A probe of new molecularly imprinted solid-phase extraction coupled with HPLC-DAD and atomic absorption spectrophotometry for quantification of tetracycline HCl, metronidazole and bismuth subcitrate in combination with their official impurities: Application in dosage form and human plasma.

The integration of molecular imprinting technique with chromatographic one has a great impact on the assay's selectivity and sensitivity. Herein, a molecularly imprinted solid-phase extraction associated with high performance liquid chromatography (MISPE-HPLC) was employed for simultaneous determination of the co-formulated drugs; tetracycline hydrochloride (TET) and metronidazole (MET), in plasma and in their anti-H-pylori drug for the first time. Two sorts of molecularly imprinted polymers (MIPs) were fabricated using TET and MET as the template molecules, while ethylene glycol dimethacrylate and methacrylic acid were used as a cross-linker and a monomer, respectively. The synthesized MIPs were identified using different techniques. The adsorption-desorption capability of each template was investigated towards its corresponding MIP. The extraction conditions of MISPE was optimized with respect to TET/MIP and MET/MIP sorbent. Bismuth subcitrate (BSC), the third co-formulated drug was analyzed in spiked human plasma using an atomic absorption spectrometric (AAS) method. The performance of the developed methods was assured as per ICH guidelines for analyzing the studied drugs in their pharmaceutical dosage form along with two of their official impurities. In addition, bioanalytical method validation was conducted where linearity was achieved at 2.0-40.0 µg mL-1, 2.0-40.0 µg mL-1 and 5.0-80.0 µg mL-1 for TET, MET and BSC, respectively.

Application of multivariate chemometrics tools for spectrophotometric determination of naphazoline HCl, pheniramine maleate and three official impurities in their eye drops.

This work is concerned with exploiting the power of chemometrics in the assay and purity determination of naphazoline HCl (NZ) and pheniramine maleate (PN) in their combined eye drops. Partial least squares (PLS) and artificial neural network (ANN) were the chosen models for that purpose where three selected official impurities, namely; NZ impurity B and PN impurities A and B, were successfully determined. The quantitative determinations of studied components were assessed by percentage recoveries, standard errors of prediction as well as root mean square errors of prediction. The developed models were constructed in the ranges of 5.0-13.0 µg mL-1 for NZ, 10.0-60.0 µg mL-1 for PN, 1.0-5.0 µg mL-1 for NZ impurity B and 2.0-14.0 µg mL-1 for two PN impurities. The proposed models could determine NZ and PN with respective detection limits of 0.447 and 1.750 µg mL-1 for PLS, and 0.494 and 2.093 µg mL-1 for ANN. The two established models were compared favorably with official methods where no significant difference observed.

Molecular imprinted polymer-based potentiometric approach for the assay of the co-formulated tetracycline HCl, metronidazole and bismuth subcitrate in capsules and spiked human plasma.

BACKGROUND: An anti-H-pylori co-formulated mixture of tetracycline HCl (TET), metronidazole (MET), and bismuth subcitrate (BSC) is recently available. Only two chromatographic and spectrophotometric methods are reported for determining those drugs simultaneously where the effect of impurities that could be present as well as the biological fluids matrix influence do not be taken into consideration. There is a need to develop an easy-to-use potentiometric technique for analysis of TET, MET, and BSC in their co-formulated capsules, in presence of some official impurities and in spiked human plasma. RESULTS: Three carbon paste electrodes (CPEs) were fabricated for this purpose. Being a solid contact ion-selective electrode, CPE suffers from the creation of a water layer affecting its stability and reproducibility. Besides, it has a common problem in differentiation between two drugs carrying the same charge (positively charged TET and MET). Water layer formation was prevented through inserting polyaniline nanoparticles (≈10.0 nm diameter) between solid contact and ion-sensing membrane in the three proposed sensors. TET and MET interference was overcome by synthesizing a corresponding molecular imprinted polymer (MIP) for each drug. The synthesized MIPs were inserted in equivalent sensing membranes and characterized using several techniques. The suggested MIPs have a noticeable enhanced sensitivity in potentiometric determination. The obtained LODs were 5.88 × 10-8, 5.19 × 10-7, and 1.73 × 10-6 M for TET, MET and BSC proposed CPEs, respectively, with corresponding slopes of 57.37, 56.20, and -57.40 mV decade-1. SIGNIFICANCE: The proposed potentiometric method makes the detection of the three cited drugs simple, fast, and feasible. This approach is the first for determining three drugs potentiometrically in one combined formulation. The obtained results were compared favorably with previously reported potentiometric methods.

