Applications of metal organic frameworks in dispersive micro solid phase extraction (D-µ-SPE).

Metal-organic frameworks (MOFs) are a fascinating family of crystalline porous materials made up of metal clusters and organic linkers. In comparison with other porous materials, MOFs have unique characteristics including high surface area, homogeneous open cavities, and permanent high porosity with variable shapes and sizes. For these reasons, MOFs have recently been explored as sorbents in sample preparation by solid-phase extraction (SPE). However, SPE requires large amounts of sorbents and suffers from limited contact surfaces with analytes, which compromises extraction recovery and efficiency. Dispersive SPE (D-SPE) overcomes these limitations by dispersing the sorbents into the sample, which in turn increases contact with the analytes. Miniaturization of the microextraction procedure, particularly the amount of sorbent reduces the amount consumed of the organic solvent and shorten the time required to attain the equilibrium state. This may explain the reported high efficiency and applicability of MOFs in dispersive micro SPE (D-µ-SPE). This method retains all the advantages of solid phase extraction while also being simpler, faster, cheaper, and, in some cases, more effective in comparison with D-SPE. Besides, D-µ-SPE requires smaller amounts of the sorbents which reduces the overall cost, and the amount of waste generated from the analytical process. In this review, we discuss the applications of MOFs in D-µ-SPE of various analytes including pharmaceuticals, pesticides, organic dyes from miscellaneous matrices including water samples, biological samples and food samples.

Unveiling Pharmacokinetics and Drug Interaction of Linagliptin and Pioglitazone HCl in Rat Plasma Using LC-MS/MS.

The linagliptin (LIN) and pioglitazone HCl (PIO) combination, currently undergoing phase III clinical trials for diabetes mellitus treatment, demonstrated significant improvements in glycemic control. However, the absence of an analytical method for simultaneous determination in biological fluids highlights a crucial gap. This underscores the pressing need for sensitive bioanalytical methods, emphasizing the paramount importance of developing such tools to advance diabetes management strategies and enhance patient care. Herein, a sensitive reverse-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry method was developed for simultaneous determination of LIN and PIO in rat plasma using alogliptin as an internal standard. Chromatographic separation was performed on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 µm) using an isocratic mobile phase system consisting of ammonium formate (pH 4.5) and methanol using an acetonitrile-induced protein precipitation technique for sample preparation. Multiple reaction monitoring in positive ion mode was used for quantitation of the precursor to production at m/z 473.2 → 419.9 for LIN, 357.1 → 134.2 for PIO, and 340.3 → 116.1 for ALO. The linearity range was 0.5 to 100 and 1 to 2000 ng/mL for LIN and PIO, respectively. The developed method was validated as per US-FDA guidelines and successfully applied to clinical pharmacokinetic and drug-drug interaction studies with a single oral administration of LIN and PIO in rat plasma. Pharmacokinetic parameters of LIN were significantly influenced by the concomitant administration of PIO and vice versa. Molecular modeling revealed the significant interaction of LIN and PIO with P-glycoprotein. Therefore, the drug-drug interaction between LIN and PIO deserves further study to improve drug therapy and prevent dangerous adverse effects.

Simple colorimetric paper-based test strip for point-of-use quality testing of ethanol-based hand sanitizers.

A novel, simple, affordable, and reliable colorimetric paper-based analytical device (PAD) was developed for the point-of-use quality testing of ethanol-based hand sanitizers, mainly against adulteration by water. The principle was based on the novel solvatochromism of methylparaben (MPB)-Fe3+ complex, where water is essential for complex formation and ethanol is necessary for MPB solubility. The intensity of the formed violet color, measured at 528 nm, showed a good correlation (R2 = 0.996) with the percentage water in the reaction media over a range from 40% to 100% (0-60% ethanol), with excellent accuracy and precision as indicated by the percent recovery within 100.00% ± 2% and %RSD of <2%. A PAD was prepared by the sequential immobilization of Fe3+ ions and MPB on chitosan-modified filter paper. The developed PAD was successfully applied for the quality testing of ethanol-based hand sanitizers using an established color index, where clearly distinct colors were observed as a function of the percentage ethanol (0-100%). The developed test strips could achieve on-site lab-quality results without expensive or sophisticated instruments using a few milligrams of FeCl3 and MPB in addition to regular filter paper. Accordingly, it can be used as a test strip for the quality checking of ethanol-based hand sanitizers by end users.

