DAD Method Research Articles

Simultaneous determination of eight adulterants in weight management supplements and herbs by HPLC–DAD and LC–MS/MS

ABSTRACTAn efficient HPLC–DAD method was developed for simultaneous determination of eight adulterants in weight management supplements and herbs. The eight adulterants were phenolphthalein, sibutramine, nuciferine, and five anthraquinone compounds including aloe-emodin, rhein, emodin, chrysophanol, and physcion. The analytes were ultrasonically extracted with 70% (v/v) methanol aqueous solution followed by centrifugation. The supernatant was subjected to HPLC analysis. A Phenomenex Luna C18 column was applied for chromatographic separation. The mobile phase was consisted of methanol and aqueous solution of 0.05% (v/v) phosphoric acid–0.025% (m/v) sodium dodecyl sulfonate. The flow rate of mobile phase was 0.8 ml min−1 with gradient elution. Clenbuterol and ibuprofen were used as internal standards. The retention times and the characteristic UV spectrograms were used for qualitative analysis. Quantifications were based on the internal standard curves. Good linearities (r > 0.9996) for all analytes were obtained with the intra- and inter-day precision (n = 6) ranging from 0.76 to 5.9% and 0.90 to 8.1%, respectively. The average recoveries from the spiked samples with different matrices varied from 73.4 to 114%. Validations were subsequently performed using LC–MS/MS. The proposed method successfully determined the target adulterants in eight commercial weight management supplements and five weight reducing herbs with satisfactory results.

Proliferative and antioxidant activity of Symphytum officinale root extract

The root of Symphytum officinale L. is commonly used in folk medicine to promote the wound healing, reduce the inflammation and in the treatment of broken bones. The objective of our investigation was to analyse the extract from S. officinale in term of its antioxidant activity and the effect on cell viability and proliferation of human skin fibroblast (HSF). Moreover, the quantification of main phenolics and allantoin was conducted using HPLC–DAD method. Five compounds were found: rosmarinic, p-hydroxybenzoic, caffeic, chlorogenic and p-coumaric acid. DPPH, FRAP and TPC assay showed the high antioxidant activity of the extract. MTT test proved the stimulatory effect on cell metabolism and viability of HSF cells. Moreover, no changes in cytoskeleton structure and cells shape were observed. The obtained results indicate that non-toxic extract from S. officinale root has strong antioxidant potential and a beneficial effect on human skin fibroblasts.

Simultaneous Determination of Six Compounds in Hedera helix L. Using UPLC-ESI–MS/MS

We previously reported a method for simultaneously determining flavonoids and saponins from ivy leaf extract using HPLC–DAD. Due to the absence of a chromophore in two triterpene saponins, α-hederin and hederacoside C, the previous method was conducted at a non-specific wavelength of 210 nm. Thus, in this study, we focused on a rapid and sensitive analysis method for determining two important triterpene saponins from ivy leaf extract using UPLC–ESI–MS/MS. The developed method was validated in terms of linearity (R 2 > 0.9997), precision (RSD < 2.95%), and accuracy (98.91–101.92%), and showed increased sensitivity with improved limit of detection when compared to the previous data. In addition, the analytical conditions of UPLC–ESI–MS/MS provided better sensitivity with a shorter analysis time when compared to the HPLC–DAD method. Using this established method, the content of the six compounds was measured in medicinal syrups containing ivy leaf extract and the feasibility of this new method was demonstrated for the use in the quality control of ivy leaf extract products.

Validated Spectrophotometric and RP-HPLC–DAD Methods for the Determination of Ursodeoxycholic Acid Based on Derivatization with 2-Nitrophenylhydrazine

