Home Simultaneous determination of lesinurad and febuxostat in commercial fixed-dose combinations using a greener normal-phase HPTLC method
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Simultaneous determination of lesinurad and febuxostat in commercial fixed-dose combinations using a greener normal-phase HPTLC method

  • Prawez Alam EMAIL logo , Faiyaz Shakeel , Sultan Alshehri , Muzaffar Iqbal , Ahmed I. Foudah , Tariq M. Aljarba , Fatma M. Abdel Bar and Mohammed H. Alqarni
Published/Copyright: April 23, 2024
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Green Processing and Synthesis
From the journal Green Processing and Synthesis Volume 13 Issue 1

Abstract

So far, no documented method for simultaneously analyzing lesinurad (LND) and febuxostat (FBX) has been reported for either traditional high-performance thin-layer chromatography (HPTLC) or a green HPTLC technique. In order to determine LND and FBX simultaneously in commercially available fixed-dose combo tablets, this study devised a normal-phase HPTLC method that is fast, sensitive, and green. The green eluents for the simultaneous analysis of LND and FBX were a mixture of ethyl acetate:ethanol:water at 70:20:10 (v/v/v) ratio. The new approach’s greenness was predicted utilizing four distinct greenness tools: the National Environmental Method Index, Analytical Eco-Scale, ChlorTox, and Analytical GREENness approaches, and the results revealed a significantly greener profile. The current method operated on a linear scale between 30 and 1,000 ng·band−1. It was confirmed that the current approach is sensitive, accurate, precise, robust, and green. The LND and FBX contents of commercially available tablet products A and B were found to be within the range of 100 ± 2%, indicating that the existing methodology for simultaneously determining LND and FBX in pharmaceutical combination products is applicable. The results of the current methodology indicated that LND and FBX could be consistently measured in pharmaceutical combination products simultaneously using the current approach.

Keywords: febuxostat; green HPTLC; greenness evaluation; lesinurad; validation

1 Introduction

The development of monosodium urate crystals in the joints and alterations in purine metabolism are the two main causes of the inflammatory form of arthritis known as gout [1,2]. The accumulation of these crystals causes hyperuricemia or elevated levels of uric acid (UA) in the blood [1,3]. The prevalence of gout and hyperuricemia are most common in elderly patients, especially postmenopausal women [4]. Febuxostat (FBX), a selective non-purine xanthine oxidoreductase (XO) inhibitor, is used in the treatment of chronic gout [5,6,7]. FBX has been reported as more tolerable and efficacious than allopurinol and preferred in the case of allopurinol-intolerant patients [8]. Lesinurad (LND), a new and specific UA transporter-1 (URAT1) inhibitor, reduces serum UA levels by enhancing its renal excretion [9]. The chemical structures of FBX and LND are shown in Figure S1. Gout patients benefit greatly from the fixed-dose combination of LND and XO inhibitors, such as FBX and allopurinol. For patients with gout-associated hyperuricemia whose serum UA levels did not reach the target level while using FBX or allopurinol alone, the USFDA approved combined dosage forms of LND and FBX or LND and allopurinol at fixed doses [9,10,11]. By increasing UA excretion and lowering urate synthesis, the combination of LND and FBX lowers serum UA in two ways. URAT1, a UA transporter responsible for reabsorbing UA from the renal tubular lumen, is inhibited by LND [12,13,14]. In individuals with gout-associated hyperuricemia, the combination of LND and FBX is preferable to FBX alone, which did not result in any clinically meaningful pharmacokinetic effects [15]. Because the FDA only permits the use of LND in combination with XO inhibitors like FBX and does not allow LND alone, the simultaneous measurement of FBX and LND is necessary in combined dosage forms.

Literature survey revealed very limited analytical procedures for determining LND and FBX simultaneously in combined dosage forms and biological samples. However, for determining LND and allopurinol concurrently in combined dosage forms and biological samples, various analytical methods, such as regular spectrometry [16], greener spectrometry [17], spectrofluorimetry approaches [18], chemometry approach [19], high-performance liquid chromatographic (HPLC) approaches [20,21,22,23], greener capillary electrophoresis approach [24], and ultra-performance hydrophilic interaction liquid chromatography and tandem mass spectrometry technique [25], have been reported. Recently, a greener high-performance thin-layer chromatography (HPTLC) method has also been reported for the simultaneous analysis of LND and allopurinol in fixed-dose combination products [26]. For measuring LND and FBX simultaneously in combined dosage forms and plasma samples of humans, synchronous and conventional micelle-enhanced spectrofluorimetry approaches were utilized [27]. For measuring LND, FBX, and diflunisal simultaneously in formulations and plasma samples of humans, a green HPLC method was also utilized [28].