All-solid-state chip utilizing molecular imprinted polymer for erythromycin detection in milk samples: Printed circuit board-based potentiometric system.

Detection of erythromycin (ERY) residues in commercial milk samples is crucial for the safety assessment. Herein, a printed circuit board was patterned as a feasible miniaturized potentiometric sensor for ERY determination in dairy samples. The proposed chip design fits to a 3.5-mm female audio plug to facilitate the potential measurements of working electrode versus reference one in this all-solid-state system. The sensor utilizes molecular imprinted polymer (MIP) for the selective recognition of the studied drug in such challenging matrix. The electrode stability is achieved through the addition of poly (3,4-ethylenedioxythiophene) nano-dispersion on its surface. The proposed device detects down to 6.6 × 10-8 M ERY with a slope of 51 mV/decade in the 1 × 10-7-1 × 10-3 M range. The results display high accuracy (99.9% ± 2.6) with satisfactory relative standard deviation for repeatability (1.6%) and reproducibility (5.0%). The effect of common antibiotic classes, namely, amphenicols, beta-lactams, fluoroquinolones, sulfonamides, and tetracyclines, can be neglected as evidenced by their calculated binding capacities towards the proposed MIP. The calculated selectivity coefficients also show a good electrode performance in the presence of naturally present inorganic ions allowing its application to different milk samples.

Resolving phenylephrine HCl and guaifenesin enantiomers on cellulose-based chiral stationary phases: Separation of four enantiomers on 50-mm column.

Chiral high performance liquid chromatographic technique usually employs polysaccharide-based stationary phases in a normal phase mode. This frequently generates large waste of organic solvents. Using shorter columns of 50 mm length as well as a mobile phase with a high water percentage are common approaches for greening this analytical technique. In this context, a new chiral chromatographic technique was developed for simultaneous enantio-separation of phenylephrine HCl and guaifenesin racemates. Four 50 mm cellulose-based columns were experimented to separate the four enantiomers in a reversed phase mode. A face centered design was then employed to optimize the mobile phase acetonitrile% and flow rate on Lux Cellulose-1 (50 × 4.6 mm, 5 µm). The simultaneous resolution of the cited drugs enantiomers was achieved using acetonitrile-water (30:70, by volume), with a flow rate of 0.5 ml min-1 . These optimized chromatographic conditions separate the enantiomers in 7 min running time, generating about 1.0 ml acetonitrile per run. The proposed method was favorably compared with other reported chiral ones in terms of waste volume generated and analysis time required.

A trimodal detection paper chip for undisclosed drug "sibutramine" in nutraceuticals.

Nutraceuticals are promoted and marketed with the stated label of being natural as well as safe herbal products. In order to enhance their effectiveness, nutraceuticals are usually adulterated with undeclared constituents. Slimming herbs may contain sibutramine (SBT) which is an FDA-banned ingredient due to its fatal outcomes. This current work's aim is to design a trimodal sensor for SBT detection in different herbal slimming formulations. Screen-printed silver and multi-walled carbon nanotube inks were employed for the potentiometric sensor. The sensor was designed to fill a reaction well in which a carbon dot-silver nanoparticle pair was applied for fluorimetric and colorimetric purposes. The trimodal sensor was designed to fit an 8 mm 2-pin LED strip connector. Potentiometric measurement took place upon application of one sample aliquot then the optical reaction proceeded next in a specified zone for optical detection. These multiple detection mechanisms achieved the required selectivity for SBT determination in the presence of other slimming products' additives. This trimodal sensor satisfied World Health Organization standards for point-of-care devices demonstrating the suggested device as a dynamic part for rapid on-site detection of undisclosed SBT.

Classical versus chemometrics tools for spectrophotometric determination of fluocinolone acetonide, ciprofloxacin HCl and ciprofloxacin impurity-A in their ternary mixture.