This work describes the development, validation, and application of two simple, accurate, and reliable methods for the determination of ursodeoxycholic acid (UDCA) in bulk powder and in pharmaceutical dosage forms. The carboxylic acid group in UDCA was exploited for the development of these novel methods. Method 1 is the colorimetric determination of the drug based on its reaction with 2-nitrophenylhydrazine hydrochloride in the presence of a water-soluble carbodiimide coupler [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride] and pyridine to produce an acid hydrazide derivative, which ionizes to yield an intense violet color with maximum absorption at 553 nm. Method 2 uses reversed-phase HPLC with diode-array detection for the determination of UDCA after precolumn derivatization using the same reaction mentioned above. The acid hydrazide reaction product was separated using a Pinnacle DB C8 column (4.6 × 150 mm, 5 μm particle size) and a mobile phase consisting of 0.01 M acetate buffer (pH 3)-methanol-acetonitrile (30 + 30 + 40, v/v/v) isocratically pumped at a flow rate of 1 mL/min. Ibuprofen was used as the internal standard (IS). The peaks of the reaction product and IS were monitored at 400 nm. Different experimental parameters for both methods were carefully optimized. Analytical performance of the developed methods were statistically validated for linearity, range, precision, accuracy, specificity, robustness, LOD, and LOQ. Calibration curves showed good linear relationships for concentration ranges 32-192 and 60-600 μg/mL for methods 1 and 2, respectively. The proposed methods were successfully applied for the assay of UDCA in bulk form, capsules, and oral suspension with good accuracy and precision. Assay results were statistically compared with a reference pharmacopeial HPLC method, and no significant differences were observed between proposed and reference methods.

HPLC–DAD Method for Simultaneous Determination of Dipyrone (Metamizole) and Caffeine in Tablets and Identification of Major Degradation Product by Direct Infusion ESI–MS

A new chromatographic method for simultaneous determination of dipyrone (metamizole) and caffeine in tablets was developed and validated. Excellent resolution and quantification were obtained using a reverse-phase Acclaim® C18 column (250.0 × 4.6 mm; 5.0 μm particles) with acetonitrile/water (65:35 v/v) as mobile phase, adjusted to pH 7.5 using drops of water:triethylamine (99:1 v/v). The elution mode was isocratic with flow rate of 1.0 mL min−1, and ultraviolet (UV) detection was performed at 273 nm. The ranges of linearity for dipyrone and caffeine were 5.0–35.0 and 1.0–7.0 μg mL−1, respectively. Intra- and interday precision showed relative standard deviation (RSD) 0.05, respectively. The method proved to be precise, sensitive, and exact with low limits of detection and quantification and average recovery of 99.3 and 100.8%. The factorial model of Youden was used to analyze the robustness of the method, while identification of critical factors was conducted by two-tailed t test with 95% confidence interval. The method was shown to be selective, detecting presence of analytes in degradation products originating from study of hydrolytic stability at different pH values (5.0, 6.0, 7.0, and 9.0) at room temperature. Identification of product degradation by direct injection electrospray ionization (ESI) mass spectroscopy (MS) revealed the presence of a fragment ion at m/z 187 and ions corresponding to [M + H]+ and [M + Na]+ at m/z 218 and 240, respectively, confirming the identity of the 4-methylaminoantipyrine product related to dipyrone.

Stability-indicating RP- HPLC -DAD method for the simultaneous estimation of Tramadol HCl and Diclofenac sodium

Stability-indicating RP- HPLC -DAD method for the simultaneous estimation of Tramadol HCl and Diclofenac sodium

Discrimination of Alismatis Rhizoma based on chromatographic fingerprint, multiple components quantification, and pattern recognition analysis

ABSTRACTA simple, sensible, and reliable HPLC–DAD method was first developed for fingerprint analysis of Alismatis Rhizoma, and then applied to analyze 85 samples from three main cultivated areas. In all, 40 common fingerprint peaks were designated, and six of which were definitely identified. Then, the combinatory analysis using similarity evaluation, principal component analysis, and orthogonal partial least square discriminant analysis revealed clear chemical consistency between samples from Fujian and Jiangxi provinces and substantial differences between those from Fujian/Jiangxi and Sichuan provinces. Furthermore, six components were dug out as potential chemical markers for distinguishing Alismatis Rhizoma from different areas, among which five were qualified for quantitative analysis. In conclusion, the combination of chemical fingerprint, multiple components quantification, and pattern recognition analysis was rather powerful and useful in discriminating Alismatis Rhizoma from different regions, which was a benefit for quality control.

