key: cord-0787171-3fo9rt6o authors: Marzouk, Hoda M.; Ibrahim, Engy A.; Hegazy, Maha A.; Saad, Samah S. title: Sustainable Liquid Chromatographic Determination and Purity Assessment of a Possible Add-on Triple-Action Over-the-counter Pharmaceutical Combination in COVID-19 date: 2022-03-19 journal: Microchem J DOI: 10.1016/j.microc.2022.107400 sha: f8322bf973ebb2fe230d34146e18b0940ff79aef doc_id: 787171 cord_uid: 3fo9rt6o Nowadays, all researchers are focused on combating the pandemic COVID-19. According to recent statistics, most patients are managed at home. An over-the-counter (OTC) triple action formula containing paracetamol (PAR), aspirin (ASP), and diphenhydramine (DIPH) is widely prescribed for pain, fever and as night-time sleep aid. For COVID-19 patients, this combination is now suggested as part of symptomatic therapy and prophylaxis. In this work, two simple liquid chromatographic approaches were designed for simultaneous determination of PAR, ASP, and DIPH in Excedrin® PM caplets, beside three specified official toxic impurities, namely, p-aminophenol, p-nitrophenol, and salicylic acid. The first method comprised high-performance thin-layer chromatographic separation coupled with densitometric quantification, on silica gel HPTLC 60 F(254) aluminium sheets as the stationary phase, ethyl acetate-methanol-aqueous ammonium hydroxide (10.0: 2.0: 0.1, by volume) as the developing system and scanning was performed at 210.0 nm. The second one is a high-performance liquid chromatography coupled with diode array detector. Successful separation of the six components was performed on XTerra C(18) column with isocratic elution of mobile phase 0.1% triethylamine acidified water: methanol (70:30, v/v) adjusted with o-phosphoric acid to pH 3.0 and methanol (90:10, v/v) with flow rate programming and detection at 210.0 nm. Validation of the proposed methods was performed according to ICH guidelines. Both methods were successfully used for quality control of the cited drugs in their marketed formulation. Moreover, the in-vitro release study was monitored using the proposed HPLC-DAD method. The greenness profile of the proposed methods was assessed and comparatively evaluated through various assessment tools, specifically; the analytical eco-scale system, national environmental method index (NEMI), green analytical procedure index (GAPI) and analytical greenness (AGREE) metric. Over-the-counter (OTC) is currently a strongly competitive market and a prominent area of focus [1] . Oral pharmaceutical combinations of analgesics and antihistaminics are widely used as night-time headache relief and sleep aid, they are among the most important sectors of OTC medications. Paracetamol (PAR) is an antipyretic and analgesic agent [2] , that can be used in palliative care for COVID-19 symptoms control according to WHO guidance [3] . Among PAR pharmacopeial related substances are p-nitrophenol (PNP) and p-aminophenol (PAP), namely, impurity F and K, respectively [2] . PAP is PAR main hydrolytic degradation product [4, 5] , in addition, it exhibits teratogenic [6] and nephrotoxic effects [7] . Similarly, PNP exhibits genotoxicity and carcinogenicity [8] . Aspirin (ASP) is a non-selective cyclo-oxygenase inhibitor with antipyretic, analgesic, and anti-inflammatory actions [2] . Because of its antiplatelet aggregation and lung damage prevention, as well as its viral replication suppression impact, ASP is also used as a prophylactic therapy in COVID-19 patients [9] . The main degradation product and pharmacopeial impurity C of ASP is salicylic acid (SAL) [2] . SAL is reported to cause toxic symptoms as gastric membrane, esophagus damage and ototoxicity [10] . Diphenhydramine (DIPH), a first-generation antihistamine [2] , is recently reported to have direct antiviral action against SARS-CoV-2 [11] . Figure 1 displays the chemical structures of the cited drugs and these related substances. Co-formulated PAR, ASP and DIPH is an effective and highly tolerated analgesic therapy that is used to treat headache, insomnia, and pain. The significance of the cited drugs arises from the recent researches on the prospects of employing them as an add-on remedy for COVID-19 patients, specifically home-treated and mild cases [12, 13] . The literature survey revealed the lack of a reported analytical method for the concurrent