Green, simple, accurate and robust univariate and chemometrics assisted UV spectrophotometric approaches have been adopted and validated for concurrent quantification of fluocinolone acetonide (FLU), ciprofloxacin HCl (CIP) together with ciprofloxacin impurity-A (CIP imp-A) in their ternary mixture. Double-divisor ratio spectra derivative (DDRD) method has been used for determination of FLU. On the other hand, the first (D1) and second (D2) derivative approaches have been applied for the quantification of CIP and CIP imp-A, respectively. For the ratio difference (RD), derivative ratio (DR), and mean centering of ratio spectra (MC) methods, CIP and its impurity A have been simultaneously determined. The acquired calibration plots were linear over the concentration range of 0.6-20.0 µg/mL, 1.0-40.0 µg/mL and 1.0-40.0 µg/mL for fluocinolone acetonide, ciprofloxacin HCl, and ciprofloxacin impurity-A, respectively. The chemometrics methods namely; partial least squares (PLS) and artificial neural networks (ANN) were used for the concurrent determination of the three adopted components via using twenty-five mixtures as calibration set and fifteen mixtures as validation one. The investigated approaches were validated in accordance with International Council for Harmonisation (ICH) guidelines, and statistically compared with the official ones. The proposed methods were acceptably applied to the examination of FLU and CIP in their pure powders and pharmaceutical ear drops.

Exploiting the power of UPLC in separation and simultaneous determination of pholcodine, guaiacol along with three specified guaiacol impurities.

Pholcodine and guaiacol are widely used together in pharmaceutical syrups for cough treatment. On the other hand, the Ultra Performance Liquid Chromatographic technique is characterized by having the power of increasing chromatographic efficiency and decreasing run time compared to the traditional High Performance Liquid Chromatographic one. In this work, this power was exploited for the simultaneous determination of pholcodine, guaiacol along with three guaiacol impurities, namely; guaiacol impurity A, guaiacol impurity B, and guaiacol impurity E. Good separation was achieved by employing Agilent Zorbax C8 column (50 × 2.1 mm) as the stationary phase, and acetonitrile: phosphate buffer pH 3.5 (40: 60, by volume) as a mobile phase. The proposed method was validated as per International Council for Harmonisation guidelines. Linear relationships, at ranges of 50-1000 µg mL-1 for pholcodine and 5-100 µg mL-1 for guaiacol and the three related impurities, were established. Finally, the proposed method was applied for pholcodine and guaiacol determination in Coughpent® syrup and compared favorably to the reported one.

Normal-Phase TLC and Gradient Reversed-Phase HPLC for the Simultaneous Determination of Enrofloxacin and Bromhexine HCl in Presence of Two of Their Official Impurities.

In this work, two chromatographic methods are developed and validated for the determination of enrofloxacin and bromhexine (BRM) HCl in the presence of two of their specified impurities, ciprofloxacin and BRM impurity C. The suggested chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high-performance liquid chromatography (HPLC). In case of TLC-densitometry, good separation was achieved by using mobile phase of n.butanol:acetone:water:glacial acetic acid:triethylamine (10:3:1:0.5:0.5, by volume) on silica gel stationary phase at 254-nm detection. The developed HPLC method used BDS HYPERSIL C18 column with a mobile phase of water:acetonitrile:methanol:triflouroacetic acid. A linear gradient elution of 75-10%, 20-50% and 5-40% for water, acetonitrile and methanol, respectively, was applied in 13 min at a flow rate of 1.5 mL min-1. These methods were sufficient to separate the four substances simultaneously, and they are validated as per International Conference on Harmonization guidelines.

Two fabricated carbon paste electrodes for novel potentiometric determination of probenecid in dosage form and human plasma.

Solid contact ion selective electrodes are extensively utilized owing to their marvelous performance over traditional liquid contact ones. The main drawback of those solid contact electrodes is aqueous layer formation which affects their constancy. Herein and to overcome this common drawback, a carbon paste electrode containing poly(3,4-ethylenedioxythiophene) was constructed and used for determination of probenecid at variant pH values. This modification decreased the potential drift down to 0.8 mV/h and improved its stability over 30 days. A Nernstian slope of - 57.8 mV/decade associated with a linear range of 1.0 × 10-6-1.0 × 10-2 mol/L was obtained. The modified carbon paste electrode successfully detected up to 8.0 × 10-7 mol/L probenecid. Results of this modified carbon paste electrode were also compared to unmodified one.

Stability indicating potentiometric method for the determination of palonosetron HCl using two different sensors.