Development and optimization of an HPLC–DAD method for quantification of six petroleum hydrocarbon compounds in aqueous samples

ABSTRACTBenzene, toluene, and xylene isomers (BTXs) and pyrene, benzo(b)fluoranthene, and benzo(a)pyrene (polycyclic aromatic hydrocarbons) are common pollutants found in many industrial effluents and in aquifers due to fossil fuels spill from underground storage reservoirs. For these reasons, the determination of these compounds has gained importance in the last decades. In this work, a simultaneous, fast, and accurate quantification of six petroleum hydrocarbon compounds (such as BTXs, pyrene, benzo(b)fluoranthene, and benzo(a)pyrene) using a high-performance liquid chromatography–diode array detector method has been demonstrated. The proposed method is suitable for the direct aqueous sample evaluation and also brings advantages, including the use of small volumes of organic solvents, with high resolution, reducing the analysis cost. The method was also checked using synthetic and real samples, including those containing surfactants, commercial gasoline, and river water samples spiked with petroleum hydrocarbon compounds.

Verification of the selectivity of a liquid chromatography method for determination of stilbenes and flavonols in red wines by mass spectrometry

Quantification of bioactive phenols, like stilbenes and flavonols (SaF), has been conducted to evaluate the nutraceutical potential of red wines. However, there is still a lack of full validated, fast and accessible liquid chromatography methods offering high selectivity and a simple procedure. We present here the use of a high-resolution mass spectrometer to evaluate the selectivity of a feasible and traditional liquid chromatography technique (HPLC–DAD) to analyze markers of aglycone SaF in red wines. The SaF compounds were tested: trans-resveratrol, trans-e-viniferin, quercetin, myricetin, and kaempferol, as well as trans-cinnamic acid, one of their precursors. System suitability and validation tests were employed for the selected conditions (octylsilane column, methanol mobile phase, and gradient elution). The validation process ensured the HPLC–DAD method was selective, linear, sensitive, precise, accurate and robust. The method was then applied to red wine samples from the Campanha Gaucha region, Southern Brazil. The real samples contained different SaF levels, showing that the method is applicable to routine use. Furthermore, this was the first SaF characterization of red wines from the Campanha Gaucha, contributing to regional and product development.

Development of an Advanced HPLC–MS/MS Method for the Determination of Carotenoids and Fat-Soluble Vitamins in Human Plasma

The concentration of carotenoids and fat-soluble vitamins in human plasma may play a significant role in numerous chronic diseases such as age-related macular degeneration and some types of cancer. Although these compounds are of utmost interest for human health, methods for their simultaneous determination are scarce. A new high pressure liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) method for the quantification of selected carotenoids and fat-soluble vitamins in human plasma was developed, validated, and then applied in a pilot dietary intervention study with healthy volunteers. In 50 min, 16 analytes were separated with an excellent resolution and suitable MS signal intensity. The proposed HPLC–MS/MS method led to improvements in the limits of detection (LOD) and quantification (LOQ) for all analyzed compounds compared to the most often used HPLC–DAD methods, in some cases being more than 100-fold lower. LOD values were between 0.001 and 0.422 µg/mL and LOQ values ranged from 0.003 to 1.406 µg/mL, according to the analyte. The accuracy, precision, and stability met with the acceptance criteria of the AOAC (Association of Official Analytical Chemists) International. According to these results, the described HPLC-MS/MS method is adequately sensitive, repeatable and suitable for the large-scale analysis of compounds in biological fluids.

Phenols and Volatiles of Istarska Bjelica and Leccino Virgin Olive Oils Produced with Talc, NaCl and KCl as Processing Aids

AbstractThe effect of processing aids (2.5 % of talc, NaCl or KCl) on oil extractability and the profile of phenolic and volatile compounds of Istarska bjelica and Leccino oils was studied. Talc significantly increased extractability in both cultivars, while salts increased extractability in Leccino cv. In the laboratory extracted oils, phenols were determined by a RP‐HPLC–DAD method, whereas volatiles were determined by SPME/GC–MS. Talc addition significantly decreased hydroxytyrosol and increased ligstroside derivatives in produced oils, but did not affect the total phenol content. Among volatile compounds, only Z‐2‐penten‐1‐ol in Leccino and 1‐pentene‐3‐one in Istarska bjelica oils significantly increased by talc addition. Salts improved transfer of most individual phenols into oil, particularly oleuropein derivatives, and increased C6 aldehydes and C5 volatiles in Leccino oils. NaCl exerted a stronger effect in increasing individual phenols and volatiles than KCl.

High-Performance Liquid Chromatography with Diode Array Detector and Electrospray Ionization Ion Trap Time-of-Flight Tandem Mass Spectrometry to Evaluate Ginseng Roots and Rhizomes from Different Regions.