Green micellar liquid chromatographic method for simultaneous determination of antihistaminic drugs tripelennamine hydrochloride and diphenhydramine in pharmaceutical gel.

A new green micellar liquid chromatographic method has been developed and validated for the simultaneous determination of diphenhydramine (DPH) and tripelennamine hydrochloride (TRP) using a micellar mobile phase consisting of 1 mM Tween 20 in phosphate buffer pH 4:isopropanol (85:15, %v/v). The method was linear in the range of 4-150 and 5-120 µg/mL for TRP and DPH, respectively. The method was successfully applied for the simultaneous determination of DPH and TRP in a laboratory-prepared gel containing all possible excipients with mean percent recoveries ± standard deviation of 100.346 ± 1.265 and 100.754 ± 1.117 for TRP and DPH, respectively. The method was validated according to the International Conference on Harmonization guidelines. The method is confirmed to have excellent greenness.

Recent Applications of Quantum Dots in Pharmaceutical Analysis.

Nanotechnology has emerged as one of the most potential areas for pharmaceutical analysis. The need for nanomaterials in pharmaceutical analysis is comprehended in terms of economic challenges, health and safety concerns. Quantum dots (QDs)or colloidal semiconductor nanocrystals are new groups of fluorescent nanoparticles that bind nanotechnology to drug analysis. Because of their special physicochemical characteristics and small size, QDs are thought to be promising candidates for the electrical and luminescent probes development. They were originally developed as luminescent biological labels, but are now discovering new analytical chemistry applications, where their photo-luminescent properties are used in pharmaceutical, clinical analysis, food quality control and environmental monitoring. In this review, we discuss QDs regarding properties and advantages, advances in methods of synthesis and their recent applications in drug analysis in the recent last years.

Homogeneous liquid-liquid microextraction coupled with HPLC/DAD for determination of nirmatrelvir and ritonavir as COVID-19 combination therapy in human plasma.

The study reports the development of a high-performance liquid chromatography/diode array detection method to measure the levels of nirmatrelvir and ritonavir in human plasma. These two antiviral medications are used for the treatment of COVID-19 and are marketed as Paxlovid®. The method employed sugaring-out induced homogeneous liquid-liquid microextraction to improve sensitivity. Optimization of the method was performed using the one variable at a time approach by adjusting several factors such as type of sugar, extractant, amount of sugar, volume of extractant, and pH of the aqueous sample to achieve the highest efficiency. The developed method was validated according to the Food and Drug Administration guidelines and demonstrated good linearity, accuracy, and precision. The range of linearity was from 1000 to 20,000 ng/mL for nirmatrelvir and 200 to 20,000 ng/mL for ritonavir with correlation coefficient values of 0.998 and 0.996, respectively. Selectivity studies revealed that no others peaks appeared in the retention times of the studied drugs. The stability of nirmatrelvir and ritonavir were also investigated through short term and three cycles of freeze-thaw, and both drugs were found stable. This analytical method could be useful for monitoring drug concentrations in patients undergoing treatment with these medications for COVID-19. In this work, for the first time, SULLME was used for the sensitive determination of nirmatrelvir and ritonavir in biological fluids. The developed method was able to determine both drugs in therapeutic levels with no need to sophisticated techniques like LC-MS. In addition to that, SULLME is considered a simple and green sample preparation in comparison with conventional sample preparation methods.

UV spectrophotometric methods for simultaneous determination of ketorolac tromethamine and olopatadine hydrochloride: Application of multiple standard addition for assay of ophthalmic solution.