This paper presents a novel potentiometric approach for the determination of palonosetron HCl using two sensors; ionophore-free and ionophore-doped ones. The two sensors successfully determined the cited drug in the range of 1 × 10-5-1 × 10-2 M with respective Nernstian slopes of 54.9 ± 0.25 and 59.3 ± 0.16 mV/decade. Incorporating calix[8]arene as an ionophore resulted in a lower detection limit (LOD = 3.1 × 10-6 M) and enhanced selectivity when compared to the ionophore-free sensor (LOD = 7.9 × 10-6 M). This modification was also associated with faster response for the ionophore-doped sensor (response time = 20 s) compared to the ionophore-free one (response time = 30 s). The two sensors showed a stable response over a pH range of 3.0-8.0. They successfully determined palonosetron HCl in presence of its oxidative degradation products. They were also used for direct determination of the drug in commercially available parenteral solution without any interference from other dosage forms' additives.

A green TLC densitometric method for the simultaneous detection and quantification of naphazoline HCl, pheniramine maleate along with three official impurities.

Impurity profiling of a pharmaceutical compound is now taking great attention during quality assessment of pharmaceuticals, as presence of small amount of impurities may affect safety and efficacy. In this work, a novel TLC chromatographic method coupled with densitometric detection was established for the simultaneous quantification of naphazoline HCl, pheniramine maleate and three of their official impurities, namely; naphazoline impurity B, pheniramine impurities; A & B. Chromatographic separation was carried out on TLC aluminum silica plates F254, as a stationary phase, using methanol: ethyl acetate: 33.0% ammonia (2.0: 8.0: 1.0, by volume), as a mobile phase. Plates were examined at 260.0 nm and International Council for Harmonisation (ICH) guidelines were followed for method's validation. Important factors, such as; composition of mobile phase and detection wavelengths were optimized. Linearity was achieved over the ranges of 2.0-50.0 µg band-1 for naphazoline, 10.0-110.0 µg band-1 for pheniramine, 0.1-10.0 µg band-1 for naphazoline impurity B and 2.0-50.0 µg band-1 for both pheniramine impurities. The proposed method was assessed in terms of accuracy, precision and robustness where satisfactory results (recovery % ≈ 100% and RSD < 2) were obtained. The method was also applied for the simultaneous determination of naphazoline HCl and pheniramine maleate, in Naphcon-A® eye drops, with respective recoveries of 101.36% and 100.94%. Method greenness was evaluated and compared to the reported HPLC one via environmental, health and safety tool. The developed method has much potential over the reported one of being simple, selective, economic and time saving for the analysis of the five cited compounds.

A sensing platform of molecular imprinted polymer-based polyaniline/carbon paste electrodes for simultaneous potentiometric determination of alfuzosin and solifenacin in binary co-formulation and spiked plasma.

Solid contact ion-selective electrodes (ISEs) have witnessed versatile applications in pharmaceutical and biological analysis however they suffer from some limitations. Besides formation of water layer, the doped ion exchanger in sensing membrane fails to distinguish between two ionic species having relatively similar lipophilicity and carrying same charges. Those shortcomings practically hampered the simultaneous determination of alfuzosin and solifenacin in their combined pharmaceutical combination. Hence, this paper was directed to develop two carbon paste electrodes allowing their simultaneous determination based on molecular imprinted polymers (MIPs). Efforts were firstly directed to stabilize the potential signals through synthesis of polyaniline (PANI) nanoparticles with 26 nm particle size as confirmed by means of UV-spectrophotometry, Zeta-sizer and transmission electron microscope. This was followed by its doping at electrode/ion selective membrane interface leading to diminished potential drift, better Nernstian slopes and lower limit of detections. Secondly, MIPs for each drug were prepared by precipitation polymerization technique and fully characterized by Fourier-transform infrared spectroscopy, field-emission scanning electron microscope, differential scanning calorimetry, surface area analysis and rebinding studies. The prepared MIPs were then incorporated in membrane cocktail and doped over PANI layer. The graved cavities inside MIPs act as synthetic host-tailored receptors that could recognize and bind specifically to each drug. The obtained Nernstian slopes were 57.16 mV/decade for alfuzosin MIP-based sensor and 58.17 mV/decade for solifenacin MIP-based one with respective LOD values of 7.9 × 10-7 M and 8.9 × 10-8 M. Moreover, no interference was ostensibly detected from dosage form excipients, plasma constituents or degradation products/official impurities allowing quantification of alfuzosin and solifenacin in their combined capsule, spiked human plasma and in presence of their degradation products.

