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

A green TLC densitometric method for the simultaneous detection and quantification of naphazoline... Impurity profiling of a pharmaceutical compound is now taking great attention during quality assessment of phar - maceuticals, 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 quantifica- tion 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. −1 −1 Linearity was achieved over the ranges of 2.0–50.0 µg band for naphazoline, 10.0–110.0 µg band for pheniramine, −1 −1 0.1–10.0 µg band for naphazoline impurity B and 2.0–50.0 µg band for both pheniramine impurities. The pro- posed 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. Keywords: Naphazoline, Pheniramine, Impurities, TLC, EHS tool may affect the quality, potency and safety of the drug Introduction product [1]. In a different manner, Thin Layer Chroma - A great attention was given, by modern pharmaceuti- tography (TLC) is one of the most familiar and adapt- cal analysis, to impurity profiling of the drug substances, able techniques used in detection and quantification of as the presence of impurities, even in trace amounts, related impurities in the pharmaceutical filed. It has sev - eral advantages including; simplicity, cost-effectiveness, *Correspondence: mahmoud.eltantawy@pharma.cu.edu.eg rapidness as well as good resolving power with accurate Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, quantification of multicomponent mixtures [2]. Kasr el Aini Street, Cairo 11562, Egypt Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kelani et al. BMC Chemistry (2022) 16:24 Page 2 of 11 Naphazoline HCl (NPZ) is chemically known as [38], spectrophotometric [39, 40], HPLC [17–19, 27, 41– 2-(naphthalen-1-ylmethyl)-4, 5-dihydro-1H-imidazole; 48] and capillary electrophoretic ones [36, 49]. hydrochloride. It has a decongestant property through NPZ and PHN are usually co-formulated together in mimicking the sympathetic  influence on alpha-adren - optic dosage forms used for eye inflammation treat - ergic receptors. It plays an important role in manag- ment. The literature survey revealed some methods for ing allergic conjunctiva, as it can reduce eye swelling their simultaneous quantification, such as HPLC [17–19, and edema by acting on those receptors in the con- 27] and one capillary electrophoresis [36]. One of the junctiva arterioles [3]. NPZ is an authorized drug in the reported HPLC methods described their determination United States (USP) [4] and British (BP) [5] pharmaco- in presence of three selected impurities [19]. As a result, peias where its determination was carried out by HPLC the aim of this work was to develop a first validated, as methods. Moreover, the BP states four specified official per ICH guidelines [50], TLC densitometric method for impurities; one of them is impurity B (NPZ impurity B). detection and quantification of NPZ, PHN, NPZ impu - On reviewing literature, NPZ has been determined as a rity B, PHN impurities; A and B, (Fig.  1). The proposed single drug or in combination using several techniques, method was successfully applied for their simultaneous namely; spectrophotometry [6–14], HPLC [15–27], TLC determination in a quinary mixture as well as in phar- [28] and capillary electrophoresis [29–36]. maceutical eye drops. Furthermore, the organic solvents Pheniramine maleate (PHN) is chemically designed used in this work were assessed and compared to that as (Z)-but-2-enedioic acid; N, N-dimethyl-3-phenyl- used in the reported HPLC one [19] by the aid of envi- 3-pyridin-2-ylpropan-1-amine. It is widely available in ronmental, health and safety (EHS) tool [51]. eye drops due to its antihistaminic and anticholinergic effect [37]. PHN is official in USP and BP whereas HPLC Methods/Experimental technique was reported for its assay. Two impurities were Instruments stated in BP for PHN, namely; A & B [5]. The literature TLC system; a Camag Linomat autosampler (Muttenzl, survey revealed different techniques for its quantification Switzerland), a Camag micro syringe (100 µL), a Camag either in single or in combined form, such as titrimetric 35/N/30319 TLC scanner with win CATS software, UV Fig. 1 Chemical structures of the five cited components Kelani  et al. BMC Chemistry (2022) 16:24 Page 3 of 11 lamp with a short wavelength at 260  nm (Desaga, Wies- plates (10 × 20  cm). A mobile phase of methanol: ethyl loch, Germany) and TLC plates (10 × 20  cm) pre-coated acetate: 33.0% ammonia (2.0: 8.0: 1.0, by volume) was with silica gel GF of 0.25 mm thickness (Merck, Darm- used for elution over 8.5  cm distance. The elution time stadt, Germany). was around 6.0 min. After that, the plates were removed, air dried and scanned at 260.0  nm. Calibration curves, Materials and chemicals for the five cited drugs, representing the polynomial rela - Pure standard tionship between peak area and corresponding concen- NPZ and PHN were kindly provided by Eva pharma tration were constructed and regression parameters were pharmaceutical company, Cairo, Egypt. Their purities computed. were assessed to be 100.12 ± 0.102% and 99.58 ± 0.124%, respectively [5]. The impurities (NPZ impurity B, PHN Assay of laboratory prepared mixtures impurities; A & B) were purchased from the German Different mixtures of NPZ, PHN and their official impu - company Alfa Aesar Company. Their certified potency rities were prepared as mentioned under solution section values were found to be 99.00%, 100.30%, and 99.70%, and mixed with different ratios. The prepared mixtures respectively. were then analyzed by the proposed method as men- tioned above. Pharmaceutical dosage form Naphcon-A eye drops (Batch no.H13949-0615); manu- Forced degradation study factured by Alcon laboratories INC (Novartis Company), Stability of the two cited drugs were studied under differ - −1 labeled to contain 0.25 & 3.0 mg  mL of