Ophthalmic preparations that contain ketorolac tromethamine (KET) and olopatadine HCl (OLO) are used to relieve seasonal allergies and allergic conjunctivitis. Simultaneous quantification of KET and OLO was held by validated and simple spectrophotometric methods. KET was determined directly from the fundamental UV absorption spectra (at 323 nm), while OLO was determined after performing either dual wavelength or ratio derivative methods. The first method was based on measuring the absorbance difference (ΔA) between 243 and 291 nm, while the second depended on generating first derivative ratio spectra using 3.0 µg/mL KET as a divisor and measuring OLO responses at 234 nm (minima). Multiple standard addition method was applied to enable the determination of OLO which is considered as the weakly absorbing species as well as the minor component in a challenging dosage form ratio (4:1). The linearity ranges of the developed methods were 3-12 µg/mL and 4-40 µg/mL for KET and OLO, respectively. Simultaneous determination of both drugs was successfully implemented to lab prepared eye drops that contain KET, OLO and benzalkonium chloride as an inactive ingredient. Greenness assessment indicates minimal impact on environment. The developed methods determined the cited drugs with % recovery ± SD of 99.63 ± 0.01 for KET, 100.90 ± 0.02 and 100.31 ± 0.01 for OLO using dual wavelength and first derivative ratio methods, respectively. Using F-test and t-test at confidence level %95 to compare between the results of the presented methods and a reported method show no significant difference which allows precise, accurate, rapid, and simple quantification of quality control samples that contain KET and OLO.

Design of experiment-oriented development of solvent-free mixed micellar chromatographic method for concomitant determination of metronidazole and ciprofloxacin hydrochloride.

A green, fast and robust solvent-free chromatographic method has been developed for concomitant analysis of ciprofloxacin HCl and metronidazole in bulk powder as well as in dosage form using levofloxacin as internal standard (I.S.). Two different designs including fractional factorial (FFD) and Box-Behnken (BBD) designs were implemented for screening and optimization steps, respectively. The optimum chromatographic separation was accomplished using mobile phase composed of 0.13 M sodium dodecyl sulfate and 0.02 M Birij-35 solution adjusted to pH 2.5 using phosphoric acid at a flow rate of 1.3 mL/min and column oven temperature of 40 °C. Chromatographic analysis was performed on X-Bridge (150 mm × 4.6 mm, 5 µm) column with UV detection at 280 nm. A linear response was acquired over the range of 0.4-50 µg/mL for both drugs. The developed method was applied for quantitation of cited drugs in commercially available tablet with mean percent recovery ± SD of 99.45 ± 0.72 and 100.13 ± 0.81 for metronidazole and ciprofloxacin respectively. The method was proven to be green as evaluated by three greenness assessment tools. The run time was 8 min, thus saving time and reagent.

Formation of plasmonic silver nanoparticles by glucosamine reduction: Application to a colorimetric sensor for determination of glucosamine in its pharmaceutical preparations.

The purpose of this study is to develop a novel method for synthesizing silver nanoparticles using glucosamine as reducing agent and to utilize the developed method for colorimetric detection and quantitative determination of the non-chromophoric drug, glucosamine. Silver nanoparticles are prepared by reducing 0.02 mol/L silver nitrate by glucosamine in 0.075 mol/L ammonia and stabilizing the nanoparticles with 0.1% polyvinylpyrrolidone and the mixture is heated at 90 °C for 5 min. The prepared silver nanoparticles dispersed in water exhibit a bright yellow color due to a localized surface plasmon resonance band at 412 nm. The principle of glucosamine sensing is based on measuring the intensity of the surface plasmon resonance band at 412 nm which is directly proportional to the concentration of glucosamine with a linearity range (1 - 9 µg/mL), limit of detection 0.33 µg/mL and limit of quantitation 1.0 µg/mL. The proposed method was validated according to the ICH guidelines, and it was found to be accurate, precise, selective, and robust. The method was applied for determination of glucosamine in Joflex® capsules using the standard addition approach with mean % recovery ± standard deviation of 100.077 ± 1.786. The method is simple, rapid, and cost-effective and can be used for determination of glucosamine in bulk and in its pharmaceutical preparations.

Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3 mL/minute. Separation and analysis were performed on X-bridge C18 (150 mm × 4.6 mm × 3.5 µm) column with diode array detector set at 230 nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125 µg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62 µg/mL for MTR and 0.92 and 2.83 µg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time.

A hybrid microcrystalline cellulose/metal-organic framework for dispersive solid phase microextraction of selected pharmaceuticals: A proof-of-concept.