Smart spectrophotometric methods for stability assessment of two co-formulated antigout drugs.

Simple, sensitive and accurate stability indicating spectrophotometric methods have been developed for the simultaneous determination of probenecid, colchicine as well as colchicine degradation product in their ternary mixture. Probenecid was firstly assayed using the double divisor ratio spectra derivative method. On the other hand, three spectrophotometric methods, namely: ratio difference, derivative ratio and mean centering of ratio spectra, have been suggested for the simultaneous quantification of colchicine and its degradation product. The obtained calibration curves were linear at 2.5-30.0 µg/mL, 0.5-25.0 µg/mL and 1.0-13.0 µg/mL for probenecid, colchicine and colchicine degradation product, respectively. The investigated methods were validated in accordance with the International Council for Harmonisation guidelines and were effectively used for quantification of probenecid and colchicine in their bulk powders and combined pharmaceutical dosage form.

Univariate versus multivariate spectrophotometric methods for the simultaneous determination of omarigliptin and two of its degradation products.

Six selective spectrophotometric techniques, four univariate and two multivariate ones were developed for the determination of the antidiabetic drug omarigliptin (OMR) along with its hydrolytic and oxidative degradation products. The proposed univariate spectrophotometric methods were ratio subtraction, first derivative, derivative ratio and ratio difference. Linearities were constructed in the range of 10.0-180.0 µg mL-1 for both OMR & its hydrolytic degradation product and 10.0-110.0 µg mL-1 for the oxidative degradation one. On the other hand, partial least squares and artificial neural networks were the chosen multivariate approaches. Their linearity ranges were 20.0-60.0 µg mL-1 for OMR and 10.0-30.0 µg mL-1 for the two degradation products. All the methods were validated, effectively applied for quantification of the intact drug in its tablet formulation and favorably compared to the reported one.

Two Validated Chromatographic Methods for Determination of Ciprofloxacin HCl, One of its Specified Impurities and Fluocinolone Acetonide in Newly Approved Otic Solution.

Two sensitive, selective and precise chromatographic methods have been established for concomitant quantification of ciprofloxacin HCl (CIP), fluocinolone acetonide (FLU) along with ciprofloxacin impurity A (CIP-imp A). The first method was thin-layer chromatography (TLC-densitometry) where separation was accomplished using TLC silica plates 60 G.F254 as a stationary phase and chloroform-methanol-33%ammonia (4.6:4.4:1, by volume) as a developing system. The obtained plates were scanned at 260 nm over concentration ranges of 1.0-40.0, 0.6-20.0 and 1.0-40.0 µg band-1 for CIP, FLU and CIP-imp A, respectively. The second method was based on high-performance liquid chromatography using a Zorbax ODS column (5 µm, 150 × 4.6 mm i.d.) where adequate separation was achieved through a mobile phase composed of phosphate buffer pH 3.6-acetonitrile (45:55, v/v) at flow rate 1.0 mL min-1 with ultraviolet detection at 254 nm. Linear regressions were obtained in the range of 1.0-40.0 µg mL-1 for CIP, 0.6-20.0 µg mL-1 for FLU and 1.0-40.0 µg mL-1 for CIP-imp A. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and were successfully applied for determination of CIP and FLU in bulk powder and newly marketed otic solution.

All solid-state miniaturized potentiometric sensors for flunitrazepam determination in beverages.

Flunitrazepam is one of the frequently used hypnotic drugs to incapacitate victims for sexual assault. Appropriate diagnostic tools should be available to victims regarding the growing concern about "date-rape drugs" and their adverse impact on society. Miniaturized screen-printed potentiometric sensors offer crucial point-of-care devices that alleviate this serious problem. In this study, all solid-state screen-printed potentiometric flunitrazepam sensors have been designed. The paper device was printed with silver and carbon ink. Formation of an aqueous layer in the interface between carbon-conducting material and ion-sensing membrane nevertheless poses low reproducibility in the solid-contact electrodes. Accordingly, poly(3,4-ethylenedioxythiophene) (PEDT) nano-dispersion was applied as a conducting hydrophobic polymer on the electrode surface to curb water accumulation. Conditioning of ion-sensing membrane in the vicinity of reference membrane has been considered carefully using special protocol. Electrochemical characteristics of the proposed PEDT-based sensor were calculated and compared favorably to PEDT-free one. The miniaturized device was successfully used for the determination of flunitrazepam in carbonated soft drinks, energy drink, and malt beverage. Statistical comparison between the proposed sensor and official method revealed no significant difference. Nevertheless, the proposed sensor provides simple and user-friendly diagnostic tool with less equipment for on-site determination of flunitrazepam.

