key: cord-0838257-nndeblxs
authors: Ibrahim, Hany; Hamdy, Abdallah M; Merey, Hanan A; Saad, Ahmed S
title: Dual-mode gradient HPLC and TLC densitometry methods for the simultaneous determination of paracetamol and methionine in the presence of paracetamol impurities
date: 2021-02-09
journal: J AOAC Int
DOI: 10.1093/jaoacint/qsab021
sha: c87b57bc8f6dcc9ddc5fdc79323a83c057eeeb22
doc_id: 838257
cord_uid: nndeblxs

BACKGROUND: Paracetamol is one of the most widely analgesic and antipyretic drugs recently integrated into the supportive therapy of COVID-19. The pharmaceuticals containing methionine with paracetamol may contribute to avoid hepatotoxicity and eventual paracetamol overdose-dependent death. OBJECTIVE: The current work purposes to develop and validate two chromatographic methods for the simultaneous determination of methionine and paracetamol in presence of two paracetamol impurities (4-nitrophenol and 4-aminophenol). METHODS: Two chromatographic methods were established and validated according to the International Conference on Harmonization guidelines. The first one was a RP-HPLC/UV method based on applying a “dual-mode” gradient elution. The separation was realized via varying both the composition of the ternary mobile phase (acetonitrile–methanol–water) and its flow rate. This strategy enabled a relatively rapid analysis with a satisfactory resolution, although the investigated compounds exhibit a significant difference in lipophilicity. The second one relied on TLC- densitometry, where the optimum separation was realized using a quaternary mobile phase system composed of butanol–dioxane–toluene–methanol (8: 2.5: 3.5: 0.3, by volume). Both methods were monitored at 220 nm. RESULTS: The developed methods were proven to be robust, accurate, specific, and appropriate for the routine analysis of paracetamol in its pure form or in pharmaceutical formulations with methionine in quality control laboratories. CONCLUSIONS: The corresponding methods are suitable to determine methionine and paracetamol in the presence of paracetamol impurities. HIGHLIGHTS: The study achieves the analysis of methionine and paracetamol in the presence of paracetamol impurities via the application of HPLC and TLC- densitometry methods.

paracetamol (PC) is often the analgesic or antipyretic of choice, especially in the elderly and in patients in whom salicylates or other NSAIDs are contra-indicated (2). Such patients include asthmatics, those with a history of peptic ulcer, and children. Although paracetamol is a safe and well-tolerated drug at the recommended doses, it is the most commonly overdosed drug inducing life-threatening toxicity and death (3). PC has been implemented recently into the supportive therapy of COVID-19, but PC may induce hepatotoxicity at high doses (4), (5).

Methionine (MT) is considered an essential amino acid and implicated in glutathione biosynthesis. Though, in case of paracetamol overdoses, it serves as an antidote via increasing glutathione levels and hence preventing significant tissue damage (6) and may contribute to avoiding hepatotoxicity and eventual overdose-dependent death (7), (8). A combination of methionine with paracetamol reduces the acute toxicity of paracetamol by 50%. They are marketed in combination as tablets to treat fever, headache, and pain (9). 4-Aminophenol (AP) was considered as the chief co-existing impurity of paracetamol in pharmaceutical preparations resulting from either degradation or synthesis (10), (11). As AP is a pharmacologically active substance owning teratogenic and nephrotoxic effects, consequently its concentration should be strictly tracked (11). 4-Nitrophenol (NP) is the precursor of the AP and considered a potential paracetamol impurity (12).

Several methods have been reported for the assay of PC (13) (14) (15) (16) (17) (18) and a recent review mentioned the different analytical techniques implicated in PC analysis (19) . Few methods have been described the assay of MT in different matrices (20) (21) (22) and in pharmaceuticals (23) 4 and the combination of both MT and PC (24) , (25) . However, to the best of our knowledge, the literature survey did not reveal any method for the determination of PC and MT mix in the presence of PC impurities.