determination and purity testing of the three components in their newly introduced pharmaceutical formulation. Yet, all of the published work were focused on the determination of PAR and ASP combination along with other compounds using HPLC [14] [15] [16] [17] [18] , UV-Spectrophotometry [19] [20] [21] , and voltammetry [22, 23] . Similarly, PAR and DIPH combination was analyzed by different spectrophotometric [24] [25] [26] , HPLC [26, 27] , and capillary gas chromatographic [28] methods. Chromatography is currently considered as a gold standard technique in pharmaceutical industry, owing to its outstanding advantages such as low operation cost, availability, good sensitivity, small sample size requirements and diversity of applications [29] [30] [31] [32] [33] . Recently, analysts aimed to apply a competent analytical methodology with improved eco-friendliness and sustainability to preserve our environment from chemical threats [31, 34, 35] . Accordingly, the aim of the current work is planned to introduce simple, selective, cost-effective, and ecofriendly HPTLC and HPLC-DAD methods for simultaneous analysis and purity testing of the cited drugs in their pure forms and pharmaceutical formulation. Ecological impact and analytical performance of the proposed methods are compared to bring a greener analytical approach to a close. In addition, the dissolution profiles of the cited active pharmaceutical ingredients (API) from the marketed product were monitored using the proposed HPLC-DAD method. Chromatographic separation was carried out using silica gel 60 F 254 HPTLC plates (20 × 10 cm) (Merck, Darmstadt, Germany) as the stationary phase. Sample application is carried out using Camag Linomat-5 autosampler by means of Camag micro-syringe (Camag, Muttenz, Switzerland). Scanning and densitometric measurements were performed by a Camag TLC scanner (model number 3S/N 1302319), operated with winCATS ® software. The adjusted scan mode was reflectance-absorbance with slit of 3.0 × 0.45 mm dimension and scanning speed of 20 mm/s. Densitograms with recorded peak areas were obtained. The separation module was Waters Alliance 2695 LC equipped with a pump integrated with a mixing system, vacuum degasser, Waters photodiode array detector 996 (PDA), and an autosampler (Milford, United States). The system was controlled with Empower2 chromatography software for data processing and manipulation. The utilized column was XTerra C 18 (100 × 4.6 mm, 5µm) manufactured by Waters TM Corporation (Milford, United States). A Jenway pH apparatus (model 3510, UK) was used to adjust the solvent's pH. The dissolution profiling was performed using VanKel VK 7000 (USA) apparatus fitted with standard USP type-II paddle and six vessels. Pure PAR was provided by El Nasr Pharmaceutical Co., Cairo, Egypt, while ASP was supplied from Al-Gomhoria Chem. Co. (Cairo, Egypt). Purities were verified using their respective official methods and found to be 99.80% ± 0.50 for PAR [2] and 100.30% ±1.87 for ASP [36] . Pure DIPH as hydrochloride salt was obtained from Wanbury Ltd. Co. (India). Its purity was evaluated and found to be 100.54% ± 0.83, using its BP method [2] . PNP, PAP and SAL were provided by Sigma-Aldrich (Darmstadt, Germany) with stated purity of ≥ 99%. Excedrin ® PM Headache caplets (B.N. 46172679), a product by GlaxoSmithKline, USA, were claimed to comprise PAR 250 mg, ASP 250 mg, and DIPH 38 mg as citrate salt per caplet. HPLC-grade methanol was obtained from Sigma-Aldrich (Darmstadt, Germany). Also, analytical-grade solvents and reagents were used including o-phosphoric acid (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt), triethylamine, ethyl acetate (S.D. Fine Chemicals Ltd., India), and aqueous ammonium hydroxide solution (25%) (Pioneer Chemical Co., Giza, Egypt). De-ionized distilled water was provided by Otsuka Pharmaceutical Co. (Cairo, Egypt). Stock standard solutions (1.0 mg/mL) of the studied drugs were prepared, separately, in methanol. Further, PAP, PNP, and SAL working standard solutions (100.0 μg/mL) were prepared by appropriate dilutions of their corresponding stock standard solutions. The prepared solutions were kept in the refrigerator and protected from light. Chromatographic separation was carried out on 20 × 10 cm Silica gel 60 F 254 HPTLC plates as stationary phase with a developing mixture system