Stability-Indicating Chromatographic Methods for the Simultaneous Determination of Probenecid and Colchicine in Their Combined Tablet.

Two stability-indicating chromatographic methods have been established and validated for concurrent determination of probenecid (PRO), colchicine (COL) along with the degradation product of colchicine (COL deg). PRO and COL were exposed to a stress stability study, which includes acidic, alkaline, oxidative, photolytic and thermal degradations. Chromatographic methods included the use of thin layer chromatography (TLC-densitometry) and high performance liquid chromatography (HPLC). In the first method, separation was achieved by using aluminum TLC plates that were precoated with silica gel G.F254 as the stationary phase and ethyl acetate-methanol-33%ammonia (8:1:1, by volume) as a mobile phase. The obtained chromatograms were scanned at 254 nm. The second method was based on HPLC using a RP- C18 column with isocratic elution. Good separation was obtained through a mobile phase comprised of phosphate buffer pH 5-acetonitrile (70:30, v/v) at a flow rate of 1.0 mL min-1 and ultraviolet detection at 254 nm. Different parameters affecting efficiency of the two methods were studied accurately for optimum separation of the three cited components. The suggested methods were validated according to the International Conference on Harmonization (ICH) guidelines and were applied for bulk powder and commercial tablets.

Quality and Stability Profile Assessment of the Recent Antidiabetic Omarigliptin by Using Different Chromatographic Methods.

In a contribution to stability profiling of the recent antidiabetic drug, omarigliptin (OMR), two stability-indicating chromatographic methods were developed and validated. Stability profiling was performed for OMR under different stress conditions as acidic, alkaline, oxidative, photolytic and thermal degradations. Structures elucidation to all formed degradation products were identified using IR and mass spectrometry. Thin Layer Chromatography (TLC) and High-Performance Liquid Chromatography (HPLC) were used. In TLC-densitometric method, aluminum TLC plates precoated with silica gel G.F254 were used as stationary phase along with methanol: ethyl acetate: 33% ammonia (2:8:1,v/v/v) as mobile phase. The obtained chromatograms were scanned at 254 nm over concertation range of 5-70 µg band-1 for OMR. The second chromatographic method was an HPLC one with diode array detection and RP-C18 column with isocratic elution. Mobile phase used was composed of phosphate buffer pH 3.5: acetonitrile (80, 20, v/v), delivered at flow rate of 1.0 mL min-1. Diode array detector was adjusted at 230 nm with linearity range of 15-180 µg mL-1 for OMR. Several factors affecting TLC and HPLC efficiency have been carefully studied. The developed methods were validated according to International Conference on Harmonization guidelines and successfully applied for assessment of OMR in bulk powder and tablets.

Determination of naphazoline HCl, pheniramine maleate and their official impurities in eye drops and biological fluid rabbit aqueous humor by a validated LC-DAD method.

A simple RP-HPLC-DAD method was developed and validated, as per the ICH guidelines, for simultaneous determination of naphazoline HCl (NPZ) & pheniramine maleate (PHN) along with three of their official impurities. Chromatographic separation was performed on a hypersil ODS column (5 mm, 250-4.6 mm i.d.) with isocratic elution using phosphate buffer pH 6.0: acetonitrile (70 : 30, v/v) as mobile phase, at a flow rate of 1.0 mL min-1 and UV detection at 260.0 nm. The developed method was found to be linear over the concentration ranges of 5.00-45.00 µg mL-1 for NPZ and NPZ impurity B and 10.00-110.00 µg mL-1, 10-70 µg mL-1 and 10-120 µg mL-1 for PHN, and PHN impurity A and B, respectively, with correlation coefficient values <0.999 for the five cited compounds. The method was confirmed to be accurate, robust and precise with RSD >2.0%. LOD and LOQ values for the five cited compounds were calculated. Moreover, the method was also validated in rabbit aqueous humor as per the US food and drug administration (FDA) bioanalytical validation guidelines. Finally, the proposed method was applied for the analysis of the two drugs along with their impurities in dosage form and spiked aqueous humor samples.