Solid phase microextraction (SPME) is considered simple, ecofriendly, sustainable, cost-effective and timesaving sample preparation mode in comparison with other sample preparation procedures. The researchers always try to develop new sorbents with higher surface area in comparison with other conventional sorbents aiming to enhance the extraction efficiency. In this work for the first time, a comparative study was performed between Ca-BTC MOF (1,3,5-benzenetricarboxylic acid, BTC; metal-organic framework, MOF) and a hybrid Ca-BTC-MCC MOF (microcrystalline cellulose, MCC) by using as model compounds seven drugs with different physicochemical properties. The evaluation of the extraction efficiency of both sorbents were obtained by means of an HPLC/DAD instrument configuration in reversed phase mode under isocratic elution mode. The results indicate that Ca-BTC MOF showed superior extraction efficiency than Ca-BTC-MCC MOF in the case of all analytes except nirmatrelvir and ritonavir. The results highlight that not only the surface area of adsorbents controlled the adsorption capacity, but also other factors have a role in extraction efficiency including morphology of adsorbent and physico-chemical properties of the analytes. It is worth mentioning that this is the first time that a comparative study was performed between Ca-BTC MOF and Ca-BTC-MCC MOF hybrid material.

Area under the curve and ratio difference spectrophotometric methods with evaluation of the greenness for simultaneous determination of olmesartan medoxomil and metoprolol succinate.

The aim of this study is to develop and validate two simple spectrophotometric methods for simultaneous determination of metoprolol succinate (MET) and olmesartan medoxomil (OLM) in tablet form. Method (I) was area under the curve (AUC) method. This approach involved the measuring of the area over a variety of wavelengths. Two wavelength ranges; 213-230 nm and 244-266 nm were chosen for determination of MET and OLM, respectively. Method (II) was ratio difference spectrophotometricmethod. For determination of MET, the ratio spectra were generated using 15 µg/mL OLM as a divisor then the peak to trough amplitudes between 221 nm and 245 nm were displayed versus the corresponding concentrations of MET. For determination of OLM, the peak-to-peak amplitudes between 247 and 293 nm were chosen and found to be directly proportional to OLM concentrations using 15 µg/mL MET as a divisor. The linearity ranges were 2-30 µg/mL and 2-25 µg/mL for MET and OLM, respectively. The assay results showed good mean %recovery ± SD as well as good agreement with that of the reported method. The developed methods were validated according to ICH guidelines. The developed methods are accurate, precise, eco-friendly and could be applied successfully to estimate OLM and MET in their combined dosage form.

Dual fluorescence-colorimetric sensor based on silver nanoparticles for determination of tobramycin in its pharmaceutical preparations.

The purpose of this study is to develop a dual fluorescence-colorimetric sensor for determination of the non-chromophoric drug, tobramycin using fluorescein-modified silver nanoparticles. Fluorescein is adsorbed on the surface of silver nanoparticles resulting in quenching of the fluorescence intensity of fluorescein at 513 nm. Upon addition of tobramycin to fluorescein-bound silver nanoparticles, tobramycin can displace fluorescein from the surface of nanoparticles resulting in nanoparticles aggregation and liberation of free fluorescein restoring its fluorescence. The interaction of tobramycin with fluorescein-bound silver nanoparticles is manifested by a decrease in the surface plasmon resonance band of silver nanoparticles at 395 nm, an increase in the fluorescence intensity of fluorescein at 513 nm and color change of the colloidal solution from yellow to light pink. These spectral effects are directly proportional to the concentration of tobramycin with a linearity range of 0.10 - 0.45 µg mL-1 and 0.05 - 0.45 µg mL-1 for the spectrophotometric and spectrofluorimetric methods, respectively. The proposed methods were applied for determination of tobramycin in Tobrin® ophthalmic solution with mean %recovery ± standard deviation of 99.036 ± 1.737 for the spectrophotometric method and 101.192 ± 1.315 for the spectrofluorimetric method. The optical sensor is simple, rapid, and cost-effective and can be used for determination of tobramycin in bulk and in its pharmaceutical preparations.

Probing the potential toxicity of trimetazidine by characterizing its interaction with human serum albumin.