No single HPTLC method – conventional or greener – has been reported for the simultaneous assessment of LND and FBX in combined dosage forms, as far as we are aware. One of the 12 principles of green analytical chemistry (GAC) is the usage of substitute ecologically acceptable solvents to lower the harmful effects of toxic/hazardous solvents on the environment [29]. A literature search revealed that the usage of greener solvents has grown dramatically during the last few decades [30,31,32,33,34]. Green analysis employing HPTLC techniques is currently applied to pharmaceutical products [35,36,37,38]. The literature describes a number of greenness methods for evaluating the greenness profiles of analytical procedures. These include the National Environmental Method Index (NEMI) [39], the Green Analytical Procedure Index (GAPI) [40], the Analytical Eco-Scale (AES) [41], Red, Green, and Blue (RGB) [42], the Environmental Assessment Tool (EAT) [43], the Analytical Method Volume Intensity (AMVI) [44], the Analytical Method GREENness Score (AMGS) [45], ChlorTox [46], and the Analytical GREENness (AGREE) [47]. To assess the greener profile of the present technique, we used four different tools: NEMI [39], AES [41], ChlorTox [46], and AGREE [47]. The present strategy aims to develop and validate a normal-phase HPTLC technology that is fast, sensitive, and green for measuring LND and FBX in pharmaceutical combined dosage forms. It is predicated on the data and observations that were previously mentioned. By using the criteria outlined in The International Council for Harmonization (ICH)-Q2-R2, the suggested approach for determining LND and FBX simultaneously was verified [48].

2 Materials and methods

2.1 Materials

Standard LND was provided by the Toronto Research Chemicals (North York, Ontario, Canada). Standard FBX and HPLC-grade ethyl acetate (EA) and ethyl alcohol (EtOH) were procured from E-Merck (Darmstadt, Germany). The purified/deionized water (H2O) was procured using Milli-Q (Lyon, France) equipment. Each brand of commercial combo tablet, A and B, had 200 mg of LND and 80 mg of FBX, which were purchased from New Delhi, India. All other used solvents and reagents were of AR grade.

2.2 Instrumentation and analytical procedures

The HPTLC system (CAMAG, Muttenz, Switzerland) was used to concurrently measure LND and FBX in pure forms and commercial tablets. The processed solutions were spotted in 6 mm bands using an Automatic TLC Sampler 4 (ATS4) Sample Applicator (CAMAG, Geneva, Switzerland). The microliter Syringe (Hamilton, Bonaduz, Switzerland) was loaded into the sample applicator. The stationary phase used in the TLC plates was glass plates (plate size: 10 × 20 cm2) pre-coated with normal-phase silica gel (particle size: 5 µm) 60F254S plates (E-Merck, Darmstadt, Germany). The glass plates resist the pressure during the HPTLC run. However, aluminum plates are sometimes tilted during the HPTLC run. Therefore, glass plates were used over aluminum plates in this work. The ternary combination of EA–EtOH–H2O (70:20:10 v/v/v) was used as the green mobile phase. In order to ascertain LND and FBX simultaneously, 150 nL·s−1 application rate was established. In an automated developing chamber 2 (ADC2) (CAMAG, Muttenz, Switzerland), the plates were developed in a linear ascending mode at an 8 cm spacing. For 30 min at 22°C, vapors from a greener mobile phase filled the development chamber. LND and FBX were simultaneously detected at a wavelength of 275 nm. The slit dimension and scanner speed were set at 4 × 0.45 mm2 and 20 mm·s−1, respectively. Three or six replications were employed for every measurement. WinCAT (version 1.4.3.6336, CAMAG, Muttenz, Switzerland) was the software used.

2.3 LND and FBX calibration curves and quality control (QC) samples

By dissolving the required amounts of each medication in the appropriate amount of the green mobile phase, separate stock solutions of LND and FBX were produced. Each drug’s final stock solution contained 100 µg·mL−1 of the substance. The green mobile phase was used to dilute different amounts of the stock solutions, resulting in the 30–1,000 ng·band−1 levels of both medications. In order to maintain uniformity in the sample preparation and HPTLC analysis, the mobile phase was used as a diluent in this study. After applying 20 µL of each concentration to TLC plates using the recommended procedure, the peak area of each concentration of LND and FBX was recorded. LND and FBX calibration plots were made by plotting the concentrations of the two compounds against the observed peak area in six replications (n = 6). To evaluate several validation parameters, three different QC samples were created.