composed of ethyl acetate-methanolaqueous ammonium hydroxide solution (10.0: 2.0: 0.1, by volume). Samples were applied in triplicates, using a Camag autosampler, as bands of 6 mm width, and 10 mm apart from the sides and bottom border of the plates. The development was performed in a pre-saturated Camag binary glass chamber for 45 min at room temperature. Ascending development, over 8 cm of the plates, was then allowed. The developed plates were removed, air dried and scanned at 210.0 nm using the previously specified instrumental conditions. The XTerra C 18 (100 mm × 4.6 mm, 5 µm) column was used for isocratic separation of studied analytes with a mixture of 0.1% triethylamine acidified water: methanol (70:30, v/v) adjusted with o-phosphoric acid to pH 3.0 and methanol (90:10, v/v) as mobile phase at room temperature. The flow rate was adjusted to 1.0 mL/min, and starting from 5.5 min was fixed at 1.5 mL/min. The mobile phase components were filtered using 0.45 µm Millipore membrane filter, degassed, and sonicated for 15 min in an ultrasonic bath prior to use. The samples were filtered and applied with 50 µL injection volume, in triplicates, into the LC system using an autosampler. The separation was successfully achieved within 12 min and detection was performed at 210.0 nm. Accurately measured volumes in the range of 1.00-15.0, 1.00-15.0, 0.20-9.00, and 0.10-3.00 µg/band of PAR, ASP, DIPH and studied impurities from their respective stock and working standard solutions were applied, separately, in triplicates as bands using the HPTLC system with the previously described chromatographic conditions. The scanning profiles were obtained, and the calibration plots were constructed relating the average peak area to the corresponding concentration of each drug. Finally, the polynomial regression equations were computed. Different volumes of each analyte were accurately transferred from their respective stock and working standard solutions to 10-mL volumetric flasks, to get solutions in concentration range of 1.00-14.0 μg/mL for PAP, 5.00-160.0 μg/mL for PAR, 2.00-160.0 μg/mL for ASP, 1.00-13.0 μg/mL for PNP, 1.00-14.0 μg/mL for SAL, and 2.00-55.0 μg/mL for DIPH. Volumes were brought to the mark with the mobile phase. Samples were prepared in mixtures and injected into the HPLC-DAD system, in triplicates, using the previously specified chromatographic conditions. The calibration curves were built by plotting the average peak areas against the respective drug concentrations. Ten Excedrin ® PM caplets, each contain labelled amounts of 250 mg PAR, 250 mg ASP, and 38 mg DIPH as citrate salt, were individually weighed, powdered and thoroughly mixed in a hand mortar. Then, accurately weighted quantity, equal to one caplet, was subsequently transferred to a 100-mL volumetric flask, by 50-mL methanol, and left in the sonicator for 45 min. The volume was completed with methanol then filtered. The prepared solution was further diluted with methanol (for HPTLC) and mobile phase (for HPLC), to achieve the appropriate concentrations within PAR, ASP and DIPH linearity ranges, and the procedures were then carried out as described under each method. The found concentrations and the %recoveries of studied drugs were computed from the respective regression equations. Definite portions of PAR, ASP and DIPH pure standards were added to an accurately weighted powdered caplets. Following that, the same extraction and dilution were carried out to perform the standard addition technique. The aforementioned HPLC-DAD procedure was applied for monitoring the dissolution behaviour of Excedrin ® PM caplets in water as per USP requirements [36] . Dissolution testing was performed by placing one caplet in a USP-dissolution apparatus II vessel, containing 900 mL water as a dissolution medium, equilibrated at 37°C ± 0.5 • C and operated at 100 rpm. Five mL samples were withdrawn at pre-determined time intervals of 10, 20, 25, 30, 45, and 60 min, and the volume was replenished with an equal volume of fresh medium. The collected samples were then filtered through a 0.22 µm syringe filter and analysed. The experiment was carried out in triplicate. The % dissolution was calculated, by means of the corresponding regression equation for each drug in the studied medium, and the in-vitro release profiles of PAR, ASP and DIPH were