The small molecular drugs pharmacodynamics and pharmacokinetics could be affected by human serum albumin (HSA) transport, so we studied the interaction between HSA and the widely used anti-ischemic agent, trimetazidine (TMZ), using different approaches. As shown by synchronous fluorescence spectroscopy, the interaction affects the microenvironment confirmation around tyrosine residues. The site-competitive experiments showed that TMZ had an affinity toward subdomain III A (site II) of HSA. The enthalpy and entropy changes (ΔH and ΔS), which were 37.75 and 0.197 K J mol-1, respectively, showed that the predominant intermolecular interactions are hydrophobic forces. According to FTIR research, the interaction between HSA and TMZ caused polypeptide carbonyl-hydrogen bonds to rearrange. The HSA esterase enzyme activity was decreased with TMZ. Docking analysis confirmed the site-competitive experiments and thermodynamic results. This study demonstrated that TMZ interacted with HSA, and the structure and function of HSA were influenced by TMZ. This study could aid in understanding the pharmacokinetics of TMZ and provide basic data for safe use.

Green microwave-prepared N and S Co-doped carbon dots as a new fluorescent nano-probe for tilmicosin detection.

A straightforward, rapid, and selective fluorescent probe for determination of tilmicosin has been developed based on novel nitrogen and sulfur co-doped CDs (NS-CD). The NS-CDs were synthesized, for the first time, through green, simple one step microwave pyrolysis in only 90 s using glucose as carbon source and l-cysteine as nitrogen and sulfur source. This proposed synthesis method was energy-efficient and resulted in NS-CDs with high production yield (54.27 wt%) and narrow particle size distribution. Greenness of NS-CDs synthesis method was assessed using EcoScale and was proven to be excellent green synthesis. The produced NS-CDs were applied as a nano-probe for determination of tilmicosin in its marketed formulation and milk based on dynamic quenching mechanism. The developed probe showed a good performance for tilmicosin detection in marketed oral solution and pasteurized milk and linearity range of 9-180 µM and 9-120 µM, respectively.

Quality by design paradigm for optimization of green stability indicating HPLC method for concomitant determination of fluorescein and benoxinate.

A green, robust and fast stability indicating chromatographic method has been developed for concomitant analysis of fluorescein sodium and benoxinate hydrochloride in the presence of their degradation products within four minutes. Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic analysis was achieved using a mixture of isopropanol and 20 mM potassium dihydrogen phosphate solution (pH 3.0) in the ratio 27:73 as mobile phase. The flow rate was 1.5 mL/min and column oven temperature was 40 °C. Chromatographic analysis was performed on Eclipse plus C18 (100 mm × 4.6 mm × 3.5 µm) column with DAD detector set at 220 nm. A linear response was acquired over the range of 2.5-60 µg/mL and 1-50 µg/mL for benoxinate and fluorescein respectively. Stress degradation studies were executed under acidic, basic, and oxidative stress conditions. The method was implemented for quantitation of cited drugs in ophthalmic solution with mean percent recovery ± SD of 99.21 ± 0.74 and 99.88 ± 0.58 for benoxinate and fluorescein respectively. The proposed method is more rapid and eco-friendly compared to the reported chromatographic methods for determination of cited drugs.

Green micellar stability-indicating high-performance liquid chromatography method for determination of rupatadine fumarate in the presence of its main impurity desloratadine: Oxidative degradation kinetics study.

A green micellar stability-indicating high-performance liquid chromatography method was developed for rupatadine fumarate determination in existence with its main impurity desloratadine. Separation was attained using Hypersil ODS column (150 × 4.6 mm, 5 µm), the micellar mobile phase consisted of 0.13 M sodium dodecyl sulfate, 0.1 M disodium hydrogen phosphate adjusted by phosphoric acid to pH 2.8 and 10% n-butanol. The column was maintained at 45◦ C and detection was carried out at 267 nm. A linear response was achieved over the range of 2-160 µg/ml for rupatadine and 0.4-8 µg/ml for desloratadine. The method was applied for rupatadine determination in alergoliber tablets and alergoliber syrup without the interference of methyl paraben and propyl paraben present as main excipients. Rupatadine fumarate revealed pronounced susceptibility to oxidation; further study of oxidative degradation kinetics was carried out. Rupatadine was found to follow pseudo-first-order kinetics when exposed to 10% H2 O2 at 60 and 80°C and the activation energy was found to be 15.69 Kcal/mol. At a lower temperature (40°C), degradation kinetics regression was best fitted as a polynomial quadratic relationship, thus rupatadine oxidation at a lower temperature tends to adopt a second-order kinetics rate. Oxidative degradation product structure was revealed using infrared and found to be rupatadine N-oxide at all temperature values.