2.4 Sample processing for determining LND and FBX simultaneously in commercially available tablets

In order to simultaneously determine LND and FBX in combination with products A and B, 20 tablets from commercially available products were taken. There were 200 mg of LND and 80 mg of FBX in each tablet’s brand. Each brand of tablet was crushed and then powdered. A quantity of fine powder was mixed with 50 mL of the green mobile phase, which was equivalent to the average mass of each product. About 50 mL of the green mobile phase was used to dilute 1 mL of each product again for the current procedure. The solutions made for each brand of tablet were sonicated for around 10 min and filtered to remove any insoluble excipients. Using current methodology, the collected solutions were subjected to evaluate LND and FBX in both products concurrently.

2.5 Validation assessment

The proposed methodology for determining LND and FBX simultaneously was verified for many parameters using the ICH-Q2-R2 standards [48]. The linear ranges of LND and FBX were established through the plotting of concentrations against the observed peak area. The LND and FBX linearity of the present method was assessed for a range of 30–1,000 ng·band−1 (n = 6).

The system suitability parameters of the proposed methodology for the simultaneous determination of LND and FBX were obtained by the calculation of retardation factor (R f), tailing factor (As), and theoretical plates/meter (N/m). The R f, As, and N/m for the proposed methodology were obtained using their published equations [38].

As percentage recoveries, the accuracy of the proposed methodology for simultaneously measuring LND and FBX was evaluated using the spiking/standard addition methodology [48]. To create low-QC (LQC) solutions of LND and FBX of 150 ng·band−1, middle-QC (MQC) solutions of 200 ng·band−1, and high-QC (HQC) solutions of 250 ng·band−1, additional 50%, 100%, and 150% LND and FBX solutions were spiked with the previously measured LND and FBX solution (100 ng·band−1). The previously mentioned LND and FBX QC levels were reassessed in order to assess the accuracy. The percentage recovery was calculated at each concentration of FBX and LND. To measure the accuracy, six replicates (n = 6) were used.

An assessment was conducted on the intra- and inter-assay precision of the current protocol for the simultaneous measurement of LND and FBX. Quantifying newly prepared LND and FBX solutions at the aforementioned QC levels on the same day (n = 6) allowed for the evaluation of intra-assay variance for LND and FBX. During the course of 3 days (n = 6), evaluation of newly prepared solutions was conducted at the aforementioned QC levels as part of the evaluation of inter-assay variation for LND and FBX for the proposed approach.

By intentionally changing the composition of the green mobile phase, the robustness for LND and FBX was derived for the current technique. Peak area and R f data changes were noted (n = 6) following the transition between EA–EtOH–H2O (72:18:10 v/v/v) and EA–EtOH–H2O (68:22:10 v/v/v) as the green mobile phase for LND and FBX.

The proposed method’s sensitivity for the simultaneous assessment of FBX and LND was measured as limit of detection (LOD) and limit of quantification (LOQ) by utilizing a standard deviation methodology. Eqs. 1 and 2 were used to get LND and FBX LOD and LOQ (n = 6) [48]:

(1) LOD = 3.3 × σ S

(2) LOQ = 10 × σ S

where S is the slope of the calibration curve for LND and FBX, and σ is the standard deviation of the intercept.

The UV-absorption spectra, 3D spectrum, and R f data of LND and FBX in commercially available products were compared to that of pure LND and FBX in order to evaluate the specificity and peak purity of the existing method for assessing LND and FBX simultaneously.

2.6 Application of current protocol in determining LND and FBX simultaneously in commercially available tablets

For the current method, the prepared samples of the commercially available tablets were deposited on normal-phase TLC plates, and the peak area was measured under the identical experimental conditions as the concurrent determination of pure LND and FBX (n = 3). For the current technique, the quantities of LND and FBX in commercially available tablets were approximated using the calibration curves for LND and FBX.