NPZ and PHN, ent conditions, namely; acidic, alkaline, oxidative, photol- respectively, and has been purchased from Egyptian ytic and thermal ones. For acidic and alkaline hydrolyses, market. a mass of 10 mg of each drug was separately dissolved in least amounts of methanol then refluxing in 1 M HCl or Chemicals and reagents methanolic solutions of 1 M NaOH at 100 °C for 2 h. For Analytical-grade chemicals were used; methanol (Alpha, oxidative studies, 0.5  mL of 5% H O aqueous solution 2 2 −1 Egypt), ethyl acetate (Otsuka, Egypt), chloroform, ace- was separately added to 10 mL of 1.0 mg  mL solutions. tone, 33.0% ammonia (El-Nasr, Egypt), hydrochloric acid The two drugs solutions were then kept at room tem - (Sigma, Germany), 30.0% hydrogen peroxide solution perature for 24 h. For photolytic study, thin layers of each (Adwic, Egypt) and sodium hydroxide pellets (Piochem, powdered drug was uniformly spread in two Petri dishes, Egypt). and exposed to UV light at 254 nm for 10 h at a distance of 15  cm. Thermal degradation was assessed through Solutions sealing each powdered drug in glass ampoules and heat- Standard solutions In 10-mL volumetric flasks, stand - ing in a thermostatic oven at 100 °C for 7 h. Finally, sam- −1 ard solutions of 20.0  mg  mL , for NPZ, PHN and two ples were periodically withdrawn for observing the forced PHN impurities, were separately prepared in methanol. degradation process. −1 For NPZ impurity B, 1.0 mg  mL standard solution was prepared. Pharmaceutical application The content of 10 Naphcon-A eye drops were emptied. Laboratory prepared mixtures Different aliquots, from 20.0  mL aliquot was transferred into a 25-mL measur- the five standard solutions, were transferred into separate ing flask. 3.0  mL methanol was added and the flask was 10-mL volumetric flasks to prepare laboratory prepared then sonicated for 20.0  min. Volume was completed to mixtures of various ratios. The volume of each flask was the mark using methanol to obtain final concentration of −1 −1 then completed to the mark using methanol.200.0 µg  mL NPZ and 2400.0  µg  mL PHN. 10.0  µLs from this solution were applied onto TLC plates. Finally, Procedures solutions were analyzed as mentioned before under con- Construction of the calibration curves struction of the calibration curves. Aliquots equivalent to 2.0–50.0  mg of NPZ, 10.0– 110.0  mg of PHN, 0.1–10.0  mg of NPZ impurity B and Results and discussion 2.0–50.0 mg of two PHN impurities (A & B) were trans- The importance of impurity detection and determination ferred from their corresponding solutions into five sets of evokes the requirement for developing simple, economi- 10-mL volumetric flasks. Volumes were then completed cal, rapid and accurate analytical techniques which can to the mark with methanol. 10.0  µL from each solution be utilized easily in quality control laboratories wherein was applied as a band with 3.0  mm length onto TLC cost and time are essential. Owing to simplicity, cost Kelani et al. BMC Chemistry (2022) 16:24 Page 4 of 11 effectiveness, time saving, no need for tedious sample NPZ impurity B, NPZ, PHN, PHN impurity A and PHN preparation and high sensitivity, TLC densitometry could impurity B (Fig.  2). Scanning profiles were obtained at be considered as one of the best options for that purpose 260.0 nm, and five calibration curves were then plotted. [2, 52, 53] Here, we present a novel TLC densitometric method for the simultaneous determination of NPZ, System suitability parameters PHN and three related official impurities (NPZ impu - To evaluate the performance of the proposed TLC rity B, PHN impurities; A & B) in their quinary mixture. method, system suitability parameters were calculated Moreover, EHS tool is applied for greenness evaluation manually [54]. The results of retardation, resolution, of this method in comparison to our previously reported capacity and tailing factors for the five components were HPLC one [19]. obtained in Table 2. Method validation Development and optimization of TLC densitometric Method’s validation was conducted in agreement to ICH method guidelines [50]. Various mobile phases were tried to get optimum separa- tion and resolution between the five cited components. Linearity and range Firstly, mixtures with different ratios of methanol and Polynomial relationships were established between the ethyl acetate have been tried, but the separation between integrated peak area and the corresponding concentra- the five cited components was not achieved. Thus, dif - −1 tion in the ranges of 2.0  − 50.0 µg band , 10.0–110.0 µg ferent solvents were added individually to the previous −1 −1 −1 band , 0.1  − 10.0 µg band and 2.0  − 50.0 µg band mixture for improving the separation between the stud- for NPZ, PHN, NPZ impurity B and the two PHN related ied components such as chloroform, acetone and ammo- impurities, respectively. nia. Table  1 summarizes the obtained resolution values during this optimization phase. It was noticed that addi- tion of ammonia to the conventional mixture enhanced Accuracy separation and resolution between the studied drugs. Accuracy was assessed by applying the previously men- Finally, a mixture of methanol–ethyl acetate–ammonia tioned procedures on pure samples with various concen- (2.0: 8.0: 1.0, by volume) was chosen for optimum suit- trations within the defined ranges. Satisfactory results ability parameters. Moreover, different wavelengths were regarding recovery % were computed in Table 3. tried for evaluating the densitometric measurement as 260.0  nm and 280.0  nm. 260.0  nm was the wavelength Precision of choice as it gave the highest sensitivity with minimum Repeatability Three separate concentrations of NPZ −1 noise for measuring the five cited components. Retarda - (15.0, 25.0, 40.0  µg band ), PHN (30.0, 50.0, 70.0  µg −1 −1 tion factor (R ) values were sequentially