Ginseng, Panax ginseng C. A. Meyer, is an industrial crop in China and Korea. The functional components in ginseng roots and rhizomes are characteristic ginsenosides. This work developed a new high-performance liquid chromatography coupled with electrospray ionization ion trap time-of-flight multistage mass spectrometry (LC–ESI-IT-TOF-MSn) method to identify the triterpenoids. Sixty compounds (1–60) including 58 triterpenoids were identified from the ginseng cultivated in China. Substances 1, 2, 7, 15–20, 35, 39, 45–47, 49, 55–57, 59, and 60 were identified for the first time. To evaluate the quality of ginseng cultivated in Northeast China, this paper developed a practical liquid chromatography–diode array detection (LC–DAD) method to simultaneously quantify 14 interesting ginsenosides in ginseng collected from 66 different producing areas for the first time. The results showed the quality of ginseng roots and rhizomes from different sources was different due to growing environment, cultivation technology, and so on. The developed LC–ESI-IT-TOF-MSn method can be used to identify many more ginsenosides and the LC–DAD method can be used not only to assess the quality of ginseng, but also to optimize the cultivation conditions for the production of ginsenosides.

Investigation on secondary metabolite content and antioxidant activity of commercial saffron powder

The secondary metabolites and the antioxidant properties of twelve commercial saffron powder samples, purchased from Italian large-scale market, were studied. An HPLC–DAD method was applied to determine the content of trans-4-digentiobioside crocetin ester (trans-crocin-4), picrocrocin, and safranal, while the antioxidant activity of methanolic extracts of saffron samples was tested using two in vitro assays, DPPH and ABTS. The samples were also subjected to alkaline hydrolysis before testing. The content of trans-crocin-4 ranged from 1.7 to 7.5/100 g, while picrocrocin content varied from 2.6 to 6.7/100 g. Using DPPH test, all saffron samples showed very low radical scavenging activity, while higher values were observed after hydrolysis. As regards ABTS assay, saffron samples exhibited higher antioxidant activity against radical than the respective hydrolyzed. Interestingly, correlations between DPPH values of hydrolyzed samples and ABTS values of methanolic extracts were obtained. Moreover, a significant correlation (R 2 = 0.5732) between ABTS values and trans-crocin-4 content of saffron samples was observed. The obtained results contribute to the characterization of saffron powder, the most used and economic but also the less studied form of saffron.

HPLC–DAD Method for the Quantification of Carbamazepine, Oxcarbazepine and their Active Metabolites in HepaRG Cell Culture Samples

A new, sensitive and fast high-performance liquid chromatography–diode-array detection assay is herein reported, for the first time, to simultaneously quantify carbamazepine (CBZ), oxcarbazepine (OXC), and the active metabolites carbamazepine-10,11-epoxide (CBZ-E) and licarbazepine (LIC) in HepaRG cell culture medium samples. Chromatographic separation of analytes (CBZ, CBZ-E, OXC, LIC) and internal standard (IS) was achieved in less than 15 min on a C18-column, at 35 °C, using a mobile phase composed of water/methanol/acetonitrile (69:25:6 v/v/v) pumped at 1 mL min−1. The analytes and IS were detected at 215 nm. The method proved to be selective, accurate (bias ± 14.6 %), precise (coefficient of variation ≤13.1 %) and linear (r 2 ≥ 0.9901) over the concentration ranges of 0.1–15 μg mL−1 for CBZ; 0.1–5 μg mL−1 for CBZ-E and OXC; and 0.1–40 μg mL−1 for LIC. Furthermore, the absolute recovery of the analytes ranged from 64.5 to 96.9 % and their stability was demonstrated in the studied conditions. This validated HPLC assay will be a suitable tool to support future in vitro metabolism profiling, drug interaction and other pharmacokinetic-based studies in HepaRG cells involving these antiepileptic drugs (CBZ and OXC) and their main metabolites.

Solid-phase extraction of flavonoids in honey samples using carbamate-embedded triacontyl-modified silica sorbent

In this study, carbamate-embedded triacontyl-modified silica (Sil-CBM-C30) is successfully prepared and used as an efficient sorbent for solid-phase extraction. The extraction performance of the resultant sorbent is evaluated with five flavonoids including myricetin, quercetin, luteolin, kaempferol and isorhamnetin. Main parameters, which affect extraction efficiencies, are carefully investigated and optimized. Comparative experiments between Sil-CBM-C30 and commercial C18 sorbents indicate that the extraction efficiencies of the former one surpass the latter one. The modification of carbamate-embedded triacontyl group on surface of silica causes analytes extracted by hydrophobic, hydrogen bonding and π–π interactions. Under optimal conditions, good linearities and satisfied LODs and LOQs are achieved. The SPE–HPLC–DAD method is successfully developed and applied for the honey sample analysis.