Deduction of the operable design space of RP-HPLC technique for the simultaneous estimation of metformin, pioglitazone, and glimepiride.

A reversed-phase RP-HPLC method was developed for the simultaneous determination of metformin hydrochloride (MET), pioglitazone (PIO), and glimepiride (GLM) in their combined dosage forms and spiked human plasma. Quality risk management principles for determining the critical method parameters (CMPs) and fractional factorial design were made to screen CMPs and subsequently, the Box-Behnken design was employed. The analytical Quality by Design (AQbD) paradigm was used to establish the method operable design region (MODR) for the developed method depended on understanding the quality target product profile (QTPP), analytical target profile (ATP), and risk assessment for different factors that affect the method performance to develop an accurate, precise, cost-effective, and environmentally benign method. The separation was carried out using a mobile phase composed of methanol: 0.05 M potassium dihydrogen phosphate buffer pH 3.7 with 0.05% TEA (78:22, v/v). The flow rate was 1.2 mL/min. DAD detector was set at 227 nm. Linagliptin (LIN) was used as an internal standard. The proposed method was validated according to The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). The assay results obtained by using the developed method were statistically compared to those obtained by the reported HPLC method, and a satisfying agreement was observed.

Coupling of synchronous fluorescence spectroscopy with derivative amplitude outcomes for simultaneous determination of metoprolol succinate and olmesartan medoxomil in combined pharmaceutical preparation: Application in spiked human plasma.

For the first time a spectrofluorimetric method had been achieved for the concurrent analysis of metoprolol succinate (MET) and olmesartan medoxomil (OLM). The approach depended on assessing the first order derivative (1D) of the synchronous fluorescence intensity of the two drugs in aqueous solution at Δλ of 100 nm. The amplitudes of 1D at 300 nm and 347 nm were measured for MET and OLM, respectively. The linearity ranges were 100-1000 ng/mL and 100-5000 ng/mL for OLM and MET, respectively. This approach is uncomplicated, repetitive, quick, and affordable. The results of analysis had been statistically verified. The validation assessments were carried out following the recommendations of The International Council for Harmonization (ICH). This technique could be employed to assess marketed formulation. The method was sensitive with limits of detection (LOD) of 32 ng/ml and 14 ng/mL for MET and OLM, respectively. Limits of quantitation (LOQ) were 99 ng/ml for MET and 44 ng/mL for OLM. So it can be applied to determine both drugs in spiked human plasma within the linearity ranges of 100-1000 ng/mL for OLM and 100-1500 ng/mL for MET.

Enhanced fluorimetric detection of diphenylpyraline HCl using micelle and cyclodextrin mediated approach: Spectrofluorimetric and micellar liquid chromatographic application for either single or combined formulation with caffeine and paracetamol.

Highly sensitive micellar spectrofluorimetric method (Method I) has been developed and validated for the determination of diphenylpyraline HCl in pharmaceutical tablets and in plasma. Sodium dodecyl sulfate improves the intensity of fluorescence of diphenylpyraline at 286 nm at pH 5 that allow its determination in plasma at nano-level. the mean percent recovery ± S.D was 99.719 ± 0.338 in plasma. In addition, Green cyclodextrin-modified micellar liquid chromatographic method (Method II) has been developed and validated for simultaneous determination of diphenylpyraline, paracetamol and caffeine using cyclodextrin micellar mobile phase consisted of 30 mM Brij*35, 0.5 mM hydroxypropyl ß-cyclodextrin and phosphate buffer pH 4: MeOH (95:5, %v/v) that allows their simultaneous determination with enhanced spectrofluorimetric detection of diphenylpyraline. Method II was effectively applied for the simultaneous determination of diphenylpyraline, paracetamol and caffeine in a ternary laboratory prepared mixture which contained all possible excipients with mean percent recoveries ± S.D of 100.176 ± 1.008, 101.166 ± 0.415 and 100.708 ± 1.836, respectively. Linearity range for Method I was 0.1-1 µg. mL-1 for diphenylpyraline and for Method II was 0.3-50, 25-350, and 0.5-50 for caffeine, paracetamol and diphenylpyraline, respectively. Method I was also applied in spiked human plasma with linearity range 0.2-0.5 µg. mL-1. The methods are verified to have excellent greenness.