at 0.18 ± 0.02, band ), NPZ impurity B (3.0, 5.0, 8.0  µg b and ), PHN −1 0.35 ± 0.02, 0.49 ± 0.02, 0.63 ± 0.02 and 0.83 ± 0.02 for impurities; A & B (15.0, 25.0, 40.0  µg b and ) were ana- Table 1 The obtained resolution values during mobile phase optimization a a a a Experiment No. Mobile phase composition Rs1 Rs2 Rs3 Rs4 1 Methanol–ethyl acetate (3.5:6.5, v/v) 1.32 1.24 1.34 0.57 2 Methanol–ethyl acetate (3.0:7.0, v/v) 1.35 1.33 1.37 0.62 3 Methanol–ethyl acetate (2.5:7.5, v/v) 1.42 1.37 1.40 0.70 4 Methanol–ethyl acetate (2.0:8.0, v/v) 1.47 1.39 1.45 0.83 5 Methanol–ethyl acetate–chloroform (2.0:8.0:1.0, v/v/v) 1.49 1.51 1.13 0.75 6 Methanol–ethyl acetate–chloroform (2.0:8.0:0.8, v/v/v) 1.52 1.53 1.22 0.78 7 Methanol–ethyl acetate–chloroform (2.0:8.0:0.5, v/v/v) 1.53 1.54 1.27 0.89 8 Methanol–ethyl acetate–chloroform (2.0:8.0:0.2, v/v/v) 1.55 1.57 1.35 0.94 9 Methanol–ethyl acetate– acetone (2.0:8.0:1.0, v/v/v) 1.46 1.44 1.21 1.13 10 Methanol–ethyl acetate– acetone (2.0:8.0:0.8, v/v/v) 1.47 1.47 1.23 1.17 11 Methanol–ethyl acetate– acetone (2.0:8.0:0.5, v/v/v) 1.49 1.48 1.27 1.22 12 Methanol–ethyl acetate– acetone (2.0:8.0:0.2, v/v/v) 1.51 1.49 1.36 1.28 Rs1, Rs2, Rs3 and Rs4 are the obtained resolutions between NPZ impurity B & NPZ, NPZ & PHN, PHN & PHN impurity A and PHN impurity A & PHN impurity B, respectively Kelani  et al. BMC Chemistry (2022) 16:24 Page 5 of 11 −1 −1 −1 Fig. 2 TLC chromatogram of NPZ (30.0 µg band ), PHN (90.0 µg band ) and three of their official impurities as NPZ impurity B (10.0 µg band ), −1 −1 PHN impurity A (40.0 µg band ) and PHN impurity B (40.0 µg band ) using a mobile phase of methanol: ethyl acetate: ammonia (2.0: 8.0: 1.0, by volume) and detection at 260.0 nm Table 2 Parameters required for system suitability tests of TLC densitometric method Parameter NPZ impurity B NPZ PHN PHN impurity A PHN impurity B R 0.18 0.35 0.49 0.63 0.83 Resolution (Rs) NA 1.50 1.58 1.40 1.64 Tailing factor ( T ) 1.50 0.80 1.30 1.20 1.20 Retention factor (k’) 4.56 1.86 1.04 0.59 0.20 Selectivity factor (α) NA 2.45 1.79 1.76 2.95 Column efficiency (N) 262.44 196.00 635.04 425.11 737.86 Height equivalent to theoretical 0.034 0.046 0.014 0.021 0.012 plate (mm) Retention factor (k’) = (1 − R )/R f f Calculation of α = k’2/k’1 c 2 Column efficiency (N) = 16 (z/w) , where z is the migration length of the spot, w is the spot width Kelani et al. BMC Chemistry (2022) 16:24 Page 6 of 11 Table 3 Regression parameters for determination of the studied drugs by the proposed TLC densitometric method Parameter NPZ impurity B NPZ PHN PHN impurity A PHN impurity B −1 −1 −1 −1 −1 Range 0.1–10.0 µg band 2.0–50.0 µg band 10.0–110.0 µg band 0.2–50.0 µg band 0.2–50.0 µg band a a a a a SlopeNo. 1 = − 172.85No. 1 = − 13.91No. 1 = − 1.82No. 1 = − 9.78No. 1 = − 11.64 a a a a a No. 2 = 3480.76No. 2 = 1389.10No. 2 = 584.57No. 2 = 943.45No. 2 = 962.46 Intercept 1343.80 3305.64 4730.10 14,708.36 11,166.09 a a a a a SE of the slopeNo. 1 = 11.13No. 1 = 0.54No. 1 = 0.10No. 1 = 0.45No. 1 = 0.59 a a a a a No. 2 = 112.36No. 2 = 26.87No. 2 = 15.50No. 2 = 23.73No. 2 = 25.15 SE of the Intercept 182.40 249.72 492.06 232.59 202.55 Specificity (mean ± SD) 99.11 ± 1.382 100.72 ± 0.221 100.25 ± 1.054 99.10 ± 1.152 98.89 ± 1.963 Accuracy 99.74 101.15 100.42 99.97 100.99 Repeatability (RSD) 1.28 1.47 1.29 0.74 1.03 Intermediate precision (RSD) 1.77 0.55 1.84 0.96 1.79 Robustness 0.98 0.78 0.84 1.07 1.45 −1 LOD (µg band ) 0.01 0.60 2.38 0.05 0.06 −1 LOQ (µg band ) 0.03 1.82 7.21 0.15 0.18 Correlation coefficient (r) 0.999 0.999 0.999 0.999 0.999 a 2 −1 Slope 1 and 2 are the coefficients of a polynomial regression, A = ax + bx + c, where A is the integrated peak area, x is the concentration of the drug (μg band ), a and b are coefficients 1 and 2, respectively, and c is the intercept Average of determinations in seven laboratory-prepared mixtures −1 Fig. 3 TLC chromatogram of 100 µg band PHN after H O treatment; R ≈ 0.49 for PHN & ≈ 0.32 for its oxidative degradation 2 2 f Kelani  et al. BMC Chemistry (2022) 16:24 Page 7 of 11 Table 4 Determination of NPZ, PHN in their dosage form and application of standard addition technique using the proposed TLC method Naphcon-A eye drop % found Standard addition technique Mean ± SD Taken Added Recovery % −1 −1 NPZ 101.36 ± 1.51 10.0 µg band 5.0 µg band 101.30 −1 10.0 µg band 101.75 −1 20.0 µg band 99.99 Mean ± SD 101.01 ± 0.914 −1 −1 PHN 100.94 ± 1.73 20.0 µg band 10.0 µg band 100.95 −1 20.0 µg band 99.02 −1 40.0 µg band 100.84 Mean ± SD 100.27 ± 1.084 Average determinations of four eye drop dosage form solution lyzed intra-daily three times. Results were obtained pre- Table 5 Statistical comparison between the results obtained by liminary to RSD calculation, Table 3. the proposed method and the official BP method Parameter TLC Official BP Intermediate precision Inter-daily analysis was also con- method [5] ducted for the formerly selected concentrations. Results NPZ PHN NPZ PHN are represented in Table 3. Mean of recoveries 101.15 100.42 99.63 99.71 Robustness SD 1.095 1.712 0.977 1.153 It was evaluated by studying the effect of deliberately Variance 1.199 2.931 0.955 1.329 changing the mobile phase composition; methanol n 5 5 5 5 a a (2.0 ± 0.2  mL), ethyl acetate (8.0 ± 0.2  mL) and ammo- Student’s t-test 2.316 (2.306) 0.778 (2.306) NA NA a a nia (1.0 ± 0.1  mL). This study was conducted on three F-test 1.26 (6.39) 2.21 (6.39) NA NA independent concentrations of NPZ (5.0, 20.0, 40.0  µg These values represent the corresponding tabulated values of t and F at −1 −1 band ), PHN (15.0, 40.0, 70.0  µg b and ), NPZ impu- p = 0.05 −1 rity B (1.0, 5.0, 8.0  µg band ) and two PHN impurities −1 (1.0, 20.0, 40.0  µg b and each). Satisfactory RSDs were corresponding to this specified impurity. This outcome is obtained, Table 3. consistent with what previously reported [20]. PHN was stable towards all