A model of dynamic adsorption–diffusion for modeling gas transport and storage in shale

Understanding gas transport and storage processes is essential for analysis of reservoir accumulation mechanisms and evaluation of a shale formation. Because organic matter such as kerogen is widely distributed in shale, it plays an important role in gas diffusion and adsorption. Although many mathematical models considering gas diffusion and adsorption have been proposed and evaluated, very few models consider the effect of organic matter on the dynamic adsorption process, and research studies systematically combining a mathematical model history-matched to experimental data is also rare. In this study, a dynamic, approaching equilibrium (hereinafter referred to as “delayed”) adsorption–diffusion (DAD) method is presented to analyze gas transport and storage processes in crushed particles of three different sizes. The delayed effect for adsorption results from the dynamic mechanisms of gas dissolution and adsorption in the semi-liquid layer of organic matter. The mathematical model for this phenomenon is based on dynamic adsorption experiments with a constant pressure condition. The general and approximate solutions for the DAD model are obtained to estimate the physical parameters through a multilevel single-linkage method. From the fitting results of the DAD method, it is found that the absolute permeability begins to decrease when particles are crushed into smaller sizes and observed that particle size plays more important role than permeability in the diffusion process for crushed shale samples. Isotherm measurements for total gas and free gas reveal that the difference in gas content between total gas and adsorbed gas become greater as pressure increases. Sensitivity analyses for the model disclose that the apparent diffusion coefficient and adsorption/desorption rate coefficient determine gas transport and storage processes together. Analysis and comparison of a diffusion model, an instantaneous adsorption–diffusion (IAD) model, and the DAD model reveal that the former two models are special forms of the DAD model, and the sequence of equilibrium times required for the gas transport and storage processes is: DAD>IAD>diffusion model.

Comparison of Four Extraction Methods for the Determination of Veterinary Pharmaceuticals in Sediment

In this work extraction by agitation (EA), ultrasound solvent extraction (USE), microwave assisted extraction (MAE) and pressurized liquid extraction (PLE) followed by high performance liquid chromatography with diode array detector (HPLC–DAD) for the determination of eight veterinary pharmaceuticals from different structural groups including one macrolide (tylosine), three anthelmintics (albendazole, febantel and levamisole), two anesthetics (lidocaine and procaine) and two steroidal hormones (hydrocortisone and dexamethasone) in sediment is described. Selected pharmaceuticals are widely used in veterinary practice in Croatia and in the whole world. Since they belong to different structure groups the usage of routine methods is not effective for their extraction from a complex sediment sample. Also, until now no article has been published for systematic extraction of named pharmaceuticals from sediment sample by different extraction methods. EA, USE, MAE and PLE have been optimized regarding different characteristic parameters such as sediment mass, extraction solvent, solvent volume, agitation frequency, power of ultrasound bath, extraction duration, extraction temperature and contact time between sediment and solvent. After extraction, pharmaceuticals were analyzed by HPLC–DAD using C18 stationary phase. HPLC–DAD–EA, HPLC-USE, HPLC–DAD-MAE and HPLC–DAD–PLE methods were validated in terms of selectivity, specificity, linearity, sensitivity, method detection limit (MDL) and quantification (MQL), repeatability, recovery and stability of a standard solution. Recoveries of selected pharmaceuticals from spiked sediment samples were generally higher than 50 %. However, dexamethasone and hydrocortisone showed lower recoveries due to their significantly different molecule structure. This paper shows the ability of simultaneous extraction and analysis of all selected pharmaceuticals by four different extraction methods in complex sediment sample followed by relatively affordable and sensitive HPLC–DAD method.