The current work intends to develop and validate robust, sensitive, and selective chromatographic methods for the simultaneous determination of MT and PC in the presence of paracetamol impurities in pharmaceuticals. A reversed-phase high-performance liquid chromatography coupled to UV-detection (RP-HPLC/UV) and thin-layer chromatography (TLC) -densitometry methods were developed and optimized for the rapid separation of the investigated compounds which displayed significant different lipophilic characters.

The obtained results were statistically analyzed and the methods were validated as per the International Conference on Harmonization (ICH) guidelines (26) .

Paracetamol and DL-methionine working standards were kindly provided by Hikma Pharmaceuticals Co, 6 th October, Giza, Egypt; their purities were reported to be 99.12 ± 1.37 and 99.65 ± 1.26%, respectively. Hepamol ® Tablets manufactured by Hikma Pharmaceuticals Co., labeled to contain 100 mg DL-methionine and 500 mg paracetamol per tablet, were purchased from the local market. AP (Acetaminophen RCK) and NP (Acetaminophen RCF) (27) were purchased from Sigma-Aldrich. Acetonitrile and methanol were HPLC-grade (Fisher-Scientific, UK). Butanol, dioxane and toluene were of analytical grade (Adwic Co, Egypt). Mobile phase system composed of water, methanol, and acetonitrile and the dual-mode gradient elution was performed via programming both mobile phase flow rate and solvents ratios. The UV-Vis detector was set at 220 nm. The column used in chromatographic separation was Zorbax ® SB C-8 column, 5µm, 150 mm× 4.6 mm (Agilent Technologies, USA). The column was equilibrated with the mobile phase for 30 min. prior to injection. 20 µL of each sample were injected into the analytical column. The separation was performed at room temperature under the specified chromatographic conditions as described in Table 1 . The mobile phase ran about 8 cm. The plates were developed by ascending chromatography to 8 cm from the spotting line, in a chromatographic chamber saturated previously with the mobile phase at room temperature for 60 min. The plates were kept at room temperature for 30 min till dryness then the bands were scanned using a UV lamp which set at 220 nm in the absorption mode at a scanning speed of 20 mm/s for densitometric determination.

The chromatographic methods were developed and validated regarding linearity, accuracy, precision, specificity, limit of detection, limit of quantitation, robustness, and system suitability test. for the HPLC method. The system suitability testing parameters were assessed regarding tailing factor, selectivity factor, resolution, and theoretical plate number.

Twenty tablets were weighted to determine the mean weight then they were finely ground. For both methods, the standard addition technique was done. The assessment was carried out on pharmaceutical instead of preparing placebo, thus, known accurate quantities of each standard were spiked into pharmaceutical formulation. The recovery assessment was carried out on three different concentration levels of addition for each standard.

The current study purposes to develop and validate two chromatographic methods for the simultaneous determination of MT and PC in the presence of PC impurities.

After evaluation of several solvent compositions, 10% aqueous methanol at which all standards exhibited good solubility under the investigated concentrations, was chosen for preparing the stock and working standard solutions. The investigated compounds exhibit a significant difference in lipophilicity (log P) and ranging from -1.85, 0.51, 0.47, and 1.93 for MT, PC, AP, and NP, respectively (28).

In the HPLC-UV method, a "dual-mode" gradient was employed to improve the resolution and shorten the time of analysis (1), (29) (30) (31) (32) (33) (34) . Changing both mobile phase composition and flow rate creates more chance to obtain optimum separation in the least possible time and provides more solutions for the separation of overlapped peaks. Furthermore, the ternary mobile phase systems permitted better separation compared to their binary counterparts (35) (36) (37) (38) (39) .

Trials for the HPLC method development were starting via using normal binary combinations of H 2 O/MeOH or H 2 O/ACN in different gradient programs but these trials suffered from either loss of resolution and/or long analysis time. The best chromatographic parameters were achieved via ternary combination of H 2 O/ACN/MeOH but still suffering a long analysis time. Though changing the flow rate via the dual-mode gradient offers a relatively short analysis time alongside no potential increase in back pressure was noticed (maximum back pressure reached was 350 bar). The flow rate was increased after emerging the third analyte, from 1.5 to 2.5 mL/min. This flow rate represents the best compromise between reducing analysis time and a tolerable increase in column backpressure. Also, optimum flow rate after 2.7 min was found to be 1.5 mL/min where the optimum resolution between PC and AP peaks was obtained.