conditions except for oxidation where Specificity ≈ 30.0% was degraded upon H O treatment (Fig. 3). It was assessed by analysis of different laboratory pre - 2 2 pared mixtures containing various ratios of the five Analysis of pharmaceutical eye drops studied components. Table  3 shows good recovery per- The two active pharmaceutical ingredients (NPZ and centages and RSDs for analyzing those mixtures. Further- PHN) were simultaneously quantified in their combined more, forced degradation study was conducted whereas dosage form. Excipients did not have an impact on the the two drugs were subjected to different stress condi - obtained TLC chromatograms. In addition, method’s tions: (1) Acidic and alkaline hydrolysis via refluxing with validity was proved using standard addition technique, 1 M HCl and 1 M NaOH for 2 h, respectively, (2) Oxida- Table 4. tive degradation through treatment of each solution with 5% H O then keeping at room temperature for 24 h, (3) 2 2 Statistical analysis Photostability on powdered drugs placed in petri dishes, Statistical comparison between results of the suggested using 254 nm UV light for 10 h, and finally (4) Dry heat TLC method and that of official HPLC ones [5] were by putting each drug powder in 100 °C oven for 7 h. The performed. The calculated values of student’s t-test and degradation study was monitored by the proposed TLC F-test indicated that there is no significant difference method. NPZ was only liable to alkaline hydrolysis giv- observed between those methods, Table 5. ing its impurity B where its spot (R ≈ 0.35) disappeared accompanied by appearance of a new one (R ≈ 0.18) f Kelani et al. BMC Chemistry (2022) 16:24 Page 8 of 11 Table 6 EHS assessment of the solvents used in this work (ethyl acetate & methanol) as well as the reported one (acetonitrile) Selected substance Safety Health Environment Total Release Fire/Explosion Reaction/ Acute toxicity Irritation Chronic toxicity Persistency Air Hazard Water Hazard potential Decomposition Acetonitrile 0.61 1.00 0.60 0.51 0.63 0.43 0.34 0.43 0.00 4.55 Ethyl acetate 0.62 1.00 0.00 0.28 0.63 0.17 0.03 0.17 0.00 2.89 Methanol 0.65 1.00 0.00 0.27 0.11 0.32 0.00 0.32 0.00 2.66 Obtained by summation of nine main categories scores Kelani  et al. BMC Chemistry (2022) 16:24 Page 9 of 11 Table 7 Comparative overview on reported HPLC and proposed TLC methods Ref. No LOD Elution time EHS score F-test NPZ PHN NPZ PHN −1 −1 a a [19] 1.29 µg mL 3.10 µg mL ≈ 30 min 4.55 (acetonitrile) 3.90 (6.39) 2.22 (6.39) −1 −1 This work 0.60 µg band 2.38 µg band ≈ 6 min 2.89 (ethyl acetate) 2.66 (methanol) This value represents the corresponding tabulated value of F at p = 0.05 draft. MAH: Conceptualization, Methodology, Software, Formal analysis, Data Greenness evaluation and methods comparison curation, Visualization, Supervision, Project administration, Funding acquisi- In order to assess and compare this work with our pre- tion, Writing—review & editing. AMH; Methodology, Software, Validation, viously reported HPLC one [19], EHS tool was applied. Formal analysis, Investigation, Funding acquisition, Project administration, Writing—original draft, Writing—review & editing. MAT; Methodology, Soft- In this tool, nine categories representing safety, health ware, Validation, Formal analysis, Investigation, Funding acquisition, Project and environmental hazards are utilized for organic administration, Writing—original draft, Writing—review & editing. All authors solvents assessment whereas the lower the calcu- read and approved the final manuscript. lated score, the greener the solvent will be [51]. The Funding calculated scores for methanol, ethyl acetate (used in Open access funding provided by The Science, Technology & Innovation this work) and acetonitrile (used in reported HPLC) Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Not applicable. revealed the dominance of the proposed method over our previously reported one in terms of environmental Availability of data and materials sustainability, Table  6. Finally, a comparative overview All data generated or analysed during this study are included in this published article. on those two methods along with a statistical F-test for their variances are shown in Table 7. Declarations Conclusion Ethics approval and consent to participate A novel simple TLC densitometric method was estab- Not applicable. lished for the simultaneous detection and quantification Consent for publication of NPZ, PHN as well as three of their official impurities Not applicable. (NPZ impurity B, PHN impurities; A & B). The proposed Competing interests method was validated in agreement to ICH guidelines. The authors declare that they have no competing interests. NPZ and PHN were successfully determined in their combined eye drops. EHS tool was utilized for green- Author details Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr ness assessment of the organic solvents used in this work el Aini Street, Cairo 11562, Egypt. Analytical Chemistry Department, Faculty as well as the previously reported HPLC one. The pro - of Pharmacy, Modern University for Technology and Information, El-hadaba posed TLC densitometric method provides simplicity, El-Wosta, Mokatam, 5th district, Cairo, Egypt. Chemistry Department, Faculty of Pharmacy, October 6 University, 6 October City, Giza, Egypt. low cost, fast analysis and environmental sustainability compared to the reported one. In addition, the capacity Received: 6 February 2022 Accepted: 25 March 2022 of the method to detect low concentrations of NPZ and PHN official impurities highlights it as a promising one for impurity profiling of those drugs. References 1. Tantawy MA, Weshahy SA, Wadie M, Rezk MR. 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A green TLC densitometric method for the simultaneous detection and quantification of naphazoline HCl, pheniramine maleate along with three official impurities