2.7 Greenness evaluation

The proposed protocol for determining LND and FBX simultaneously was assessed for its greenness profile utilizing four different approaches: NEMI [39], AES [41], ChlorTox [46], and AGREE [47]. Preliminary judgment based on persistent, bioaccumulative, and toxic (PBT), waste, hazardous, and corrosive is obtained using NEMI [39]. AES is a semi-quantitative technique that considers instruments, waste, and each step of the analytical process. It is expected that substances requiring minimal to no reagent use, low energy, and no waste will have an ideal analysis with 100 points. Penalty points are awarded and deducted from 100 in total if any of these standards are violated [41]. The ChlorTox scale is computed using Eq. 3 [46] in accordance with the ChlorTox technique

(3) ChlorTox = CH sub CH CH Cl 3 × m sub

where m sub is the mass of the substance of interest needed for a single analysis, CHCHCl3 is the chemical hazard of standard chloroform, and CHsub is the chemical risks of the substance of interest. The safety data sheet from Sigma Aldrich (St. Louis, MO, USA) was used in conjunction with the weighted hazards number (WHN) methodology to determine the values of CHsub and CHCHCl3 [46]. With the help of WHN methodology and safety data sheet from Sigma Aldrich (St. Louis, MO, USA), CHsub values for the substance of interests like EA and EtOH were obtained using Eq. 4:

(4) C H sub = ( 1 × N cat 1 ) + ( 0.75 × N cat 2 ) + ( 0.5 × N cat 3 ) + ( 0.25 × N cat 4 )

where N cat1, N cat2, N cat3, and N cat4 represent the toxicity numbers under the categories of 1, 2, 3, and 4, respectively. For substance EA, N cat1 = 0, N cat2 = 2, N cat3 = 1, and N cat4 = 0 were taken the safety data sheet of Sigma Aldrich (St. Louis, MO, USA).

Hence , for EA , C H sub = ( 1 × 0 ) + ( 0.75 × 2 ) + ( 0.5 × 1 ) + ( 0.25 × 0 ) = 2

For substance EtOH, N cat1 = 0, N cat2 = 2, N cat3 = 0, and N cat4 = 0 were taken from the safety data sheet of Sigma Aldrich (St. Louis, MO, USA).

Hence , for EtOH , C H sub = ( 1 × 0 ) + ( 0.75 × 2 ) + ( 0.5 × 0 ) + ( 0.25 × 0 ) = 1.5

For chloroform, N cat1 = 1, N cat2 = 4, N cat3 = 3, and N cat4 = 1 were taken from the safety data sheet of Sigma Aldrich (St. Louis, MO, USA).

Hence , C H CH Cl 3 = ( 1 × 1 ) + ( 0.75 × 4 ) + ( 0.5 × 3 ) + ( 0.25 × 1 ) = 5.75

The values of m sub for a single analysis are mentioned in in Section 3. Finally, the ChlorTox values were derived using Eq. 3.

The AGREE scale for the proposed methodology for estimating LND and FBX simultaneously was assessed using the AGREE methodology [47]. The AGREE scales for the proposed methodology were derived utilizing the AGREE: The Analytical Greenness Calculator (version 0.5, Gdansk University of Technology, Gdansk, Poland, 2020). The values were based on 12 distinct GAC criteria and ranged from 0.0 to 1.0.

3 Results and discussion

3.1 Method development

The development of an appropriate analytical approach for the simultaneous assessment of LND and FBX involved an examination of both binary and ternary mixtures of green mobile phases. The percentage of EA in the range of 50–90% (v/v) was assessed to be the green mobile phases among binary mixtures of EA and EtOH.

As the green mobile phases, ternary combinations, with varying ratios of EA, EtOH, and H2O, such as EA–EtOH–H2O (50:40:10 v/v/v), EA–EtOH–H2O (60:30:10 v/v/v), EA–EtOH–H2O (70:20:10 v/v/v), and EA–EtOH–H2O (80:10:10 v/v), were studied. Figure 1 shows the saturated chambers used for the development of all the greener solvent systems that were evaluated.

Figure 1 The representative TLC image for standard LND, FBX, and commercially available fixed-dose combination tablet brands A and B established utilizing EA–EtOH–H2O (70:20:10 v/v/v) as the eco-friendly solvent system for the current method.
Figure 1

The representative TLC image for standard LND, FBX, and commercially available fixed-dose combination tablet brands A and B established utilizing EA–EtOH–H2O (70:20:10 v/v/v) as the eco-friendly solvent system for the current method.