Development of Basin-scale PMP Estimation Method Using Grid-based Rain Search Method

This study aims to develop a PMP estimation method in a basin-scale by considering both temporal and spatial characteristics and to compare the existing PMP estimation method with the developed method in this study. Since the movement of heavy rainfall is predictable like typhoons, DAD analysis method is usually utilized to storm centered DAD method and fixed areal DAD method. However, utilizing DAD in bigger basin area or longer rainfall duration induces more errors in estimation of average areal rainfall and thus reducing the accuracy of the results. Therefore, new DAD analysis method is necessary to improve the accuracy. In this study, by utilizing the new DAD analysis method, PMP was estimated through the processes of: firstly, converting point rainfall data to distributed areal rainfall data; secondly, a Grid-based rainfall search method was developed through the utilization of Average Point-tracking; and lastly, the PMP was calculated by considering moisture maximization. PMP estimation from using the new method was compared with the existing PMP as well as the areal maximum probable precipitation. As a result, even though the new estimate is lower than the exiting PMP, it was larger than the areal maximum probable precipitation (200-Year 48-Hour Areal Rainfall Frequency). Therefore, the estimated PMP can be utilized for the flood estimation.

A SINGLE HPLC-DAD METHOD FOR SIMULTANEOUS ANALYSIS OF PARACETAMOL, PHENYLEPHRINE, CAFFEINE AND LEVOCETIRIZINE IN BULK POWDER AND TABLET FORMULATION: APPLICATION TO IN-VITRO DISSOLUTION STUDIES

ABSTRACT Present work describes development and validation of HPLC–DAD method for the analysis of a complex mixture consisting phenylephrine hydrochloride (PHE), paracetamol (PAR), caffeine (CAF) and levocetirizine (LEV). Chromatographic separation was achieved using a Kinetex-C18 column involving gradient elution, the mobile phase composed of 10mM phosphate buffer (pH 3.3) and methanol. The three step gradient program started with step-1 initially with 2% (by volume) methanol and 98 % phosphate buffer (pH 3.3) for first 5 minutes. In step-2 methanol concentration changed linearly to 80% in next 12 minutes the analysis was concluded by step-3 changing methanol to 2% in next 8 minutes. The method was linear in concentration ranges of 50 to 200 % of labeled claims with r 2 near to 1. Developed method proved to be precise for inter and intra-day studies the %RSD values were < 2 , accuracy studies by standard addition at 80, 100 and 120% levels resulted in recoveries of standard near to 100%. The method was selective with no interferences from placebo; robustness of method was proved by no significant alteration in system suitability parameters. The LOD values for PHE, PAR, CAF and LEV were 0.13, 0.51, 0.05 and 0.05 and LOQ were 0.39, 1.53, 0.15 and 0.15 respectively. The study concluded a validated HPLC method for analysis of complex mixture consisting PHE, PAR, CAF and LEV in bulk, tablet and dissolution samples in simulated gastric fluid (SGF).

Development and Validation of Chromatographic Methods for Resolving Ternary Mixture of Diflunisal, Naproxen and DiflunisalToxic Impurity

This search article describes accurate, precise and sensitive TLC-densitometric and HPLC– DAD methods which have been developed and validated for simultaneous determination of Diflunisal (DIF) and Naproxen (NAP) in their binary mixture and in the presence of Diflunisal toxic impurity, Biphenyl-4-ol (BIP). The developed TLC-densitometric method depended on separation of the three components on a stationary phase of silica gel plates 60 F254 using a solvent mixture consisting of methylene chloride: methanol: ethyl acetate: acetic acid (9: 0.5: 0.5: 0.05, by volume) and then scanning at 240 nm. The linearity ranges were found to be 0.2-2, 0.2-1 and 0.2-2 µg/band for DIF, NAP and BIP, respectively. The developed HPLC DAD method depended on using Zorbax Eclipse C18 column as a stationary phase and a mixture of methanol: water (55: 45, by volume, pH=4 with orthophosphoric acid) as a mobile phase, delivered at constant flow rate of 1 mL/min and UV detection at 215 nm. Linear relationships were obtained between the peak area and the concentration in the ranges of 3-40, 3-40 and 3-25 µg/mL for DIF, NAP and BIP, respectively. Factors affecting the developed methods have been studied and optimized. Furthermore, method's validation has been carried out according to International Conference on Harmonization guidelines. The proposed methods were successfully applied for determination of DIF and NAP in Maxipan ® tablets. Also they were statistically compared with the reported method using student's-t and F-tests and no significant difference was obtained. The reported methods were the first developed stability indicating methods for resolving the proposed mixture and their toxic impurity.