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Abstract

Impurity profiling of a pharmaceutical compound is now taking great attention during quality assessment of phar - maceuticals, 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 quantifica- tion 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. −1 −1 Linearity was achieved over the ranges of 2.0–50.0 µg band for naphazoline, 10.0–110.0 µg band for pheniramine, −1 −1 0.1–10.0 µg band for naphazoline impurity B and 2.0–50.0 µg band for both pheniramine impurities. The pro- posed 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. Keywords: Naphazoline, Pheniramine, Impurities, TLC, EHS tool may affect the quality, potency and safety of the drug Introduction product [1]. In a different manner, Thin Layer Chroma - A great attention was given, by modern pharmaceuti- tography (TLC) is one of the most familiar and adapt- cal analysis, to impurity profiling of the drug substances, able techniques used in detection and quantification of as the presence of impurities, even in trace amounts, related impurities in the pharmaceutical filed. It has sev - eral advantages including; simplicity, cost-effectiveness, *Correspondence: mahmoud.eltantawy@pharma.cu.edu.eg rapidness as well as good resolving power with accurate Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, quantification of multicomponent mixtures [2]. Kasr el Aini Street, Cairo 11562, Egypt Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kelani et al. BMC Chemistry (2022) 16:24 Page 2 of 11 Naphazoline HCl (NPZ) is chemically known as [38], spectrophotometric [39, 40], HPLC [17–19, 27, 41– 2-(naphthalen-1-ylmethyl)-4, 5-dihydro-1H-imidazole; 48] and capillary electrophoretic ones [36, 49]. hydrochloride. It has a decongestant property through NPZ and PHN are usually co-formulated together in mimicking the sympathetic  influence on alpha-adren - optic dosage forms used for eye inflammation treat - ergic receptors. It plays an important role in manag- ment. The literature survey revealed some methods for ing allergic conjunctiva, as it can reduce eye swelling their simultaneous quantification, such as HPLC [17–19, and edema by acting on those receptors in the con- 27] and one capillary electrophoresis [36]. One of the junctiva arterioles [3]. NPZ is an authorized drug in the reported HPLC methods described their determination United States (USP) [4] and British (BP) [5] pharmaco- in presence of three selected impurities [19]. As a result, peias where its determination was carried out by HPLC the aim of this work was to develop a first validated, as methods. Moreover, the BP states four specified official per ICH guidelines [50], TLC densitometric method for impurities; one of them is impurity B (NPZ impurity B). detection and quantification of NPZ, PHN, NPZ impu - On reviewing literature, NPZ has been determined as a rity B, PHN impurities; A and B, (Fig.  1). The proposed single drug or in combination using several techniques, method was successfully applied for their simultaneous namely; spectrophotometry [6–14], HPLC [15–27], TLC determination in a quinary mixture as well as in phar- [28] and capillary electrophoresis [29–36]. maceutical eye drops. Furthermore, the organic solvents Pheniramine maleate (PHN) is chemically designed used in this work were assessed and compared to that as (Z)-but-2-enedioic acid; N, N-dimethyl-3-phenyl- used in the reported HPLC one [19] by the aid of envi- 3-pyridin-2-ylpropan-1-amine. It is widely available in ronmental, health and safety (EHS) tool [51]. eye drops due to its antihistaminic and anticholinergic effect [37]. PHN is official in USP and BP whereas HPLC Methods/Experimental technique was reported for its assay. Two impurities were Instruments stated in BP for PHN, namely; A & B [5]. The literature TLC system; a Camag Linomat autosampler (Muttenzl, survey revealed different techniques for its quantification Switzerland), a Camag micro syringe (100 µL), a Camag either in single or in combined form, such as titrimetric 35/N/30319 TLC scanner with win CATS software, UV Fig. 1 Chemical structures of the five cited components Kelani  et al. BMC Chemistry (2022) 16:24 Page 3 of 11 lamp with a short wavelength at 260  nm (Desaga, Wies- plates (10 × 20  cm). A mobile phase of methanol: ethyl loch, Germany) and TLC plates (10 × 20  cm) pre-coated acetate: 33.0% ammonia (2.0: 8.0: 1.0, by volume) was with silica gel GF of 0.25 mm thickness (Merck, Darm- used for elution over 8.5  cm distance. The elution time stadt, Germany). was around 6.0 min. After that, the plates were removed, air dried and scanned at 260.0  nm. Calibration curves, Materials and chemicals for the five cited drugs, representing the polynomial rela - Pure standard tionship between peak area and corresponding concen- NPZ and PHN were kindly provided by Eva pharma tration were constructed and regression parameters were pharmaceutical company, Cairo, Egypt. Their purities computed. were assessed to be 100.12 ± 0.102% and 99.58 ± 0.124%, respectively [5]. The impurities (NPZ impurity B, PHN Assay of laboratory prepared mixtures impurities; A & B) were purchased from the German Different mixtures of NPZ, PHN and their official impu - company Alfa Aesar Company. Their certified potency rities were prepared as mentioned under solution section values were found to be 99.00%, 100.30%, and 99.70%, and mixed with different ratios. The prepared mixtures respectively. were then analyzed by the proposed method as men- tioned above. Pharmaceutical dosage form Naphcon-A eye drops (Batch no.H13949-0615); manu- Forced degradation study factured by Alcon laboratories INC (Novartis Company), Stability of the two cited drugs were studied under differ - −1 labeled to contain 0.25 & 3.0 mg  mL of NPZ and PHN, ent conditions, namely; acidic, alkaline, oxidative, photol- respectively, and