The optimum separation was achieved via a ternary mobile phase constituted of methanolacetonitrile-water. The percentage of each component and the mobile phase flow rate were modified with time as described in Table 1 . Figure 1a and 1b show representative chromatograms realized with standards without and with the impurities, respectively.

In the TLC method, the optimum mobile phase composition was (butanol-dioxanetoluene-methanol; 8:2.5:3.5:0.3 by volume) revealing satisfactory chromatographic separation between PC and MT without and with PC impurities as presented in Figures 2a, 2b, 3 and 4. MT demonstrated a relatively high affinity towards the polar silica and showing a good separation from PC and its impurities.

Validation was conducted according to ICH guidelines (26) and the validation parameters were assessed for the two proposed methods as shown in Table 2 . For inter-day precision testing, RSD was found to be 0.09 for MT and 1.16 for PC in the TLC method and 1.37, 0.98 for MT and PC, respectively, in the HPLC method. These data suggest that both methods are precise. Table 2 .

(e) LOD and LOQ.-LOD and LOQ were calculated for each method as mentioned before and the results were represented in Table 2 . and detector wavelength (220 ± 4 nm) in the HPLC method. For the TLC method, a small variation in the percent of mobile phase components (butanol, 56 ± 1%), and time (60 ± 5 min) required for saturation with mobile phase were performed. The two methods were found robust as the investigated parameters did not reveal considerable differences in results regarding RSD, Table 3 .

System suitability parameters including tailing factor, selectivity factor, resolution of peaks, and the number of theoretical plates were computed for the HPLC method and successfully fulfilled FDA recommendations (40) as shown in Table 4 .

Both methods were employed for the determination of MT and PC in Hepamol ® tablet.

Standard addition technique was used to assess the validity of the developed methods to determine MT and PC selectively in presence of formulation excipients and additives, where satisfactory results were found, Table 5 .

One-way ANOVA Statistical comparison at 95% confidence interval (41) was performed on the recovery percent data acquired from the application of the two described methods on pharmaceuticals as shown in Table 6 . The comparison showed that there was no significant difference between results obtained from the developed methods and the manufacturer's 12 method. The proposed methods can be used accurately for the assessment of MT and PC in their binary mixes and in pharmaceutical preparations.

Two robust, precise and selective impurity indicating chromatographic methods were developed and validated for the determination of MT and PC in its pharmaceutical preparation without interference from PC impurities. The methods were validated as per the ICH guidelines. Results reveal that the dual-mode gradient in combination with a ternary mobile phase enabling good resolution of different lipophilic compounds within a relatively short analysis time. The TLC method displayed good chromatographic parameters and validated as per the ICH. However, the HPLC method was found to be advantageous concerning the analysis time than the TLC method. Both methods were found appropriate to be employed as impurity indicating methods for determination of paracetamol in pure form or in pharmaceutical dosage forms with methionine in quality control laboratories. a Accuracy (mean of percent recoveries ± SD) assessed using a minimum of nine determinations over a minimum of three concentration levels covering the specified range. b The intraday (n = 3), RSD on three different concentration levels within the specified range for each standard repeated three times within the same day.

c The interday (n = 3), RSD on three different concentration levels within the specified range for each standard repeated three times in three consecutive days.

d Specificity (mean of percent recoveries ± SD) of recovery percent data of the laboratory prepared mixes. Table 6 . One-way ANOVA statistical analysis within 95% confidence interval on recovery percent data obtained from the manufacturer's method and application of the two corresponding methods on pharmaceutical preparation.

One Way ANOVA 

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ICH, (2005) ICH, Topic Q2 (R1) Validation of Analytical Procedures : Text and Methodology, International Conference on Harmonization

(28) VCCLAB, Virtual Computational Chemistry Laboratory

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Center of Drug Evaluation and Research (FDA) reviewer guidance on Validation of Chromatographic Methods

The Science and Practice of Pharmacy

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