then plotted. For regular drug analysis, chromatography is a well-established and gold standard technique in quality control laboratories all over the world. Resolution and quantification of drugs in presence of pharmacopeial impurities with extremely similar structures and thus predicted to have comparable physical and chemical characteristics is a key challenge in the development and validation of the analytical procedures. Moreover, designing analytical processes in such a manner that results in a considerable reduction or elimination of the use and/or generation of harmful substances is a current drive [31, 37] . So, this work aims at developing and validating first sustainable, selective, and robust analytical methods for simultaneous estimation and purity assessment of PAR, ASP, and DIPH in their new OTC pharmaceutical combination. The suggested HPLC-DAD method was exploited for monitoring the dissolution profiles of the cited drugs in the combined formulation as well. Finally, the suggested approaches' ecological and health implications were assessed using several green evaluation indicators. HPTLC-densitometric procedure was developed and optimized taking in account the use of green solvents and excluding toxic and hazardous ones (i.e., acetonitrile, toluene, and chloroform). Trials started using various eco-friendly developing systems such as ethyl acetateethanol, ethyl acetate-butanol and ethyl acetate-methanol in varying ratios (4:6, 5:5, 6:4, 7:3, 8:2, and 9:1 v/v), respectively. However, the results were not acceptable due to observed tailing and incomplete separation of the studied compounds. Then, glacial acetic acid, aqueous ammonium hydroxide, and triethylamine were tried as pH modifiers to the developing mixture of ethyl acetate-methanol. DIPH was retained at the baseline in acidic pH. On the other hand, improved separation was attained upon using alkaline pH in the developing system. Aqueous ammonium hydroxide was preferred over triethylamine owing to its less environmental hazardous in addition to better bands symmetry. Different volumes of aqueous ammonium hydroxide solution were tried, and the optimal ratio of ammonia was found to be (0.1, by volume) regarding both resolution and selectivity. Increasing the aqueous ammonium hydroxide ratio has led to increased retention of ASP at the baseline. Fig. 2a . For evaluating the chromatographic system's suitability, different parameters were calculated for the six components [38] . All of the calculated criteria were within the acceptable ranges, Table 1 . The developed approach was designed with the chief goal of developing a robust, simple, green and efficient method for quantitative analysis of PAR, ASP and DIPH, simultaneously, along with three of their official impurities with optimum resolution in a reasonable analysis time. Method development was conducted using Waters XTerra C 18 (100 × 4.6 mm, 5 µm) column. Several organic solvents were tried namely, acetonitrile, methanol and ethanol, and acidified water with o-phosphoric acid as the aqueous phase with varying ratios, pH, and flow rates. For the sake of method greenness, acetonitrile was excluded as an organic modifier. Whereas, methanol was selected, as it gives promising separation with stable baseline. In addition, triethylamine was examined, as a mobile phase additive, for peak enhancement. Fig. 2b . In order to guarantee chromatographic system's performance, System suitability parameters were computed. Results were in good agreement to the acceptable limits [39] . In chemical laboratories, green analytical chemistry principles are well-known. To properly evaluate the environmental influence of chemical processes, specialized assessment tools are needed [40] . The desire to replace conventional pharmaceutical analytical methods, which depend on the usage of hazardous chemicals, with more eco-friendly green ones without affecting performance features has grown in light of the importance of environmental protection. [41] . Four tools were presented to verify and appraise the greenness of the proposed methods. We also provided a comprehensive benchmark to compare their environmental impacts. These tools are the analytical eco-scale, national environmental methods index (NEMI), green analytical procedure index (GAPI), and analytical greenness (AGREE) metric. A