has been purchased from Egyptian ytic and thermal ones. For acidic and alkaline hydrolyses, market. a mass of 10 mg of each drug was separately dissolved in least amounts of methanol then refluxing in 1 M HCl or Chemicals and reagents methanolic solutions of 1 M NaOH at 100 °C for 2 h. For Analytical-grade chemicals were used; methanol (Alpha, oxidative studies, 0.5  mL of 5% H O aqueous solution 2 2 −1 Egypt), ethyl acetate (Otsuka, Egypt), chloroform, ace- was separately added to 10 mL of 1.0 mg  mL solutions. tone, 33.0% ammonia (El-Nasr, Egypt), hydrochloric acid The two drugs solutions were then kept at room tem - (Sigma, Germany), 30.0% hydrogen peroxide solution perature for 24 h. For photolytic study, thin layers of each (Adwic, Egypt) and sodium hydroxide pellets (Piochem, powdered drug was uniformly spread in two Petri dishes, Egypt). and exposed to UV light at 254 nm for 10 h at a distance of 15  cm. Thermal degradation was assessed through Solutions sealing each powdered drug in glass ampoules and heat- Standard solutions In 10-mL volumetric flasks, stand - ing in a thermostatic oven at 100 °C for 7 h. Finally, sam- −1 ard solutions of 20.0  mg  mL , for NPZ, PHN and two ples were periodically withdrawn for observing the forced PHN impurities, were separately prepared in methanol. degradation process. −1 For NPZ impurity B, 1.0 mg  mL standard solution was prepared. Pharmaceutical application The content of 10 Naphcon-A eye drops were emptied. Laboratory prepared mixtures Different aliquots, from 20.0  mL aliquot was transferred into a 25-mL measur- the five standard solutions, were transferred into separate ing flask. 3.0  mL methanol was added and the flask was 10-mL volumetric flasks to prepare laboratory prepared then sonicated for 20.0  min. Volume was completed to mixtures of various ratios. The volume of each flask was the mark using methanol to obtain final concentration of −1 −1 then completed to the mark using methanol.200.0 µg  mL NPZ and 2400.0  µg  mL PHN. 10.0  µLs from this solution were applied onto TLC plates. Finally, Procedures solutions were analyzed as mentioned before under con- Construction of the calibration curves struction of the calibration curves. Aliquots equivalent to 2.0–50.0  mg of NPZ, 10.0– 110.0  mg of PHN, 0.1–10.0  mg of NPZ impurity B and Results and discussion 2.0–50.0 mg of two PHN impurities (A & B) were trans- The importance of impurity detection and determination ferred from their corresponding solutions into five sets of evokes the requirement for developing simple, economi- 10-mL volumetric flasks. Volumes were then completed cal, rapid and accurate analytical techniques which can to the mark with methanol. 10.0  µL from each solution be utilized easily in quality control laboratories wherein was applied as a band with 3.0  mm length onto TLC cost and time are essential. Owing to simplicity, cost Kelani et al. BMC Chemistry (2022) 16:24 Page 4 of 11 effectiveness, time saving, no need for tedious sample NPZ impurity B, NPZ, PHN, PHN impurity A and PHN preparation and high sensitivity, TLC densitometry could impurity B (Fig.  2). Scanning profiles were obtained at be considered as one of the best options for that purpose 260.0 nm, and five calibration curves were then plotted. [2, 52, 53] Here, we present a novel TLC densitometric method for the simultaneous determination of NPZ, System suitability parameters PHN and three related official impurities (NPZ impu - To evaluate the performance of the proposed TLC rity B, PHN impurities; A & B) in their quinary mixture. method, system suitability parameters were calculated Moreover, EHS tool is applied for greenness evaluation manually [54]. The results of retardation, resolution, of this method in comparison to our previously reported capacity and tailing factors for the five components were HPLC one [19]. obtained in Table 2. Method validation Development and optimization of TLC densitometric Method’s validation was conducted in agreement to ICH method guidelines [50]. Various mobile phases were tried to get optimum separa- tion and resolution between the five cited components. Linearity and range Firstly, mixtures with different ratios of methanol and Polynomial relationships were established between the ethyl acetate have been tried, but the separation between integrated peak area and the corresponding concentra- the five cited components was not achieved. Thus, dif - −1 tion in the ranges of 2.0  − 50.0 µg band , 10.0–110.0 µg ferent solvents were added individually to the previous −1 −1 −1 band , 0.1  − 10.0 µg band and 2.0  − 50.0 µg band mixture for improving the separation between the stud- for NPZ, PHN, NPZ impurity B and the two PHN related ied components such as chloroform, acetone and ammo- impurities, respectively. nia. Table  1 summarizes the obtained resolution values during this optimization phase. It was noticed that addi- tion of ammonia to the conventional mixture enhanced Accuracy separation and resolution between the studied drugs. Accuracy was assessed by applying the previously men- Finally, a mixture of methanol–ethyl acetate–ammonia tioned procedures on pure samples with various concen- (2.0: 8.0: 1.0, by volume) was chosen for optimum suit- trations within the defined ranges. Satisfactory results ability parameters. Moreover, different wavelengths were regarding recovery % were computed in Table 3. tried for evaluating the densitometric measurement as 260.0  nm and 280.0  nm. 260.0  nm was the wavelength Precision of choice as it gave the highest sensitivity with minimum Repeatability Three separate concentrations of NPZ −1 noise for measuring the five cited components. Retarda - (15.0, 25.0, 40.0  µg band ), PHN (30.0, 50.0, 70.0  µg −1 −1 tion factor (R ) values were sequentially at 0.18 ± 0.02, band ), NPZ impurity B (3.0, 5.0, 8.0  