metabolites were confirmed to be in the sample and several novel metabolites of pyrilamine were discovered and tentatively identified.
A procedure is described for determining unconjugated paracetamol in plasma and urine. The method, based on gas chromatography of the unchanged drug, is more rapid than a spectrophotometric assay and has a limit of detection of 2 μg/ml.Plasma and urine levels are reported after therapeutic and overdose ingestion.
A rapid, sensitive and simple spectrophotometric method is proposed for the determination of hydrolysis products of paracetamol (PRL) and phenacetin (PHN) with sodium 1,2-naphthoquinone-4-sulphonate and cetyltrimethyl ammonium bromide (CTA) in alkaline medium. The absorbances are measured at 570 and 500 nm and the molar absorptivities found to be 1.118 x 10(4) and 4.54 x 10(3) l mol-1 cm-1 for PRL and PHN, respectively. The coloured species conforms to Beer's law over the range 1-20 micrograms ml-1 for PRL and 2-24 micrograms ml-1 of PHN. The sensitivity is enhanced by the addition of CTA. The method is successfully employed for determination of PRL or PHN in various pharmaceutical preparations and laboratory made tablets and results have been statistically compared with those obtained by the official method.