Unsatisfactory chromatographic peaks of LND and FBX with unacceptable As for LND (As > 1.40) and FBX (As > 1.40) were obtained in all binary combinations of EA and EtOH. As for LND (As > 1.25) and FBX (As > 1.30) were unreliable, and the ternary combinations EA–EtOH–H2O (50:40:10 v/v/v), EA–EtOH–H2O (60:30:10 v/v/v), and EA–EtOH–H2O (80:10:10 v/v) likewise showed bad chromatographic signals of LND and FBX. The green solvent system EA–EtOH–H2O (70:20:10 v/v/v) was discovered to provide intact, well-separated chromatographic peaks of LND at R f = 0.35 ± 0.01 and FBX at R f = 0.54 ± 0.02 (Figure 2). Furthermore, projections of As values of 1.06 and 1.05 for LND and FBX, respectively, were made; these are all incredibly dependable figures. Therefore, it was decided that, in order to determine LND and FBX simultaneously in commercially available tablets using the current approach, the final solvent solution would be EA–EtOH–H2O (70:20:10 v/v/v). The densitometry mode recording of LND and FBX spectral bands revealed the greatest response at 275 nm. A comprehensive simultaneous determination of the FBX and LND, therefore, took place at 275 nm.

Figure 2 Representative chromatogram of standards LND (R f = 0.35) and FBX (R f = 0.54).
Figure 2

Representative chromatogram of standards LND (R f = 0.35) and FBX (R f = 0.54).

3.2 Validation assessment

A number of parameters were derived for the simultaneous estimation of LND and FBX using the ICH-Q2-R2 protocol [48]. Table 1 displays the findings of the linearity evaluation of the LND and FBX calibration plots using the present method. The calibration curves for the LND and FBX were linear in the 30–1,000 ng·band−1 range. The coefficient of determination (R 2) for LND and FBX was estimated to be 0.9970 and 0.9959, respectively. The correlation coefficient (R) for LND and FBX were found to be 0.9984 and 0.9979, respectively. The values of R 2 and R were highly significant (p < 0.05) for LND and FBX. These results demonstrated a substantial correlation between measured responses and LND and FBX concentrations. These findings demonstrated that the existing approach was linear enough to determine LND and FBX concurrently.

Table 1

Linearity assessment for the concurrent measurement of LND and FBX by the current protocol (mean ± SD; n = 6)

Parameters LND FBX
Linear range (ng·band−1) 30–1,000 30–1,000
Regression equation y = 21.08x + 1160.4 y = 19.403x + 1112.9
R 2 0.9970 0.9959
R 0.9984 0.9979
SE of slope 0.33 0.30
SE of intercept 2.47 2.40
95% CI of slope 19.62–22.53 18.10–20.70
95% CI of intercept 1149.73–1171.06 1102.55–1123.24
LOD ± SD (ng·band−1) 0.95 ± 0.01 1.01 ± 0.02
LOQ ± SD (ng·band−1) 2.84 ± 0.03 3.03 ± 0.06

R 2: determination coefficient; R: correlation coefficient; x: LND or FBX concentration; y: LND or FBX peak area; SE: standard error; CI: confidence interval; LOD: limit of detection; LOQ: limit of quantitation.

The system appropriateness considerations for the present methodology are shown in Table 2. For the current technique, R f, As, and N/m were derived to be adequate for determining LND and FBX simultaneously.

Table 2

System appropriateness criteria for LND and FBX for the present approach (mean ± SD; n = 3)

Parameters LND FBX
R f 0.35 ± 0.01 0.54 ± 0.02
As 1.06 ± 0.03 1.05 ± 0.02
N/m 5178 ± 5.62 5084 ± 5.17

The accuracy of the current method was evaluated by calculating the percentage recovery for the simultaneous calculation of LND and FBX. The findings of accuracy evaluation for the present technique are shown in Table S1. The percentage recoveries of LND and FBX at three different QC solutions were found to be, respectively, 99.5–101.1% and 99.5–101.5% with the aid of the current method. These outcomes showed that the proposed methodology could accurately measure the LND and FBX concurrently.

The intra/inter-assay precision of the proposed protocol was evaluated for determining LND and FBX simultaneously, and the data were reported as the % of the coefficient of variation (% CV). Table 3 displays the results of both precisions for the simultaneous determination of LND and FBX using the proposed methodology. The % CVs of LND and FBX for the intra-day fluctuation were determined to be 0.7–0.9% and 0.8–1.0%, respectively. The inter-day variance % CVs for LND and FBX were derived to be 0.8–0.9% and 0.8–1.1%, respectively. Each of these outcomes demonstrated the precision of the proposed method for concurrently determining LND and FBX.