semi-quantitative evaluation was achieved upon using analytical eco-scale by assigning penalty points based on the impact of different method parameters, including the utilized chemicals, instrumental energy consumption, waste generation, and occupational risk [42] . The analytical eco-score is computed by subtracting the total parameters penalty points from 100 as the perfect green method base value. Table 2 displays the analytical eco-scores greater than 75, emphasizing the proposed methods as excellent ones. The NEMI is a qualitative approach used to evaluate the total environmental safety of the analytical methodology [43] . The NEMI pictogram is represented by a simple circle divided into four quadrants: the first comprises persistent, bio-accumulative, and toxic chemicals, the second represents the hazardousness of chemicals, while the third and fourth embraces the corrosiveness of the mobile phase, and the amounts of waste generated, respectively [44] . Concerning the proposed HPTLC-densitometry, and HPLC-DAD methods, the US EPA Toxic Release Inventory (TRI) chemical list did not list ethyl acetate as PBT nor hazardous [45] . Conversely, methanol is present in the TRI list. The pH of the mobile phase used was ~9 and 3 for each method, respectively, so the pH is not corrosive (pH not <2 or >12). The generated waste is less than 50 grams per sample. All quarters of the proposed method pictogram are colored green except the hazardous quarter due to methanol, as illustrated in Table 2 . The GAPI tool has been newly introduced as an indicator for assessing the green characteristics of the entire analytical measures beginning from sample preparation and collection to final determination step [46] . A visual pictogram comprised of five pentagrams, with a color code ranging from green to yellow to red, assigned to low, medium, and high ecological impact for each step, is used. The proposed methods showed green covered sections with fewer red shaded ones, as presented in Table 2 . Finally, the recently dedicated AGREE tool is used for the comprehensive evaluation of the analytical methodology greenness. It is a free downloadable software that creates a colored pictogram, covering the GAC's twelve fundamentals that are transformed to a final zero-one score [47] . A characteristic circular pictogram appears in the final assessment report, automatically and is divided into 12 sections with the ability to adjust the width of each section based on its significance. The section color ranged from deep red (=0) to deep green (=1) [47] . Table 2 shows the obtained AGREE pictograms with an overall score of 0.69 and 0.65 for the developed HPTLC-densitometry and HPLC-DAD methods, in order. Method validation parameters were assessed according to the ICH guidelines to assure the validity of the proposed analytical methods [48] . Working calibration range was established considering the correlation between the mean peak area and the corresponding concentration of each drug. Table 3 displays the linearity ranges of the proposed methods and their computed regression parameters. Accuracy of the developed methods was assured by the acceptable mean %recoveries obtained upon analyzing each drug at five different concentration levels covering the linearity ranges, Table 3 . Three different chosen concentrations of each drug were analyzed in triplicates, on the same day and on three consecutive days, to assess the method's intra-day and inter-day precision, respectively. The attained percentage relative standard deviation (RSD%) were less than 2, Table 3 . Good separation of the studied drugs along with cited impurities demonstrates the selectivity of the proposed methods, Fig. 2 . In addition, the good % recoveries of PAR, ASP and DIPH in their co-formulation ensures the absence of chromatographic interference from common caplets excipients, Table 4 . Specificity was further assured via winCATS spectral correlation tool (for HPTLC) or online by DAD (for HPLC) by monitoring the purity for each eluting drug peak. LOD and LOQ were calculated for the official impurities using the slope of standard calibration curve and standard deviation of residuals. The achieved LOD and LOQ values reflect the proposed methods' good sensitivity, Table 3 . Robustness of the developed methods was ascertained by calculating RSD% upon performing