µg b and ), PHN −1 0.35 ± 0.02, 0.49 ± 0.02, 0.63 ± 0.02 and 0.83 ± 0.02 for impurities; A & B (15.0, 25.0, 40.0  µg b and ) were ana- Table 1 The obtained resolution values during mobile phase optimization a a a a Experiment No. Mobile phase composition Rs1 Rs2 Rs3 Rs4 1 Methanol–ethyl acetate (3.5:6.5, v/v) 1.32 1.24 1.34 0.57 2 Methanol–ethyl acetate (3.0:7.0, v/v) 1.35 1.33 1.37 0.62 3 Methanol–ethyl acetate (2.5:7.5, v/v) 1.42 1.37 1.40 0.70 4 Methanol–ethyl acetate (2.0:8.0, v/v) 1.47 1.39 1.45 0.83 5 Methanol–ethyl acetate–chloroform (2.0:8.0:1.0, v/v/v) 1.49 1.51 1.13 0.75 6 Methanol–ethyl acetate–chloroform (2.0:8.0:0.8, v/v/v) 1.52 1.53 1.22 0.78 7 Methanol–ethyl acetate–chloroform (2.0:8.0:0.5, v/v/v) 1.53 1.54 1.27 0.89 8 Methanol–ethyl acetate–chloroform (2.0:8.0:0.2, v/v/v) 1.55 1.57 1.35 0.94 9 Methanol–ethyl acetate– acetone (2.0:8.0:1.0, v/v/v) 1.46 1.44 1.21 1.13 10 Methanol–ethyl acetate– acetone (2.0:8.0:0.8, v/v/v) 1.47 1.47 1.23 1.17 11 Methanol–ethyl acetate– acetone (2.0:8.0:0.5, v/v/v) 1.49 1.48 1.27 1.22 12 Methanol–ethyl acetate– acetone (2.0:8.0:0.2, v/v/v) 1.51 1.49 1.36 1.28 Rs1, Rs2, Rs3 and Rs4 are the obtained resolutions between NPZ impurity B & NPZ, NPZ & PHN, PHN & PHN impurity A and PHN impurity A & PHN impurity B, respectively Kelani  et al. BMC Chemistry (2022) 16:24 Page 5 of 11 −1 −1 −1 Fig. 2 TLC chromatogram of NPZ (30.0 µg band ), PHN (90.0 µg band ) and three of their official impurities as NPZ impurity B (10.0 µg band ), −1 −1 PHN impurity A (40.0 µg band ) and PHN impurity B (40.0 µg band ) using a mobile phase of methanol: ethyl acetate: ammonia (2.0: 8.0: 1.0, by volume) and detection at 260.0 nm Table 2 Parameters required for system suitability tests of TLC densitometric method Parameter NPZ impurity B NPZ PHN PHN impurity A PHN impurity B R 0.18 0.35 0.49 0.63 0.83 Resolution (Rs) NA 1.50 1.58 1.40 1.64 Tailing factor ( T ) 1.50 0.80 1.30 1.20 1.20 Retention factor (k’) 4.56 1.86 1.04 0.59 0.20 Selectivity factor (α) NA 2.45 1.79 1.76 2.95 Column efficiency (N) 262.44 196.00 635.04 425.11 737.86 Height equivalent to theoretical 0.034 0.046 0.014 0.021 0.012 plate (mm) Retention factor (k’) = (1 − R )/R f f Calculation of α = k’2/k’1 c 2 Column efficiency (N) = 16 (z/w) , where z is the migration length of the spot, w is the spot width Kelani et al. BMC Chemistry (2022) 16:24 Page 6 of 11 Table 3 Regression parameters for determination of the studied drugs by the proposed TLC densitometric method Parameter NPZ impurity B NPZ PHN PHN impurity A PHN impurity B −1 −1 −1 −1 −1 Range 0.1–10.0 µg band 2.0–50.0 µg band 10.0–110.0 µg band 0.2–50.0 µg band 0.2–50.0 µg band a a a a a SlopeNo. 1 = − 172.85No. 1 = − 13.91No. 1 = − 1.82No. 1 = − 9.78No. 1 = − 11.64 a a a a a No. 2 = 3480.76No. 2 = 1389.10No. 2 = 584.57No. 2 = 943.45No. 2 = 962.46 Intercept 1343.80 3305.64 4730.10 14,708.36 11,166.09 a a a a a SE of the slopeNo. 1 = 11.13No. 1 = 0.54No. 1 = 0.10No. 1 = 0.45No. 1 = 0.59 a a a a a No. 2 = 112.36No. 2 = 26.87No. 2 = 15.50No. 2 = 23.73No. 2 = 25.15 SE of the Intercept 182.40 249.72 492.06 232.59 202.55 Specificity (mean ± SD) 99.11 ± 1.382 100.72 ± 0.221 100.25 ± 1.054 99.10 ± 1.152 98.89 ± 1.963 Accuracy 99.74 101.15 100.42 99.97 100.99 Repeatability (RSD) 1.28 1.47 1.29 0.74 1.03 Intermediate precision (RSD) 1.77 0.55 1.84 0.96 1.79 Robustness 0.98 0.78 0.84 1.07 1.45 −1 LOD (µg band ) 0.01 0.60 2.38 0.05 0.06 −1 LOQ (µg band ) 0.03 1.82 7.21 0.15 0.18 Correlation coefficient (r) 0.999 0.999 0.999 0.999 0.999 a 2 −1 Slope 1 and 2 are the coefficients of a polynomial regression, A = ax + bx + c, where A is the integrated peak area, x is the concentration of the drug (μg band ), a and b are coefficients 1 and 2, respectively, and c is the intercept Average of determinations in seven laboratory-prepared mixtures −1 Fig. 3 TLC chromatogram of 100 µg band PHN after H O treatment; R ≈ 0.49 for PHN & ≈ 0.32 for its oxidative degradation 2 2 f Kelani  et al. BMC Chemistry (2022) 16:24 Page 7 of 11 Table 4 Determination of NPZ, PHN in their dosage form and application of standard addition technique using the proposed TLC method Naphcon-A eye drop % found Standard addition technique Mean ± SD Taken Added Recovery % −1 −1 NPZ 101.36 ± 1.51 10.0 µg band 5.0 µg band 101.30 −1 10.0 µg band 101.75 −1 20.0 µg band 99.99 Mean ± SD 101.01 ± 0.914 −1 −1 PHN 100.94 ± 1.73 20.0 µg band 10.0 µg band 100.95 −1 20.0 µg band 99.02 −1 40.0 µg band 100.84 Mean ± SD 100.27 ± 1.084 Average determinations of four eye drop dosage form solution lyzed intra-daily three times. Results were obtained pre- Table 5 Statistical comparison between the results obtained by liminary to RSD calculation, Table 3. the proposed method and the official BP method Parameter TLC Official BP Intermediate precision Inter-daily analysis was also con- method [5] ducted for the formerly selected concentrations. Results NPZ PHN NPZ PHN are represented in Table 3. Mean of recoveries 101.15 100.42 99.63 99.71 Robustness SD 1.095 1.712 0.977 1.153 It was evaluated by studying the effect of deliberately Variance 1.199 2.931 0.955 1.329 changing the mobile phase composition; methanol n 5 5 5 5 a a (2.0 ± 0.2  mL), ethyl acetate (8.0 ± 0.2  mL) and ammo- Student’s t-test 2.316 (2.306) 0.778 (2.306) NA NA a a nia (1.0 ± 0.1  mL). This study was conducted on three F-test 1.26 (6.39) 2.21 (6.39) NA NA independent concentrations of NPZ (5.0, 20.0, 40.0  µg These values represent the corresponding tabulated values of t and F at −1 −1 band ), PHN (15.0, 40.0, 70.0  µg b and ), NPZ impu- p = 0.05 −1 rity B (1.0, 5.0, 8.0  µg band ) and two PHN impurities −1 (1.0, 20.0, 40.0  µg b and each). Satisfactory RSDs were corresponding to this specified impurity. This outcome is obtained, Table 3. consistent with what previously reported [20]. PHN was stable towards all conditions except for oxidation where Specificity ≈ 