Table 3

Evaluation of intra/inter-day precision of LND and FBX for the proposed methodology (mean ± SD; n = 6)

Conc. (ng·band−1) Intraday precision Interday precision
Conc. (ng·band−1) ± SD Standard error CV (%) Conc. (ng·band−1) ± SD Standard error CV (%)
LND
150 152.61 ± 1.42 0.57 0.9 149.81 ± 1.46 0.59 0.9
200 203.12 ± 1.69 0.69 0.8 198.41 ± 1.72 0.70 0.8
250 254.32 ± 1.96 0.80 0.7 247.95 ± 2.03 0.82 0.8
FBX
150 149.05 ± 1.61 0.65 1.0 151.64 ± 1.73 0.70 1.1
200 198.84 ± 1.83 0.74 0.9 204.61 ± 1.93 0.78 0.9
250 248.72 ± 2.16 0.88 0.8 255.74 ± 2.27 0.92 0.8

CV: coefficient of variance.

The proposed method’s robustness for the assessment of LND and FBX simultaneously was obtained by implementing deliberate, small adjustments to the recommended mobile phase. Table S2 displays the outcomes of the robustness assessment carried out with the current technique. It was observed that the % CVs for LND and FBX were 0.8–0.9%. The values of LND and FBX R f were discovered to be 0.33–0.37 and 0.53–0.55, respectively. The R f data of LND and FBX, as well as the slight variations in peak response, suggested the proposed method’s robustness.

The current protocol’s sensitivity for determining LND and FBX simultaneously was evaluated as “LOD and LOQ.” The derived values of “LOD and LOQ” for LND and FBX utilizing the current method are displayed in Table 1. The “LOD and LOQ” for LND were determined to be 0.95 ± 0.01 and 2.84 ± 0.03 ng·band−1, respectively, using the current protocol. The “LOD and LOQ” for FBX were determined to be 1.01 ± 0.02 and 3.03 ± 0.06 ng·band−1, respectively, using the current protocol. These results showed that the current protocol for determining LND and FBX simultaneously was highly sensitive.

The proposed methodology for determining LND and FBX simultaneously was assessed for their specificity and peak purity by comparing the R f data, UV-absorption spectra, and 3D spectrum of LND and FBX in commercially available tablet products to that of pure LND and FBX. Figure 3 shows the overlapping UV-absorption spectra of the commercially available tablet products A and B, as well as the standards LND and FBX. Figure S2 shows the 3D spectrum of pure LND, FBX, and commercially available tablets.

Figure 3 UV-absorption spectrum of pure LND and FBX and LND and FBX in commercially available tablets.
Figure 3

UV-absorption spectrum of pure LND and FBX and LND and FBX in commercially available tablets.

The peak purities of pure LND and FBX and LND and FBX in commercially available tablets were assessed by comparing the spectra at the peak start (S), peak apex (M), and peak end (E) positions of the spot [49,50]. The calculated values of r(S,M) and r(M,E) of pure LND and FBX, and commercially available tablet products A and B were calculated to be greater than 0.99, suggesting the peaks’ homogeneity [51,52]. The highest chromatographic response was seen at a wavelength of 275 nm for LND and FBX in standards and commercially available tablets. The identical UV-absorption spectra, 3D spectrum, R f data, and wavelengths discovered in standards and commercially available tablets demonstrated the proposed method’s specificity for simultaneously estimating LND and FBX.

3.3 Application of current protocol in determining LND and FBX simultaneously in commercially available tablets

The recommended protocol was used to determine LND and FBX simultaneously in commercially available tablets, replacing standard liquid chromatography techniques. Commercially accessible tablet products A and B were identified by comparing their chromatographic peaks at R f = 0.35 ± 0.01 for LND and R f = 0.54 ± 0.02 for FBX to that of pure LND and FBX using the proposed methodology. The LND and FBX chromatographic peaks from commercially available products A (Figure S3a) and B (Figure S3b) are shown in Figure S3. These peaks matched those in the LND and FBX standards precisely. In addition, one additional chromatography signal (peak 2 in Figure S3a and S3b) was detected in commercially available products A and B. This additional signal might be associated with the excipient peak in both formulations. The presence of additional peaks indicated that the current methodology could be utilized to measure LND and FBX simultaneously in the presence of formulation excipients. The amount of LND in commercially available products A and B was measured utilizing the current methodology, and the results were 99.24 ± 1.33% and 101.38 ± 1.37%, respectively. The amount of FBX in commercially available products A and B was found to be 98.84 ± 1.24% and 100.79 ± 1.33%, respectively, using the current method. These results proved that the current methodology is suitable for measuring LND and FBX simultaneously in commercially available products.