minor changes in the optimized experimental conditions. System suitability parameters were not significantly influenced, and the % RSD values were within the accepted range, less than 2%, Table 3 . PAR, ASP, and DIPH were efficiently assayed in their co-formulation, Excedrin ® PM caplets by the validated methods, keeping in consideration the wide disparities in their concentrations. Sample preparation was performed employing single extraction step with methanol, showing no interference from caplets excipients. Furthermore, the standard addition technique was performed, and the proposed methods validity was successfully implemented, Table 4 . The good results and minimal sample manipulation steps as well turn the attention on the worth applicability of the proposed methods as routine and ecofriendly quality control protocols of the three drugs. Pharmaceutical formulation was assayed by the developed methods near the expiry date. The samples showed traces of SAL (ASP synthetic precursor and main degradation product), Fig. S1 . Accordingly, the suggested methodologies were found to be both selective and sensitive for detecting the likely found impurities and/or degradation product(s) in the pharmaceutical formulation. The development of drug formulations should include the establishment of in-vitro dissolution profile to ensure batch-to-batch consistency and to correlate the in-vitro/in-vivo pattern. In-vitro release of the cited drugs was monitored in water as the dissolution medium [36] . Drug release profiles were constructed by plotting the percentage of drug dissolved versus time, Fig 3. In the current study, the percentage of studied drugs' release from Excedrin ® PM caplets in water were greater than 75% after 45 min for PAR, ASP, and DIPH. Accordingly, the specified acceptance criteria expressed as quantity (Q) of active substance dissolved in definite time is fulfilled [36] . To ensure the results of the proposed methods, statistical comparison was carried out, after computing the t and F-values, between the obtained results and those resulted from applying the official methods [2, 36] . The calculated t and F-values were found to be less than the respective theoretical ones, presenting no remarkable difference between the proposed and the applied official methods, Table S1. Paracetamol, aspirin, and diphenhydramine are newly co-formulated in a novel triple action formula recently used as COVID-19 OTC remedy. Novel, sustainable, reliable, and costeffective chromatographic methods were developed and validated for separation and estimation of the three cited drugs together with three of their potential official impurities. The use of safe and less hazardous solvents was greatly taken in consideration. The greenness profile was evaluated via four common assessment metrics; analytical eco-scale system, NEMI, GAPI and AGREE tools. Additionally, in-vitro dissolution profiles were successfully monitored for the cited drugs from caplet dosage form using the proposed HPLC method. The proposed methods' capacity to detect trace quantities of potential impurities also makes it a good choice for impurity profiling of the cited pharmaceutical formulation. Table 1 . System suitability parameters of the proposed HPTLC-densitometric and HPLC-DAD methods. a Retardation factor (R f ) = distance travelled by the analyte/distance travelled by the solvent front. b Capacity factor (k') = (1 -R f ) / R f . c Selectivity (α) = k' 2 /k' 1 calculated for each of two successive peaks. d Resolution (R s ) = Rf 2 -Rf 1 / 0.5 (w 1 + w 2 ), where R f is the retardation factor, and w is the peak width calculated for each of two successive peaks for HPTLC and = 2(t RB -t RA) / ( W B+ W A ) for HPLC. a Regression equation for HPLC: A = a + bc, where 'A' is the average peak area and 'c' is the concentration (μg/mL). b Coefficient 1 and 2 are the coefficients of X 2 and X, respectively. Following a polynomial regression: A = b1x 2 + b2x + a, where 'A' is the average peak area, 'c' is the concentration (μg/band), 'b1' and 'b2' are coefficients 1 and 2, respectively and 'a' is the intercept. 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Credit Author statement Conceptualization, Methodology, Writing-review and editing, Supervision. Engy A. Ibrahim: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Writing-original