30.0% was degraded upon H O treatment (Fig. 3). It was assessed by analysis of different laboratory pre - 2 2 pared mixtures containing various ratios of the five Analysis of pharmaceutical eye drops studied components. Table  3 shows good recovery per- The two active pharmaceutical ingredients (NPZ and centages and RSDs for analyzing those mixtures. Further- PHN) were simultaneously quantified in their combined more, forced degradation study was conducted whereas dosage form. Excipients did not have an impact on the the two drugs were subjected to different stress condi - obtained TLC chromatograms. In addition, method’s tions: (1) Acidic and alkaline hydrolysis via refluxing with validity was proved using standard addition technique, 1 M HCl and 1 M NaOH for 2 h, respectively, (2) Oxida- Table 4. tive degradation through treatment of each solution with 5% H O then keeping at room temperature for 24 h, (3) 2 2 Statistical analysis Photostability on powdered drugs placed in petri dishes, Statistical comparison between results of the suggested using 254 nm UV light for 10 h, and finally (4) Dry heat TLC method and that of official HPLC ones [5] were by putting each drug powder in 100 °C oven for 7 h. The performed. The calculated values of student’s t-test and degradation study was monitored by the proposed TLC F-test indicated that there is no significant difference method. NPZ was only liable to alkaline hydrolysis giv- observed between those methods, Table 5. ing its impurity B where its spot (R ≈ 0.35) disappeared accompanied by appearance of a new one (R ≈ 0.18) f Kelani et al. BMC Chemistry (2022) 16:24 Page 8 of 11 Table 6 EHS assessment of the solvents used in this work (ethyl acetate & methanol) as well as the reported one (acetonitrile) Selected substance Safety Health Environment Total Release Fire/Explosion Reaction/ Acute toxicity Irritation Chronic toxicity Persistency Air Hazard Water Hazard potential Decomposition Acetonitrile 0.61 1.00 0.60 0.51 0.63 0.43 0.34 0.43 0.00 4.55 Ethyl acetate 0.62 1.00 0.00 0.28 0.63 0.17 0.03 0.17 0.00 2.89 Methanol 0.65 1.00 0.00 0.27 0.11 0.32 0.00 0.32 0.00 2.66 Obtained by summation of nine main categories scores Kelani  et al. BMC Chemistry (2022) 16:24 Page 9 of 11 Table 7 Comparative overview on reported HPLC and proposed TLC methods Ref. No LOD Elution time EHS score F-test NPZ PHN NPZ PHN −1 −1 a a [19] 1.29 µg mL 3.10 µg mL ≈ 30 min 4.55 (acetonitrile) 3.90 (6.39) 2.22 (6.39) −1 −1 This work 0.60 µg band 2.38 µg band ≈ 6 min 2.89 (ethyl acetate) 2.66 (methanol) This value represents the corresponding tabulated value of F at p = 0.05 draft. MAH: Conceptualization, Methodology, Software, Formal analysis, Data Greenness evaluation and methods comparison curation, Visualization, Supervision, Project administration, Funding acquisi- In order to assess and compare this work with our pre- tion, Writing—review & editing. AMH; Methodology, Software, Validation, viously reported HPLC one [19], EHS tool was applied. Formal analysis, Investigation, Funding acquisition, Project administration, Writing—original draft, Writing—review & editing. MAT; Methodology, Soft- In this tool, nine categories representing safety, health ware, Validation, Formal analysis, Investigation, Funding acquisition, Project and environmental hazards are utilized for organic administration, Writing—original draft, Writing—review & editing. All authors solvents assessment whereas the lower the calcu- read and approved the final manuscript. lated score, the greener the solvent will be [51]. The Funding calculated scores for methanol, ethyl acetate (used in Open access funding provided by The Science, Technology & Innovation this work) and acetonitrile (used in reported HPLC) Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Not applicable. revealed the dominance of the proposed method over our previously reported one in terms of environmental Availability of data and materials sustainability, Table  6. Finally, a comparative overview All data generated or analysed during this study are included in this published article. on those two methods along with a statistical F-test for their variances are shown in Table 7. Declarations Conclusion Ethics approval and consent to participate A novel simple TLC densitometric method was estab- Not applicable. lished for the simultaneous detection and quantification Consent for publication of NPZ, PHN as well as three of their official impurities Not applicable. (NPZ impurity B, PHN impurities; A & B). The proposed Competing interests method was validated in agreement to ICH guidelines. The authors declare that they have no competing interests. NPZ and PHN were successfully determined in their combined eye drops. EHS tool was utilized for green- Author details Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr ness assessment of the organic solvents used in this work el Aini Street, Cairo 11562, Egypt. Analytical Chemistry Department, Faculty as well as the previously reported HPLC one. The pro - of Pharmacy, Modern University for Technology and Information, El-hadaba posed TLC densitometric method provides simplicity, El-Wosta, Mokatam, 5th district, Cairo, Egypt. Chemistry Department, Faculty of Pharmacy, October 6 University, 6 October City, Giza, Egypt. low cost, fast analysis and environmental sustainability compared to the reported one. In addition, the capacity Received: 6 February 2022 Accepted: 25 March 2022 of the method to detect low concentrations of NPZ and PHN official impurities highlights it as a promising one for impurity profiling of those drugs. References 1. Tantawy MA, Weshahy SA, Wadie M, Rezk MR. 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BMC Chemistry – Springer Journals

Published: Apr 4, 2022

Keywords: Naphazoline; Pheniramine; Impurities; TLC; EHS tool

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