3.4 Greenness evaluation

Pharmaceutical assays can be evaluated for their greenness utilizing various greenness metric tools, including NEMI [39], GAPI [40], AES [41], RGB [42], EAT [43], AMVI [44], AMGS [45], ChlorTox [46], and AGREE [47]. The greenness of the proposed protocol was evaluated in the current work utilizing four different approaches: NEMI [39], AES [41], ChlorTox [46], and AGREE [47]. The initial evaluation is obtained by the usage of NEMI. The NEMI approach calls for drawing four quarter circles, each of which is either left blank or colored green to represent one of the following requirements [39]: PBT, corrosive, hazardous, and waste. The representative diagram for the NEMI of the current technique is displayed in Figure S4. Since none of the solvents used are toxic, PBT, or corrosive and generate little waste, the present process resulted in four green circles.

AES is a practical semi-quantitative technique that considers all phases of analysis, waste, and tools. The results of AES scales with penalty points are shown in Table 4. Excellent greenness was indicated by an AES rating of more than 75, acceptable greenness was indicated by a scale of less than 75 but more than 50, and inadequate greenness was indicated by a scale of less than 50 [41]. The proposed protocol’s AES scale was derived to be 89, indicating an exceptional greenness profile. The AES scale for synchronous and conventional micelle-enhanced spectrofluorimetric methods has been reported as 88 [27]. The AES scale for an HPLC method has been reported as 87 [28]. All reported methods to determine LND and FBX simultaneously were found to be identical to the current approach based on AES scales [27,28].

Table 4

Penalty point evaluation and AES analysis for the suggested methodology’s environmental friendliness in comparison to reported HPLC methods

Reagents/instruments/waste Penalty points
Synchronous spectrofluorimetry [27] Micelle-enhanced spectrofluorimetry [27] HPLC [28] Present HPTLC
EA — — — 4
EtOH — — — 4
H2O — — — 0
Methanol 6 6 — —
Acetonitrile — — 4 —
Borate buffer (0.2 M) 0 0 — —
Phosphate buffer (30 mM) — — 0 —
Cetrimide — 0 — —
Sodium hydroxide — — 1 —
Phosphoric acid — — 1 —
Instruments 0 0 1 0
Waste 6 6 6 3
Total penalty points 12 12 13 11
AES scale 88 88 87 89

The findings of the individual green solvent ChlorTox scores together with the total ChlorTox for the proposed methodology are displayed in Table 5. The suggested method’s calculated total ChlorTox score was 0.96 g, indicating that it was both environmentally friendly and comparatively safe [46].

Table 5

ChlorTox scale assessment for the suggested approach, which was obtained using the WHN model, in terms of relative dangers with regard to chloroform (CHsub/CHCHCl3 )

Stage Solvent/reagent Relative hazard (CHsub/CHCHCl3 ) m sub (mg) ChlorTox (g) Total ChlorTox (g)
Sample preparation EA 0.34 1,400 0.47
EtOH 0.26 400 0.10
HPTLC analysis EA 0.34 1,400 0.47 0.96
EtOH 0.26 400 0.10

The most popular greenness metric approach for assessing greenness is the AGREE methodology, which considers all 12 GAC principles [47]. The overall AGREE scale and weights assigned to each GAC principle for the current procedure are shown in Figure 4. An AGREE scale of less than 0.75 but greater than 0.50 indicated sufficient greenness, an AGREE scale of less than 0.50 indicated inadequate greenness, and an AGREE score of greater than 0.75 suggested excellent greenness [47]. According to the current technique, 0.81 would be the overall AGREE scale. The AGREE results once again illustrated the excellent green characteristics of the current approach. The overall results of all greenness approaches indicate that the current method to determine LND and FBX simultaneously in commercially available fixed-dose combination tablets has an excellent greener profile.

Figure 4 Overall Analytical GREEnness (AGREE) scale and weightage for individual components of GAC for the present methodology.
Figure 4

Overall Analytical GREEnness (AGREE) scale and weightage for individual components of GAC for the present methodology.

4 Conclusions

There are limited analytical procedures to determine LND and FBX simultaneously in pharmaceutical products and physiological samples. To determine LND and FBX concurrently, there are no conventional or green HPTLC methods available in the literature. This work developed and validated a fast, sensitive, and green HPTLC method to measure LND and FBX simultaneously in commercially accessible products. The current approach for determining LND and FBX simultaneously is linear, highly sensitive, accurate, precise, robust, and green. The LND and FBX contents of commercially available products were effectively ascertained using the current approach. The NEMI, AES, ChlorTox, and AGREE evaluation findings demonstrate the exceptional greenness of the current technique for analyzing LND and FBX concurrently. All of these results showed that LND and FBX in commercially available products may be routinely determined using the existing approach.

Acknowledgments

Authors are thankful to the Researchers Supporting Project number (RSPD2024R1040), King Saud University, Riyadh, Saudi Arabia for supporting this work. The authors also thank Prince Sattam bin Abdulaziz University for supporting this work via project number (PSAU/2024/R/1445). Sultan Alshehri would like to express sincere gratitude to AlMaarefa University, Riyadh, Saudi Arabia for providing funding to conduct this research.

  1. Funding information: This work was funded by the Researchers Supporting Project number (RSPD2024R1040), King Saud University, Riyadh, Saudi Arabia, and Prince Sattam bin Abdulaziz University via project number (PSAU/2024/R/1445).

  2. Author contributions: PA: conceptualization, supervision, Project administration, Methodology, investigation, funding acquisition; FS: methodology, software; data curation, formal analysis, funding acquisition, writing original draft; SA: validation, funding acquisition, visualization, resources; MI: formal analysis, data curation, validation; AIF: methodology, investigation, validation; TMA: methodology, investigation, validation; FMAB: formal analysis, software, data curation, validation; MHA: methodology, investigation, validation. finally, all the authors have read, edited, and approved the final version of the manuscript.

  3. Conflict of interest: Authors state no conflict of interest.

  4. Data availability statement: All data generated or analyzed during this study are included in this published article.

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Received: 2024-01-04
Accepted: 2024-03-07
Published Online: 2024-04-23

© 2024 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  109. Green synthesis, characterization, and in vitro and in vivo biological screening of iron oxide nanoparticles (Fe3O4) generated with hydroalcoholic extract of aerial parts of Euphorbia milii
  110. Novel microwave-based green approach for the synthesis of dual-loaded cyclodextrin nanosponges: Characterization, pharmacodynamics, and pharmacokinetics evaluation
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  112. Green synthesis and characterization of CuO/ZnO nanocomposite using Musa acuminata leaf extract for cytotoxic studies on colorectal cancer cells (HCC2998)
  113. Review Articles
  114. Materials-based drug delivery approaches: Recent advances and future perspectives
  115. A review of thermal treatment for bamboo and its composites
  116. An overview of the role of nanoherbicides in tackling challenges of weed management in wheat: A novel approach
  117. An updated review on carbon nanomaterials: Types, synthesis, functionalization and applications, degradation and toxicity
  118. Special Issue: Emerging green nanomaterials for sustainable waste management and biomedical applications
  119. Green synthesis of silver nanoparticles using mature-pseudostem extracts of Alpinia nigra and their bioactivities
  120. Special Issue: New insights into nanopythotechnology: current trends and future prospects
  121. Green synthesis of FeO nanoparticles from coffee and its application for antibacterial, antifungal, and anti-oxidation activity
  122. Dye degradation activity of biogenically synthesized Cu/Fe/Ag trimetallic nanoparticles
  123. Special Issue: Composites and green composites
  124. Recent trends and advancements in the utilization of green composites and polymeric nanocarriers for enhancing food quality and sustainable processing
  125. Retraction
  126. Retraction of “Biosynthesis and characterization of silver nanoparticles from Cedrela toona leaf extracts: An exploration into their antibacterial, anticancer, and antioxidant potential”
  127. Retraction of “Photocatalytic degradation of organic dyes and biological potentials of biogenic zinc oxide nanoparticles synthesized using the polar extract of Cyperus scariosus R.Br. (Cyperaceae)”
  128. Retraction to “Green synthesis on performance characteristics of a direct injection diesel engine using sandbox seed oil”
https://doi.org/10.1515/gps-2023-0264
Keywords for this article
febuxostat; green HPTLC; greenness evaluation; lesinurad; validation
Creative Commons
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